Name: organotypic slice cultures for studies of postnatal neurogenesis
Uploaded: 2015-03-04T13:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

NOTE: All animal procedures conformed to the animal health and welfare guidelines of the Department of Comparative Medicine at the University of Toronto.
1. Preparation of Hippocampal Slices
<ol>
	<li>Sterilize the following instruments using the dry autoclave at 125 &#176;C: Scalpel handle (#3) (2), Standard pattern forceps, large (1), Small dissector scissor (angled to side) (1), Micro spoon (spoon and flat spatula ends) (1), Micro-spatulas (rounded and rounded tapered ends) (2), Fine pain...

<ol>
	<li>Buchs, P. A., Stoppini, L., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+modifications+associated+with+synaptic+development+in+area+CA1+of+rat+hippocampal+organotypic+cultures.">Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures.</a> Brain research. Developmental Brain Research. 71 (1), 81-91 (1993).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Opitz-Araya, X., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures.">Organotypic hippocampal slice cultures.</a> Journal of Visualized Experiments. (48), (2011).</li><li>Muller, D., Buchs, P. A., Stoppini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+synaptic+development+in+hippocampal+organotypic+cultures.">Time course of synaptic development in hippocampal organotypic cultures.</a> Developmental Brain Research. 71 (1), 93-100 (1993).</li><li>Rio, J. A., Heimrich, B., Soriano, E., Schwegler, H., Frotscher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+glial+fibrillary+acidic+protein-immunoreactive+glial+cells+in+organotypic+slice+cultures+of+rat+hippocampus.">Proliferation and differentiation of glial fibrillary acidic protein-immunoreactive glial cells in organotypic slice cultures of rat hippocampus.</a> Neuroscience. 43 (2-3), 335-347 (1991).</li><li>Ziemka-Nalecz, M., Stanaszek, L., Zalewska, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen-glucose+deprivation+promotes+gliogenesis+and+microglia+activation+in+organotypic+hippocampal+slice+culture:+involvement+of+metalloproteinases.">Oxygen-glucose deprivation promotes gliogenesis and microglia activation in organotypic hippocampal slice culture: involvement of metalloproteinases.</a> Acta Neurobiologiae Experimentalis. 73 (1), 130-142 (2013).</li><li>Subramanian, 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=Transcription+factor+Lhx2+is+necessary+and+sufficient+to+suppress+astrogliogenesis+and+promote+neurogenesis+in+the+developing+hippocampus.">Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (27), E265-E274 (2011).</li><li>Strassburger, M., Braun, H., Reymann, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+treatment+with+the+p38+mitogen-activated+protein+kinase+inhibitor+SB239063+is+neuroprotective,+decreases+the+number+of+activated+microglia+and+facilitates+neurogenesis+in+oxygen-glucose-deprived+hippocampal+slice+cultures.">Anti-inflammatory treatment with the p38 mitogen-activated protein kinase inhibitor SB239063 is neuroprotective, decreases the number of activated microglia and facilitates neurogenesis in oxygen-glucose-deprived hippocampal slice cultures.</a> European Journal Of Pharmacology. 592 (1-3), 55-61 (2008).</li><li>Sadgrove, M. P., Chad, J. E., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kainic+acid+induces+rapid+cell+death+followed+by+transiently+reduced+cell+proliferation+in+the+immature+granule+cell+layer+of+rat+organotypic+hippocampal+slice+cultures.">Kainic acid induces rapid cell death followed by transiently reduced cell proliferation in the immature granule cell layer of rat organotypic hippocampal slice cultures.</a> Brain Research. 1035 (2), 111-119 (2005).</li><li>Wise-Faberowski, L., Robinson, P. N., Rich, S., Warner, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+glucose+deprivation+in+an+organotypic+hippocampal+slice+model+of+the+developing+rat+brain:+the+effects+on+N-methyl-D-aspartate+subunit+composition.">Oxygen and glucose deprivation in an organotypic hippocampal slice model of the developing rat brain: the effects on N-methyl-D-aspartate subunit composition.</a> Anesthesia and Analgesia. 109 (1), 205-210 (2009).</li><li>Cho, S., Wood, A., Brain Bowlby, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=slices+as+models+for+neurodegenerative+disease+and+screening+platforms+to+identify+novel+therapeutics.">slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics.</a> Current Neuropharmacology. 5 (1), 19-33 (2007).</li><li>Noraberg, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures+for+studies+of+brain+damage,+neuroprotection+and+neurorepair.+Current+Drug+Targets.">Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Current Drug Targets.</a> CNS And Neurological Disorders. 4 (4), 435-452 (2005).</li><li>Berdichevsky, Y., 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=PI3K-Akt+signaling+activates+mTOR-mediated+epileptogenesis+in+organotypic+hippocampal+culture+model+of+post-traumatic+epilepsy.">PI3K-Akt signaling activates mTOR-mediated epileptogenesis in organotypic hippocampal culture model of post-traumatic epilepsy.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 33 (21), 9056-9067 (2013).</li><li>Koyama, R., 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=GABAergic+excitation+after+febrile+seizures+induces+ectopic+granule+cells+and+adult+epilepsy.">GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.</a> Nature Medicine. 18 (8), 1271 (2012).</li><li>Staley, K. J., White, A., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+spikes:+harbingers+or+causes+of+epilepsy.">Interictal spikes: harbingers or causes of epilepsy.</a> Neuroscience Letters. 497 (3), 247-250 (2011).</li><li>Lee, H., Lee, D., Park, C. H., Ho, W. K., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+mediates+the+network+activity-dependent+facilitation+of+axonal+outgrowth+from+the+newborn+granule+cells+in+the+early+postnatal+rat+hippocampus.">GABA mediates the network activity-dependent facilitation of axonal outgrowth from the newborn granule cells in the early postnatal rat hippocampus.</a> The European Journal Of Neuroscience. 36 (6), 2743-2752 (2012).</li><li>Raineteau, O., 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=Conditional+labeling+of+newborn+granule+cells+to+visualize+their+integration+into+established+circuits+in+hippocampal+slice+cultures.">Conditional labeling of newborn granule cells to visualize their integration into established circuits in hippocampal slice cultures.</a> Molecular and Cellular Neurosciences. 32 (4), 344-355 (2006).</li><li>Raineteau, O., Rietschin, L., Gradwohl, G., Guillemot, F., Gahwiler, B. H. <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+hippocampal+slice+cultures.">Neurogenesis in hippocampal slice cultures.</a> Molecular And Cellular Neurosciences. 26 (2), 241-250 (2004).</li><li>Kamada, M., 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=Intrinsic+and+spontaneous+neurogenesis+in+the+postnatal+slice+culture+of+rat+hippocampus.">Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampus.</a> The European Journal Of Neuroscience. 20 (10), 2499-2508 (2004).</li><li>Kim, H., Kim, E., Park, M., Lee, E., Namkoong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+culture+from+the+adult+mouse+brain:+a+versatile+tool+for+translational+neuropsychopharmacology.">Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.</a> Progress in Neuro-Psychopharmacology &amp; Biological Psychiatry. 41, 36-43 (2013).</li><li>Legradi, A., Varszegi, S., Szigeti, C., Gulya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+hippocampal+slices+as+in+vitro+models+for+neurodegeneration:+Studies+on+cell+viability+and+apoptotic+processes.">Adult rat hippocampal slices as in vitro models for neurodegeneration: Studies on cell viability and apoptotic processes.</a> Brain Research Bulletin. 84 (1), 39-44 (2011).</li><li>Sadgrove, M. P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. 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=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. 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=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, 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=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., 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=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, 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=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

