Review ArticleOrganotypic Cultures as a Model to Study Adult Neurogenesis inCNS Disorders

Fabio Cavaliere Monica Benito-Muntildeoz and Carlos Matute

Departamento de Neurociencias Achucarro Basque Center for Neuroscience Universidad del Paıs Vasco (UPVEHU)and Centro de Investigacion Biomedica en Red en Enfermedades Neurodegenerativas (CIBERNED) 48940 Leioa Spain

Correspondence should be addressed to Fabio Cavaliere fabiocavaliereehueus

Received 17 December 2015 Accepted 22 March 2016

Academic Editor Weixiang Guo

Copyright copy 2016 Fabio Cavaliere et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Neural regeneration resides in certain specific regions of adult CNS Adult neurogenesis occurs throughout life especially fromthe subgranular zone of hippocampus and the subventricular zone and can be modulated in physiological and pathologicalconditions Numerous techniques and animal models have been developed to demonstrate and observe neural regeneration butin order to study the molecular and cellular mechanisms and to characterize multiple types of cell populations involved in theactivation of neurogenesis and gliogenesis investigators have to turn to in vitromodels Organotypic cultures best recapitulate the3D organization of the CNS and can be explored taking advantage of many techniques Here we review the use of organotypiccultures as a reliable and well defined method to study the mechanisms of neurogenesis under normal and pathological conditionsAs an example we will focus on the possibilities these cultures offer to study the pathophysiology of diseases like Alzheimer diseaseParkinsonrsquos disease and cerebral ischemia

1 Introduction

The use of organotypic cultures in neuroscience researchcovers the experimental gap between the in vitro and in vivomodels It provides an opportunity to cultivate CNS tissuefor weeks or months giving open accessibility to complexcellular systems Organotypic cultures are mainly preparedfrom P3ndashP10 animals (rats or mice) or with some exceptionfrom adult CNS tissues (eg [1]) Young postnatal animalsalready possess essential cytoarchitecture and are easy tohandle with respect to the embryonic tissue and nerve cellssurvive in the explantsmore than in adult slices Neverthelessthe neurodegenerative diseases linked to ageing and adultbrain present characteristics one cannot model with culturesfrom young animals Particularly young brain differs fromthe adult brain in terms of synaptic development and geneticandmetabolic profilesThe first attempt to culture brain slicesfrom adult rats and mouse failed because of their ability toreduce the thickness Slices from young or perinatal animalcan reduce their thickness from 350ndash400mm to 100mm after1 or 2 weeks of incubation while the mature adult slices

nearly kept their thickness over a two-week cultivation periodwith consequent necrosis of the central cellular layers (see [1]for a review) Several technical clues were used to overcomeand implement the technique to culture organotypic slicesfrom adult Progressive reduction of serum in the culturemedium allowed increasing cell viability in 6ndash8-week-oldmice cultures [2] It is still unclear why complete withdrawalof serum resulted successfully in prolonging cell viability butone can speculate that negative effects of serum might becaused by excessive neurotrophic and energy resources [3]

The advantage of using organotypic cultures derives fromtheir usefulness in experiments that require long-term sur-vival such as live recording [4 5] or pharmacology (chronicdrug application) as well as electrophysiology and optogenet-ics [6]

The first attempt to maintain CNS slice tissues in long-term cultures has been the ldquoroller tuberdquo technique [7] Thistechnique finally characterized more in detail by Gahwilerand Knopfel [8 9] was developed on the basis of experiencesbased on multitude of studies with explants culture [10]

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 3540568 6 pageshttpdxdoiorg10115520163540568

2 Stem Cells International

CortexHippocampusentorhinal cortex

HippocampusSVZstriatumcortex

Cerebellum

(a)

cp

lv

sn

cx

Substantia nigrastriatumSVZcortex

(b)

