Name: combined mechanical and enzymatic dissociation of mouse brain hippocampal tissue
Uploaded: 2021-10-21T13:30:00
Description: Watch this Scientific Journal Video about Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue at JoVE.com

We thank Aimee Rogers for providing hands-on training and continued product support. We thank Dr. Amanda Burke for ongoing troubleshooting and clarifying discussions. We thank Meredith Joheim and the UAMS Science Communication Group for the grammatical editing and formatting of this manuscript.&#160;This study was supported by NIH R25GM083247 and NIH 1R01CA258673 (A.R.A).

Experiments were conducted in accordance with the ethical standards approved by the Institutional Animal Care and Use Committee at UAMS. 6-month-old female C57Bl6/J wild-type mice were purchased and group-housed (4 mice per cage) under a constant 12 h light/dark cycle.
1. Preparation of reagents
<ol>
	<li>Prepare fixable live/dead stain stock solution. Reconstitute the fluorescent stain with 20 &#181;L of dimethyl sulfoxide (DMSO).</li>
	<li>Wrap the vial in foil, label it as &#34;Reconstitu...

<ol>
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L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mammalian+neural+stem+cells+and+neurogenesis:+Five+decades+later.">Adult mammalian neural stem cells and neurogenesis: Five decades later.</a> Cell Stem Cell. 17, 385-395 (2015).</li><li>Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+microglia+on+neurogenesis.">Effects of microglia on neurogenesis.</a> Glia. 63, 1394-1405 (2015).</li><li>Mu, Y., Lee, S. W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+in+adult+neurogenesis.">Signaling in adult neurogenesis.</a> Current Opinion in Neurobiology. 20, 416-423 (2010).</li><li>Fares, J., Diab, Z. B., Nabha, S., Fares, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+adult+hippocampus:+history,+regulation,+and+prospective+roles.">Neurogenesis in the adult hippocampus: history, regulation, and prospective roles.</a> International Journal of Neuroscience. 129, 598-611 (2018).</li><li>Tosun, M., Semerci, F., Maletic-Savatic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+stem+cells+in+the+hippocampus.">Heterogeneity of stem cells in the hippocampus.</a> Advances in Experimental Medicine and Biology. 1169, 31-53 (2019).</li><li>Abbott, L. C., Nigussie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+in+the+mammalian+dentate+gyrus.">Adult neurogenesis in the mammalian dentate gyrus.</a> Journal of Veterinary Medicine Series C: Anatomia Histologia Embryologia. 49, 3-16 (2020).</li><li>Rodrigues, G. M., Fernandes, T. G., Rodrigues, C. A., Cabral, J. M., Diogo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+human+induced+pluripotent+stem+cell-derived+neural+precursors+using+magnetic+activated+cell+sorting.">Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.</a> Methods in Molecular Biology. 1283, 137-145 (2015).</li><li>Ko, I. K., Kato, K., Iwata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+analysis+of+multiple+surface+markers+expressed+on+rat+neural+stem+cells+using+antibody+microarrays.">Parallel analysis of multiple surface markers expressed on rat neural stem cells using antibody microarrays.</a> Biomaterials. 26, 4882-4891 (2005).</li><li>Jager, L. 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=Effect+of+enzymatic+and+mechanical+methods+of+dissociation+on+neural+progenitor+cells+derived+from+induced+pluripotent+stem+cells.">Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells.</a> Advances in Medical Sciences. 61 (1), 78-84 (2017).</li><li>Volovitz, I., 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+non-aggressive,+highly+efficient,+enzymatic+method+for+dissociation+of+human+brain-tumors+and+brain-tissues+to+viable+single-cells.">A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells.</a> BMC Neuroscience. 17, 1-10 (2016).</li><li>Adult brain dissociation kit: mouse and rat. Miltenyi Biotec Available from: <a target="_blank" href="https://www.miltenyibiotec.com/upload/assets/IM0011920.pdf">https://www.miltenyibiotec.com/upload/assets/IM0011920.pdf</a> (2020)</li><li>Cell surface flow cytometry staining protocol. Miltenyi Biotec Available from: <a target="_blank" href="https://www.miltenyibiotec.com/US-en/applications/all-protocols/cell-surface-flow-cytometry-straining-protocol-pbs-bsa-1-50.html">https://www.miltenyibiotec.com/US-en/applications/all-protocols/cell-surface-flow-cytometry-straining-protocol-pbs-bsa-1-50.html</a> (2021)</li><li>Finak, G., Jiang, W., Gottardo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CytoML+for+cross-platform+cytometry+data+sharing.">CytoML for cross-platform cytometry data sharing.