Name: low-density primary hippocampal neuron culture
Uploaded: 2017-04-18T12:30:00
Description: Watch this Scientific Journal Video about Low-Density Primary Hippocampal Neuron Culture at JoVE.com

Dette arbejde blev støttet af CIHR MOP-142.209 til TJS.

Alle eksperimenter og protokoller anvender forsøgsdyr blev godkendt af University of Manitoba dyreetik udvalg og var i overensstemmelse med retningslinjerne fra den canadiske Rådet om Animal Care. 1. Fremstilling af Instrumenter, Buffere og opløsninger <ol><li> Der steriliseres ved autoklavering alle dissektion udstyr, glas Pasteur-pipetter, pipettespidser, filterapparat, og deioniseret vand. </li><li> Fremstil en 20% (vægt / volumen; 1,1 M) lager glucoseopløsning i deioniseret vand o...

<ol>
	<li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126, 397-425 (1977).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Adamantidis, 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=Optogenetics:+10+years+after+ChR2+in+neurons--views+from+the+community.">Optogenetics: 10 years after ChR2 in neurons--views from the community.</a> Nat Neurosci. 18, 1202-1212 (2015).</li><li>Ho, V. M., Lee, J. A., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+synaptic+plasticity.">The cell biology of synaptic plasticity.</a> Science. 334, 623-628 (2011).</li><li>Huganir, R. L., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPARs+and+synaptic+plasticity:+the+last+25+years.">AMPARs and synaptic plasticity: the last 25 years.</a> Neuron. 80, 704-717 (2013).</li><li>Luscher, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+AMPA+receptor+cycling+in+synaptic+transmission+and+plasticity.">Role of AMPA receptor cycling in synaptic transmission and plasticity.</a> Neuron. 24, 649-658 (1999).</li><li>Varoqueaux, F., 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=Neuroligins+determine+synapse+maturation+and+function.">Neuroligins determine synapse maturation and function.</a> Neuron. 51, 741-754 (2006).</li><li>Huettner, J. E., Baughman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+identified+neurons+from+the+visual+cortex+of+postnatal+rats.">Primary culture of identified neurons from the visual cortex of postnatal rats.</a> J Neurosci. 6, 3044-3060 (1986).</li><li>Lester, R. A., Quarum, M. L., Parker, J. D., Weber, E., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+6-cyano-7-nitroquinoxaline-2,3-dione+with+the+N-methyl-D-aspartate+receptor-associated+glycine+binding+site.">Interaction of 6-cyano-7-nitroquinoxaline-2,3-dione with the N-methyl-D-aspartate receptor-associated glycine binding site.</a> Mol Pharmacol. 35, 565-570 (1989).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35, 567-576 (1993).</li><li>Goslin, K., Asmussen, H., Banker, G., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+low-density+culture.">Rat hippocampal neurons in low-density culture.</a> Culturing nerve cells. 2, 339-370 (1998).</li><li>Hanisch, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+as+a+source+and+target+of+cytokines.">Microglia as a source and target of cytokines.</a> Glia. 40, 140-155 (2002).</li></ol>

Mens metoden &quot;sandwich&quot; med dyrkning neuroner er blevet godt beskrevet andetsteds 2, 11, der er flere trin i hele den protokol, der er ganske vanskeligt at beskrive i tekst alene, hvilket kan føre til frustration for efterforskerne, der ønsker at vedtage det. Fremgangsmåden kan opdeles i tre brede arbejdsgange: glial kultur, dækglas forberedelse og neuron kultur og vedligeholdelsesomkostninger. Hver af de tre præparate...