Figure 1 Different organotypic slices can be obtained from P2 to P7 postnatal forebrain or cerebellum by coronal sections of 350ndash400 120583m(a) In (b) the brain sectioning to obtain 45∘ transversal sections of s nigrastriatumSVZcortex (sn substantia nigra cp caudate putamenlv lateral ventricle cx cortex) used to model PD is shown

In the roller tube cultures the tissue is embedded in aplasma clot and attached on a glass coverslip The coverslipwith the embedded slice tissue is located in a tube that under-goes continuous slow rotation in a cell culture incubatorTheoxygenation is maintained by continuous exchange of liquid-gas interface generated by the slow rotation The techniquewas successively modified several times (eg [11 12]) butthe roller tube technique always yields very thin cultures(from an initial 400 120583m to about 50 120583m) with consequentialpreferential use for experiments that require optimal opticalconditions (eg electron microscopy or electrophysiology)

At the beginning of the 90s Stoppini and colleagues[13] published a new method to cultivate organotypic slicesIn this method brain slices were placed on a semiporousmembrane and cultivated at the air-liquid interface Theabsence of clot facilitates the studies of synaptic reorga-nization and became a useful tool to study plasticity andsprouting already during the first days of culture The realadvantage of this technique is that cultures are easily preparedand offer great advantages when a 3D structure is desired(from an initial 400120583m thickness slices are cultivated upto 100ndash150 120583m) As described more in detail below theair-liquid interface has become a key instrument to studyadult neurogenesis Organotypic slices can be obtained fromdifferent brain regions (as described in Figure 1) but forthe study of adult neurogenesis in normal and pathologicalconditions the hippocampal region containing the SGZ andthe slices containing the SVZ are most preferred Thus forexample neurogenesis in Alzheimer disease or Parkinsonrsquosdisease can be studied in hippocampusentorhinal cortexor s nigrastriatumSVZcortex slices [14 15] Organotypiccultures match the tridimensional space where neural pro-genitors migrate to reach maturation in vivo In the paperby Vergni and colleagues [16] we ideally represented theslice culture comprising subventricular zone as the spatialextension to elaborate amathematicalmodel to describe neu-roblasts activation and migration following oxygen and glu-cose deprivation

2 Adult Neurogenesis in CNS

Neurogenesis in the normal adult brain occurs mainly in thedentate gyrus from the subgranular zone of hippocampus(SGZ) and in the olfactory bulb from the subventricularzone of lateral ventricle (SVZ) In both cases the neurogenicniches the SGZ and SVZ host multipotent cells that give riseto neuroblasts or glioblasts throughout life The stem cells inthe neurogenic niches (type 1 in the SGZor B cells in the SVZ)give rise to the more proliferative transit-amplifying progeni-tors (type 2 or C cells) that in turn give rise to neuroblast (type3 or A cells) or glioblasts [17]The study of adult neurogenesisin the SGZ is relevant especially for the study of repairmecha-nism of neurodegeneration related to loss of memory or neu-ropsychiatric disorders (for a review see [18]) New neurob-lasts generated in the SGZmigrate to the granular zone of thedentate gyrus covering short distances whereas neuroblastsgenerated in the SVZ move through the rostral migratorystream (RMS) to the olfactory bulb (OB) where finally theymigrate radially and differentiate into new neurons

Adult neural stem cells (NSC) are a specialized formof glia deriving from the embryonic radial glia [19] Theypossess a regional pattern within the SVZ which is relatedto their embryonic origin and reflect the ability to generatedifferent types of OB interneurons (eg granule cells TH+or calbindin+ periglomerular cells see [20]) The neuro-genic moiety is guaranteed by different subpopulation ofmultipotent cells characterized by the expression of differentmarkers in different temporal stages as shown inTable 1 NSCcan be activated and neurogenesis can be induced underdifferent conditions A predominant role in vivo is coveredby the blood vessel and local vascular plexus that bringtrophic factors or stress molecule signals This was recentlydemonstrated by Katsimpardi et al [21] who potentiatedthe neurogenesis of old animals after transfusion of bloodfrom younger animals The absence of local circulation inthe organotypic cultures is fixed by using different trophicfactors in the medium that stimulate intrinsic signals mainly