Following CldU (or BrdU) administration, the timeline of application of pharmacological agents can be chosen to target newborn DGCs during particular developmental windows. For example, a hypothetical agent can be applied during the second week post-CldU injection, which is proposed to coincide with the age of immature neurons that are at a developmental stage where GABA is depolarizing. Future studies using this protocol could adapt the pharmacological agent and the window of exposure to &#8220;tailor&#8221; the approac...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="26">
			<td height="46" rowspan="2" style="height:47px;width:265px;">Name of Reagent/ Equipment</td>
			<td rowspan="2" style="width:131px;">Company</td>
			<td rowspan="2" style="width:135px;">Catalog Number</td>
			<td rowspan="2" style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">5-chloro-2&#39;-deoxyuridine (CldU)</td>
			<td style="width:131px;">MP Biomedicals</td>
			<td align="right" style="width:135px;">105478</td>
			<td style="width:239px;">Hazardous, Carcinogenic</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Cell culture inserts, 30mm diameter, 0.4&#181;m pore size</td>
			<td style="width:131px;">Thermo scientific&#160;</td>
			<td align="right" style="width:135px;">140660</td>
			<td style="width:239px;">Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Conical Centrifuge tubes (sterile)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">14-432-22</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Dissector scissors (angled to side)</td>
			<td style="width:131px;">Fine Science Tools&#160;</td>
			<td style="width:135px;">14082-09</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Minimum essential medium (MEM)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">11095; liquid</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Eclipse Ni-U fluorescent microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Glue for tissue</td>
			<td style="width:131px;">Krazy Glue</td>
			<td style="width:135px;">KG585</td>
			<td style="width:239px;">Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Hank&#8217;s Balanced Salt Solution (HBSS) (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">14025-092</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Horse Serum Heat Inactivated (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">16050-122</td>
			<td style="width:239px;">Make 50 mL aliquots and store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Kimwipes</td>
			<td style="width:131px;">Kimberly-Clarke</td>
			<td style="width:135px;">TW 31KYPBX</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:265px;">Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Petri Dish (100mm x 15mm) and (60mm x 15mm)</td>
			<td style="width:131px;">Fisher Brand</td>
			<td style="width:135px;">FB0875712 and FB0875713A</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel blades #11</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10011-00</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel handle #3</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10003-12</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Serological Pipettes</td>
			<td style="width:131px;">Sorfa Medical Plastic Co.</td>
			<td style="width:135px;">P8050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Standard Pattern forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Sterile vacuum filter</td>
			<td style="width:131px;">Thermo-Scientific</td>
			<td style="width:135px;">565-0020</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Surgical Scissors</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">14054-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Syringe driven filter unit</td>
			<td style="width:131px;">Millipore-Millex</td>
			<td style="width:135px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Tissue chopper with moveable stage</td>
			<td style="width:131px;">Stoelting&#160;</td>
			<td align="right" style="width:135px;">51425</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Fine tip paintbrush</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures for studies of postnatal neurogenesis

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

Determining if organotypic cultures would be suitable for adult neurogenesis research required that they satisfy two main criteria: 1) that slices maintain characteristic morphological features of hippocampal slices after 10-21 days in vitro (DIV), and 2) that newborn DGCs can be quantified using standard immunohistochemical techniques commonly employed in adult neurogenesis research. Regarding the first criterion, Figure 1A and 1B highlight the preserved hippocampal morphology....

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

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

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

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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

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

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...