Hjernen er organiseret i indviklede netværk af neuroner. Bidrag individuelle neuroner til netværksaktivitet og hjernens funktion kan studeres ved selektiv ændring af deres molekylære sammensætning og perturbance af deres fysiologiske egenskaber. Genetisk og kemisk manipulation af individuelle neuroner er velsagtens lettere i dyrkede neuroner end i intakt hjernevæv, er behæftet med sidstnævntes celleheterogenitet og kompleksitet. Neuroner i kultur udvikle veldefineret aksonal og dendritiske dorne og danner omfattende synaptiske forbindelser med hinanden. Mens neuron kultur fra voksent dyr eller fra andre regioner i nervesystemet er muligt, embryonale hippocampuskulturer ofte foretrækkes på grund af deres definerede pyramideformet cellepopulation og relativt lave glial tæthed 1, 2. Hippocampale neuroner dyrket ved lav densitet i kultur er særligt amenable til studiet af subcellulær lokalisering, protein trafficking, neuronal polaritet og synapse udvikling. Neuroner i kultur er også blevet ekstensivt anvendt i at studere molekylære processer i synaptisk plasticitet 3, 4, 5, 6. Neuron kultur præparater fra mus med globale genetiske sletninger, som ikke overlever postnatalt har været særligt nyttigt til at studere cellulære og synaptiske roller visse gener 7. Som i hjernen, dyrkede hippocampale neuroner er afhængige af trofisk støtte fra gliaceller. Dette komplicerer deres kultur, og har ført til udviklingen af ​​flere forskellige metoder, hvorved denne støtte gives. En almindeligt anvendt metode involverer plating neuroner direkte på et monolag af gliaceller 8 eller tillader forurenende gliaceller fra den erhvervede hippocampal væv til at formere sig og danne et monolag under neuronerne 9. Selv om denne fremgangsmåde har fundet en vis succes, urenhed af den resulterende neuronal kultur er ufordelagtig for billeddannelse eksperimenter. En anden almindeligt anvendt fremgangsmåde til neuron kultur er at forlade-out glial fødelag helt, og i stedet tilvejebringe trofisk støtte i form af et defineret vækstmedium 10. Her beskriver vi den &quot;sandwich&quot; eller &quot;Banker&quot; metode til neuron kultur 2, 11. Denne fremgangsmåde involverer udpladning af de hippocampale neuroner på dækglas, som derefter ophængt over et monolag af gliaceller adskilt af paraffin perler. Dette letter langsigtede kultur af en homogen population af neuroner uden at forurene glia samtidig med at trofisk støtte fra den underliggende glial monolag. Når neuroner er af tilstrækkelig alder eller modenhed,Neuron dækglas kan vendes ud af den glial skålen og anvendes ved billeddannelse eller funktionelle assays.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dissection Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors</td>
			<td>Roboz</td>
			<td>RS-5650</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors</td>
			<td>Roboz</td>
			<td>RS-5605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps (#5)</td>
			<td>Roboz</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps (#PP)</td>
			<td>Roboz</td>
			<td>RS-4950</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tissue Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (2.5%)</td>
			<td>Gibco</td>
			<td>15-090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25-200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinnex Filter Holder, 25&#160;mm</td>
			<td>EMD Millipore</td>
			<td>SX0002500</td>
			<td>Used as cell strainer. Assemble first with filter and autoclave</td>
		</tr>
		<tr>
			<td>Isoflorane</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>CP0406V2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>1492</td>
			<td></td>
		</tr>
		<tr>
			<td>Grade 105 Lens Cleaning Tissue</td>
			<td>GE Healthcare</td>
			<td>2105-841</td>
			<td>Used as cell strainer. Assemble first in filter holder and autoclave</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes with cotton filter</td>
			<td>VWR</td>
			<td>14672-412</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15-630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution without Calcium, Magnesium, Phenol Red (HBSS, 10x)</td>
			<td>Gibco</td>
			<td>14-185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>VWR</td>
			<td>14672-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas (Dnase)</td>
			<td>Sigma-Aldrich</td>
			<td>DN25-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Butane bunsen burner</td>
			<td>Wall-Lenk Mfg. Co.</td>
			<td>Model 65</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tissue Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish (100 mm)</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish (60 mm)</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Can be used in place of BGS, but each lot must be tested due to inter-lot variation. Heat-inactivation of serum is recommended.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>Gibco</td>
			<td>11-095-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21-103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25-030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Can be used in place of L-Glutamine in the NBG medium</td>
		</tr>
		<tr>
			<td>Culture Dish (60 mm)</td>
			<td>Corning, Inc</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Flasks (75 cm^2)</td>
			<td>Greiner Bio-One</td>
			<td>658170</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytarabine (Ara-C)</td>
			<td>Sigma-Aldrich</td>
			<td>C3350000</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Growth Serum (BGS)</td>
			<td>HyClone</td>
			<td>SH3054103</td>
			<td>Heat-inactivation is recommended before use.</td>
		</tr>
		<tr>
			<td>B27 Supplement (50x)</td>
			<td>Gibco</td>
			<td>17-504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic Vials</td>
			<td>VWR</td>
			<td>89094-806</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-2-Amino-5-phosphonopentanoic acid (APV)</td>
			<td>Sigma-Aldrich</td>
			<td>A5282</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Coverslip Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate decahydrate (borax)</td>
			<td>Sigma-Aldrich</td>
			<td>B9876-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine Hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoplast Paraffin Wax</td>
			<td>Fisher</td>
			<td>22-900-700</td>
			<td></td>
		</tr>
		<tr>
			<td>Gravity Convection Oven</td>
			<td>VWR</td>
			<td>89511-404</td>
			<td>Used for alternative coverslip cleaning method discussed in protocol</td>
		</tr>
		<tr>
			<td>Ultrasonic Bath (Sonicator)</td>
			<td>Fisher Scientific</td>
			<td>15337400</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric Acid</td>
			<td>Anachemia</td>
			<td>62786-460</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Staining Racks</td>
			<td>Thomas Scientific</td>
			<td>8542E40</td>
			<td>Used for alternative coverslip cleaning method discussed in protocol</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Glaswarenfabrik Karl Hecht&#160;GmbH</td>
			<td>1001/18</td>
			<td>Manufacturer is very important, as neurons do not adhere well to lower quality glass&#160;</td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B0252</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Syringe Filters</td>
			<td>VWR</td>
			<td>28145-477</td>
			<td>Used with BD syringe for filter-sterilization</td>
		</tr>
		<tr>
			<td>Syringe&#160;</td>
			<td>BD</td>
			<td>302832</td>
			<td>Used with VWR sterile syringe filters for fliter-sterilization</td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Scientific</td>
			<td>15-460-16Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Sterile Centrifuge Tubes</td>
			<td>ThermoScientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Sterile Centrifuge Tubes</td>
			<td>ThermoScientific</td>
			<td>339652</td>
			<td></td>
		</tr>
	</tbody>
</table>