</a> Cytometry Part A: The Journal of the International Society for Analytical Cytology. 93, 1189-1196 (2018).</li><li>Tura&#231;, G., 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=Combined+flow+cytometric+analysis+of+surface+and+intracellular+antigens+reveals+surface+molecule+markers+of+human+neuropoiesis.">Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.</a> PLoS One. 8, 1-14 (2013).</li><li>Schwarz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescence+activated+cell+sorting+to+examine+cell-type-specific+gene+expression+in+rat+brain+tissue.">Using fluorescence activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.</a> Journal of Visualized Experiments: JoVE. (99), e52537 (2015).</li><li>Brummelman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=application+and+computational+analysis+of+high-dimensional+fluorescent+antibody+panels+for+single-cell+flow+cytometry.">application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry.</a> Nature Protocols. 14, 1946-1969 (2019).</li><li>Recommended controls for flow cytometry. Abcam Available from: <a target="_blank" href="https://www.abam.com/protocols/recommended-conntrols-for-flow-cytometry">https://www.abam.com/protocols/recommended-conntrols-for-flow-cytometry</a> (2021)</li><li>. Fixing cells with paraformaldehyde (PFA) for flow cytometry Available from: <a target="_blank" href="https://flowcytometry.utoronto.ca/wp-content/uploads/2016/02/FixingCells_PFA.pdf">https://flowcytometry.utoronto.ca/wp-content/uploads/2016/02/FixingCells_PFA.pdf</a> (2021)</li><li>Stewart, J. C., Villasmil, M. L., Frampton, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+fluorescence+intensity+of+selected+leukocyte+surface+markers+following+fixation.">Changes in fluorescence intensity of selected leukocyte surface markers following fixation.</a> Cytometry Part A: The Journal of the International Society for Analytical Cytology. 71, 379-385 (2007).</li><li>Armand, E. J., Li, J., Xie, F., Luo, C., Mukamel, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing+of+brain+cell+transcriptomes+and+epigenomes.">Single-cell sequencing of brain cell transcriptomes and epigenomes.</a> Neuron. 109, 11-26 (2021).</li><li>Ho, H., 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+guide+to+single-cell+transcriptomics+in+adult+rodent+brain:+The+medium+spiny+neuron+transcriptome+revisited.">A guide to single-cell transcriptomics in adult rodent brain: The medium spiny neuron transcriptome revisited.</a> Frontiers in Cellular Neuroscience. 12, 1-16 (2018).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+technologies+in+studying+brain+immune+response.">Novel technologies in studying brain immune response.</a> Oxidative Medicine and Cellular Longevity. 2021, 6694566 (2021).</li><li>Liu, H., 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=DNA+methylation+atlas+of+the+mouse+brain+at+single-cell+resolution.">DNA methylation atlas of the mouse brain at single-cell resolution.</a> bioRxiv. , (2020).</li><li>Mattei, 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=Enzymatic+dissociation+induces+transcriptional+and+proteotype+bias+in+brain+cell+populations.">Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations.</a> International Journal of Molecular Sciences. 21, 1-20 (2020).</li><li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+microdissected+mouse+brain+tissue+for+artifact+free+single-cell+RNA+sequencing.">Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing.</a> STAR Protocols. 2 (2), 100590 (2021).</li><li>Delmonte, O. M., Fleisher, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+Surface+markers+and+beyond.">Flow cytometry: Surface markers and beyond.</a> The Journal of Allergy and Clinical Immunology. 143, 528-537 (2019).</li><li>O'Connor, J. E., 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=The+relevance+of+flow+cytometry+for+biochemical+analysis.">The relevance of flow cytometry for biochemical analysis.</a> IUBMB Life. 51, 231-239 (2001).</li><li>Menon, V., Thomas, R., Ghale, A. 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Several steps in this neural dissociation protocol require proficient technique and dexterity&#8211;perfusion, supernatant aspiration, and myelin removal. Throughout the perfusion process, the internal organs must remain intact (aside from removing the diaphragm and clipping the heart); this includes avoiding the upper chambers of the heart with the butterfly needle. While the amount of saline with heparin needed varies, transparent fluid flowing from the heart indicates the process is complete. The brain must be complet...

