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.

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

Research

JoVE Journal

Neuroscience

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 ...

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein G&alpha;olf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

Brain Imaging Investigation of the Neural Correlates of Emotion Regulation

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress has been made in understanding conscious/deliberate emotion regulation (ER), less is known about non-conscious/automatic ER and the associated neural correlates. This is in part due to the problems inherent in the unitary concepts of automatic and conscious processing1. Here, we present a protocol that allows investigation of the neural correlates of both deliberate and automatic ER using functional magnetic resonance imaging (fMRI). This protocol allows new avenues of inquiry into various aspects of ER. For instance, the experimental design allows manipulation of the goal to regulate emotion (conscious vs. non-conscious), as well as the intensity of the emotional challenge (high vs. low). Moreover, it allows investigation of both immediate (emotion perception) and long-term effects (emotional memory) of ER strategies on emotion processing. Therefore, this protocol may contribute to better understanding of the neural mechanisms of emotion regulation in healthy behaviour, and to gaining insight into possible causes of deficits in depression and anxiety disorders in which emotion dysregulation is often among the core debilitating features.

We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.

The ability to control/regulate emotions is an important coping mechanism in the face of emotionally stressful situations. Although significant progress ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.