low-density primary hippocampal neuron culture

Evnen til at probe struktur og fysiologi af individuelle nerveceller i kultur er afgørende for studiet af neurobiologi, og giver mulighed for fleksibilitet i genetisk og kemisk manipulation af de enkelte celler eller definerede netværk. Sådan lethed af manipulation er enklere i det reducerede dyrkningssystem sammenlignet med det intakte hjernevæv. Mens mange metoder til isolering og vækst af disse primære neuroner findes, hver har sine egne begrænsninger. Denne protokol beskriver en fremgangsmåde til dyrkning med lav massefylde og høj renhed gnaver embryoniske hippocampale neuroner på dækglas, som derefter ophængt over et monolag af gliaceller. Denne &#39;sandwich kultur&#39; giver mulighed for eksklusiv langsigtet vækst af en population af neuroner samtidig med at trofisk støtte fra den underliggende glial monolag. Når neuroner er af tilstrækkelig alder eller modenhed kan neuron dækglas vendes ud af den glial skålen og anvendes ved billeddannelse eller funktionelle assays. neuroner grown ved denne metode typisk overleve i flere uger og udvikle omfattende dorne, synaptiske forbindelser og netværk egenskaber.

I denne &quot;sandwich&quot; metode til primær nerve cellekultur, hippocampusneuroner (figur 3) vokser på et leje af gliaceller (figur 1) adskilt af paraffin perler (figur 2). Dette sikrer, at neuroner selektivt vokse på dækglas med minimal forurening glial celle, men modtager tilstrækkelig trofisk støtte fra glia vokser på Vævsdyrkningsskålen. Typisk kan neuroner opretholdes i kultur i&gt; 3 uger og udvikle omfattende arborisat...

Watch this Scientific Journal Video about Low-Density Primary Hippocampal Neuron Culture at JoVE.com

Low-Density Primary Hippocampal Neuron Culture

Denne artikel beskriver protokollen for dyrkning lav massefylde primære hippocampale neuroner vokser på dækglas inverterede over en glial monolag. Neuron og gliale lag er adskilt af paraffin perler. Neuronerne dyrket ved denne fremgangsmåde er egnede til høj opløsning optisk billeddannelse og funktionelle assays.

Low-Density Primær Hippocampal neuronkultur

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Mus baghjernen<em&gt; Ex Vivo</em&gt; Kultur til Uddannelse Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Primary Culture of Mouse Dopaminergic Neurons

Primær Kultur Mouse dopaminerge neuroner

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Immunhistokemisk Visualisering af Hippocampal Neuron aktivitet efter Spatial Læring i en musemodel af forstyrrelser i nervesystemets udvikling

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

En forenklet metode til Ultra Low Density, Long-Term Primary Hippocampal Neuron Kultur

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral Ganglion Neuron Eksplantering Kultur og Elektrofysiologi på Multi Elektrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...