The hippocampus was first described by a Bolognese anatomist, Giulio Cesare Aranzio, in the 1500&#39;s1. In naming this newfound structure, Aranzio was likely inspired by its uncanny resemblance to the seahorse of the genus Hippocampus1. The hippocampus is involved in stress responses but is widely known for its role in learning and memory. More specifically, the hippocampus is responsible for the encoding and retrieval of declarative and spatial memory1.
The hippocampus, or hippocampus proper, is divided into the CA1 (cornu ammonis), CA2, and CA3 subfields1. Compared to the rest of the nervous system, the hippocampus has several unique defining characteristics, including its plasticity and potential for ongoing neurogenesis2. Neurogenesis is the process of the proliferation and differentiation of neural stem cells, followed by their integration into the pre-existing neuronal network. Neurogenesis is restricted to the subgranular zone of the dentate gyrus and subventricular zone of the lateral ventricles (and the olfactory bulbs)3. While neurogenesis is abundant in embryogenesis, it is a lifelong process3,4. As such, this discussion will focus on adult neurogenesis in the hippocampus.
The subventricular and subgranular zones are neurogenic niches containing ependymal and vascular cells, as well as immature and mature lineages of neural stem cells5. Microglia contribute to these niches as immune cells to regulate neurogenesis6. Neural progenitor cells are nonstem cell progenies of neural stem cells7. Three types of neural progenitors are present in the subventricular zone: radial glia-like type B cells, type C transit-amplifying progenitors, and type A neuroblasts3,8. The slowly dividing type B neural progenitor cells in the subventricular zone can differentiate into rapidly dividing type C cells8. Subsequently, type C cells differentiate into type A cells8. These neuroblasts migrate through the rostral migratory stream to the olfactory bulb before differentiating into interneurons or oligodendrocytes9. These olfactory bulb interneurons are key to olfactory short-term memory, and associative learning, whereas the oligodendrocytes myelinate axons of the corpus callosum9. The majority of adult neurogenesis occurs in the subgranular zone of the dentate gyrus, where radial type 1 and nonradial type 2 neural progenitors are found3. Most neural progenitor cells are destined to become dentate granule neurons and astrocytes10. Connected by gap junctions, astrocytes form networks to modulate plasticity, synaptic activity, and neuronal excitability5. As the primary excitatory neuron of the dentate gyrus, granule cells provide input from the entorhinal cortex to the CA3 region11.
Neural stem cell populations can be isolated using immunomagnetic or immunofluorescent isolation strategies12,13. Neural tissue is particularly difficult to dissociate; efforts to do so often result in samples with poor cell viability and/ or fail to produce the necessary single-cell suspension for downstream analysis. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination of techniques14,15. In a study evaluating neural dissociation methods, the viability and quality of manual mechanical dissociation by pipette trituration versus combinations of pipette trituration and digestion with various enzymes were compared15. Quality was graded based on the amount of cell clumps and DNA or subcellular debris in the prepared suspension15. Suspensions of glial tumors subjected to manual mechanical dissociation alone had significantly lower cell viability than treatments with dispase or a combination of DNase, collagenase, and hyaluronidase15. Volovitz et al. acknowledged the variation in viability and quality between the different methods and emphasized that inadequate dissociation may reduce the accuracy of downstream analysis15.
In a separate study, the authors compared over 60 different methods and combinations of dissociation of cultured neuronal cells14. These methods included eight different variations of manual mechanical dissociation by pipette trituration, a comparison of incubation with five individual enzymes at three different intervals, and various combinations of mechanical dissociation with enzymatic digestion or the combination of two enzymes14. None of the mechanical methods yielded a single-cell suspension14. Four of the single enzyme treatments, ten of the combination enzymatic treatments, and four of the combinations of mechanical dissociation with enzymatic digestion yielded a single-cell suspension14. Enzymatic digestion with TrypLE followed by Trypsin-EDTA most effectively dissociated samples14. Incidentally, samples treated with TrypLE and/or Trypsin-EDTA tended to form gelatinous clumps14. While this study was performed on cultured cells, it speaks to the shortcomings of pipette trituration or enzymatic digestion alone.
Side-by-side comparisons of manual versus automated mechanical dissociation are lacking. However, one group ran flow cytometry to compare manual and semi-automated mechanical dissociation of whole mouse brains in conjunction with commercial papain or trypsin enzymatic dissociation kits16. Processing with the dissociator more consistently yielded viable cells16. Following dissociation, the authors also isolated Prominin-1 cells, neuronal precursor cells, and microglia16. For two of the three isolated cell populations, the purity of the isolated cells was slightly higher when samples were processed with the dissociator, as compared to manually16. Rei&#223; et al. noted that person-to-person variability in pipetting technique hinders reproducibility of viable cell population yield in tissue dissociation16. The authors concluded that automated mechanical dissociation standardizes sample processing16.
The method of dissociation outlined in this manuscript is a combination of fully automated mechanical dissociation and enzymatic digestion, using solutions accompanying a commercial adult brain dissociation kit17. Unlike standard protocols, this optimized protocol reduces sample manipulation, yields a highly viable single-cell suspension, and is intended for processing minimal amounts of starting tissue.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>02-682-003</td>
			<td>Basix, assorted color</td>
		</tr>
		<tr>
			<td>15 mL Falcon Tubes</td>
			<td>Becton Dickinson Labware Europe</td>
			<td>352009</td>
			<td>Polystyrene</td>
		</tr>
		<tr>
			<td>25 mL Serological Pipets</td>
			<td>Fisher Scientific</td>
			<td>14-955-235</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Vacuum Filter/ Storage Bottle System</td>
			<td>Corning</td>
			<td>431097</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Adult Brain Dissociation Kit</td>
			<td>Miltenyi Biotec</td>
			<td>&#160;130-107-677</td>
			<td>Contains Enzyme P, Buffer Z, Buffer Y, Enzyme A, Buffer A, Debris Removal Solution</td>
		</tr>
		<tr>
			<td>Aluminum Foil</td>
			<td>Fisher Scientific</td>
			<td>01-213-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-ACSA-2-PE-Vio770, mouse, clone REA969</td>
			<td>Miltenyi Biotec</td>
			<td>130-116-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Myelin Basic Protein</td>
			<td>Sigma-Aldrich</td>
			<td>M3821-100UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PSA-NCAM-PE, human, mouse and rat, Clone 2-2B</td>
			<td>Miltenyi Biotec</td>
			<td>130-117-394</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-VioBlue, mouse, Clone REA592</td>
			<td>Miltenyi Biotec</td>
			<td>130-113-810</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 Antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-111-541</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Hot Plate Stirrer</td>
			<td>Fisher Scientific</td>
			<td>11-100-100SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>BD</td>
			<td></td>
			<td>(v10.7.0)</td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco DPBS (1X)</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Beaker</td>
			<td>Fisher Scientific</td>
			<td>02-555-25A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Fresenius Kabi</td>
			<td>504011</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>ThermoFisher</td>
			<td>L34965</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stir Bar</td>
			<td>Fisher Scientific</td>
			<td>14-513-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Noyes Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15012-12</td>
			<td>Dissection</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>441244-3KG</td>
			<td>Prilled, 95%</td>
		</tr>
		<tr>
			<td>Pipette tips GP LTS 20 &#181;L 960A/10</td>
			<td>Rainin</td>
			<td>30389270</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips GP LTS 250 &#181;L 960A/10</td>
			<td>Rainin</td>
			<td>30389277</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips RT LTS 1000 &#181;L FL 768A/8</td>
			<td>Rainin</td>
			<td>30389213</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainin Pipet-Lite XLS (2, 20, 200, 1000 &#956;L)</td>
			<td>Rainin</td>
			<td>30386597</td>
			<td></td>
		</tr>
		<tr>
			<td>RBXMO FITC XADS</td>
			<td>Fisher Scientific</td>
			<td>A16167</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Ice Bucket with Lid</td>
			<td>Fisher Scientific</td>
			<td>07-210-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Tubes with Cell Strainer Cap</td>
			<td>Falcon</td>
			<td>100-0087</td>
			<td></td>
		</tr>
		<tr>
			<td>S1 Pipet Fillers</td>
			<td>ThermoFisher Scientific</td>
			<td>9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula &#38; Probe</td>
			<td>Fine Science Tools</td>
			<td>10090-13</td>
			<td>Dissection &#38; Perfusion</td>
		</tr>
		<tr>
			<td>Surflo Winged Infusion Set 23 G x 3/4&#34;</td>
			<td>Termuno</td>
			<td>SV-23BLK</td>
			<td>Butterfly needle</td>
		</tr>
		<tr>
			<td>Test Tube Rack</td>
			<td>Fisher Scientific</td>
			<td>14-809-37</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Legend XTR Centrifuge</td>
			<td>ThermoFisher</td>
			<td>discontinued</td>
			<td>Or other standard table top centrifuge</td>
		</tr>
		<tr>
			<td>Variable-Flow Peristaltic Pump</td>
			<td>Fisher Scientific</td>
			<td>13-876-2</td>
			<td>Low-flow model</td>
		</tr>
		<tr>
			<td>VetFlo Starter Kit for Mice</td>
			<td>Kent Scientific</td>
			<td>VetFlo-MSEKIT</td>
			<td>Anesthesia mask, tubing, induction chamber, charcoal canisters</td>
		</tr>
		<tr>
			<td>VetFlo Vaporizer Single Channel Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>VetFlo-1210S</td>
			<td>0.2&#8211;4 LPM</td>
		</tr>
		<tr>
			<td>Vi-CELL XR Cell Viability Analyzer</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>731196</td>
			<td>Cell Counting</td>
		</tr>
		<tr>
			<td>Vi-CELL XR 4 Bags of Sample Vials</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>383721</td>
			<td>Cell Counting</td>
		</tr>
	</tbody>
</table>

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.

Samples were processed with a flow cytometer at a core facility, and the resulting data were evaluated with a software package for flow analysis. Previously, compensation controls were analyzed-the live/dead stain and negative control. If multiple fluorochromes are used, fluorescence minus one (FMO) controls and single-stain controls should be prepared for each antibody. Compensation for spectral overlap for the experimental samples was calculated based on the analyzed controls. For cell population identification, a hier...

Watch this Scientific Journal Video about Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue at JoVE.com

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

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 ...

Successfully dissociating small quantities of neural tissue can equip labs to gain structure-specific insight into treatment efficacy, cellular function, as well as disease and treatment mechanisms of action. This neural dissociation protocol consistently yields a highly viable and actual single-cell suspension. In addition, we can process samples that are only a fraction of those that the commercial kits are intended for.Proper planning and preparation are key to a successful outcome for this technique. Running several practice rounds to familiarize yourself with the protocol would be beneficial. After anesthetizing a six-month-old female C57BL/6J mouse, pinch the lower abdomen and lift the skin using forceps.Then using scissors, cut through the fur and skin to the bottom of the ribcage. Make two diagonal incisions beginning below the ribcage and moving toward each shoulder. Then carefully resect the diaphragm and ribcage to expose the heart.After carefully removing any connective tissue around the heart, use scissors to clip the right atrium. Then turn off the isoflurane flow to the breather. Holding the heart steady with forceps and with the bevel of the butterfly needle facing up, pierce the left ventricle while keeping the needle level and parallel to the animal.Next, holding the needle in place, turn on the pump and perfuse at least 30 milliliters of the saline or heparin solution until the fluid leaving the heart is opaque and the liver and lungs pale in color. After perfusion, turn off the pump, remove the needle, and transfer the mouse to the dissection area. After decapitation, cut the fur from the back of the head up to the eyes and peel the skin back to expose the skull.Next, clip the skull between the eyes and make two cuts at the back of the skull at 10 and 2 o'clock positions. Then make one long cut along the midsagittal line of the skull to the original cut between the eyes. Next, using forceps, peel the two halves of the skull away to the sides.Then use a spatula to remove the brain and place it in a 60 millimeter glass Petri dish filled with cold DPBS and kept on ice. Using a scalpel or razor, separate each hemisphere and remove the olfactory bulbs and cerebellum. Then remove the midbrain until the hippocampus is exposed.Next, secure the brain with forceps. Then using a second set of forceps, gently tease the hippocampus out of each hemisphere. Transfer both hippocampi to a labeled 1.5 milliliter tube containing cold DPBS and place the tube on ice.Using forceps, transfer the hippocampi tissue pieces to a C tube and add 30 microliters of enzyme mix 2 into the tube. After twisting the cap and tightening until it clicks, place the tube in a dissociator and run the appropriate program. While the program is running, pre-wet a 70 micron cell strainer placed on a 50 milliliter conical tube with two milliliters of BSA buffer.After the dissociation program is complete, add four milliliters of BSA buffer to the dissociated tissue and filter the mixture through the cell strainer on the 50 milliliter conical tube. Next, add 10 milliliters of DPBS to the C tube. Then close the tube, swirl the solution gently, and filter it through the cell strainer on the 50 milliliter conical tube.Centrifuge the filtered cell suspension, discard the supernatant without disturbing the pellet and store the pellet. For debris removal, resuspend the pellet with 1, 550 microliters of cold DPBS and transfer the suspension to a labeled 50 milliliter conical tube. Then add 450 microliters of cold debris removal solution and pipette up and down.Gently overlay the cell suspension with one milliliter of cold DPBS, keeping the tip against the wall of the conical tube. Repeat the procedure until the total overlay is two milliliters. Next, centrifuge the suspension at 3, 000 times G for 10 minutes with full acceleration and full break.Aspirate the topmost layer, then sweep the pipette tip back and forth to aspirate the white middle layer. Remove as much of the middle layer as possible without disturbing the bottommost layer. Next, add two milliliters of cold DPBS and pipette up and down to mix.Then centrifuge the suspension at 1, 000 times G for 10 minutes with full acceleration and full break. After centrifugation, discard the supernatant and resuspend the pellet in one milliliter BSA buffer. After cell counting, centrifuge the remaining cell suspension and resuspend the pellet in 50 microliters of diluted live dead stain.Then transfer the sample to a labeled flow tube and incubate at room temperature for 8 to 10 minutes in the dark. After incubation, add 500 microliters of BSA buffer and repeat the centrifugation step. Then discard the supernatant, leaving a small amount of buffer in the tube.The primary gate excluded debris in the forward scatter versus side scatter plots and the dead cells were subsequently excluded. The second gate excluded cells positive for myelin basic protein. And of the remaining cells, density plots of cells positive for each fluorochrome were created.The frequency of each neuronal cell population was calculated out of the third gate. Samples processed with manual mechanical dissociation and enzymatic digestion yielded a substantially lower population of cells of interest, whereas both fresh and fixed samples processed with automated mechanical dissociation and enzymatic digestion showed a several-fold higher population of cells of interest. While the perfusion and debris removal steps can be challenging initially, maintaining a smooth and steady hand can help ensure success.

combined-mechanical-enzymatic-dissociation-mouse-brain-hippocampal

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish.
Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Three-photon microscopy enables high-contrast fluorescence imaging deep in living biological tissues, such as mouse and zebrafish brains, with high spatiotemporal resolution.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo ...

Murine Embryonal Hypothalamus Extraction, Collection, and Tissue Dissociation

Start by fire-polishing three pasture pipettes using a bunsen burner, creating decreasing diameters. Next, sterilize the abdomen of a euthanized pregnant mouse dam with 70%ethanol. Cut open the abdominal cavity from the pubic synthesis to the xiphoid process of the rib cage.Now, extract the uterine horn from the abdominal cavity and place it in a 100-millimeter plate filled with ice-cold HBSS solution. Extract and separate all embryos from the washed uterus using fine forceps. Quickly decapitate the extracted mice embryos and place the heads in a 60-millimeter Petri dish filled with HBSS.Place a pair of fine forceps in the eye cavity to hold the brain. With another pair, peel the skin and skull until the brain is visible. Scoop the brain out of the olfactory bulbs from the skull and flip it upside down.Observe the cortex on the ventral side and the hypothalamus on the dorsal side. Use curved forceps to remove the layer of meninges and blood vessels until the brain appears white and clear, and separate the hypothalamic area from the rest of the brain. Cut the hypothalamus into three to four small fine pieces, and use a pipette to transfer the pieces into a 15-milliliter tube.Next, fill the tube with six milliliters of HBSS. Then add Enzyme Mix 1 once the tissue has settled and the supernatant removed. Incubate the tube in a 37-degree Celsius water bath for 15 minutes, agitating every five minutes to re-suspend the tissue.Now, add 30 microliters of Enzyme Mix 2 and dissociate the tissue by pipetting up and down 10 times with a large diameter pasture pipette. After an additional 10-minute incubation, add the remaining 15 microliters of Enzyme Mix 2. Utilizing two fire polished pipettes of decreasing diameter, pipette the tissue 10 times.Immediately add 10 milliliters of HBSS with 0.5%BSA to the tube. Then centrifuge the tube at 300 G for 10 minutes at room temperature. Aspirate the supernatant and re-suspend the cell pellet in a one milliliter of HBSS with 0.5%BSA.