Stem Cells International 3

Table 1 SVZ neural stem cell characterization by cellular marker expression

GFAP CD133 EGFR Nestin DCX 120573III tubulin NeuN PDGFR ProliferationB cells

qNSC + + + ++ minus minus minus minus minus minus minus

aNSC + + + ++ ++ + minus minus minus + +

C cellsTransit amplifying + minus + + + + minus minus minus ++ + + +

A cellsNeuroblast minus minus minus + + + + + minus minus ++

Immature neurons minus minus minus minus minus + + + + minus +

Glioblast + + + minus minus + minus minus minus + + + +

Proliferation is expressed by BrdU incorporation ability (Codega et al 2014) [17] Quiescent neural stem cells qNSC and activated neural stem cells aNSC

transcription factors for example sox2 olig2 or the bmpfamily [22 23]

3 Techniques Used to StudyNeurogenesis in OC

From a technical point of view the organotypic cultures rep-resent a versatile tool to study neurogenesis or cell regenera-tion In vivo neurogenesis is a multistep process that involvesproliferation migration and differentiation of neural stemcells as well as integration into preexisting network andfunctionality [24] Each of thementioned steps can be assayedin an organotypic slice The method more used for studyingthe cell proliferation is the labelling with cell duplicationmarkers The most used ones are the nucleotide analogue5-bromodeoxyuridine (BrdU) or 5-iododeoxyuridine (IdU)and the nuclear protein Ki67 BrdU and IdU incorporate intothe duplicating DNA (during the S phase) whereas Ki67protein is a nuclear protein expressed in all phases of cellduplication all of which are subsequently visualized byimmunofluorescence In addition the combination of BrdUand IdU can be used for time window experiments andcell characterization (for a review see [25]) Infection of flu-orescent proteins with retroviruses is often used for timelapse experiments Time set of different infections allowedthe researcher to perform connectivity and lineage studies onnewly generated cells directly in vitro [12 26]

To study cell differentiation BrdUIdU immunofluores-cence can be combined with antibodies for the differentmarkers of differentiation shown in Table 1 Differentiationcan be assayed also in organotypic cultures derived fromtransgenic animal expressing fluorescent reporter genesThisis the case of reporter mouse lines in which neural stem andprogenitor cells express various fluorescent proteins (GFPCFPnuc H2B-GFP DsRedTimer and mCherry) under thecontrol of the nestin promoter [27] With these animals onecan follow all processes of differentiation and proliferationand evaluate the changes induced by various neurogenic andantineurogenic stimuli [28] Using the fluorescent reportergenes is convenient also to verify the final integration of newlygenerated cells into a preexisting network by electrophysiol-ogy

Organotypic cultures can be used especially to study themechanisms of integration of new cells into preexisting cir-cuitries In the model proposed by Toslashnnesen et al [29] theytransplanted in vitro cultured GFP-TH neurospheres over-expressing embryonic Wnt5a into striatal organotypic slicesThey observed neuronal differentiation (expression of neu-ronal markers and spontaneous firing of action potentials)synapse formation and functional expression of dopamineD2 autoreceptors In the same work the authors could acti-vate or inactivate optogenetically grafted cells demonstratingbidirectional synaptic interactions between grafted cells andhost neurons and extensive synaptic connectivity within thegraft

4 Organotypic Cultures as a Model forCNS Neurodegeneration and Studieson Neurogenesis

41 Ischemia Cerebral ischemia is generated by the lossof oxygen and nutrient in the brain In vivo ischemia isgenerated in mice by occlusion of the middle cerebral arterythat generates the damage of specific areas (cortex striatumand hippocampus) degeneration of neurons and activa-tion of microglia In organotypic slices ischemic damage ismodelled by oxygen and glucose deprivation (OGD) Theneuronal damage generated is divided in a central corewhere neurons die by necrosis and a surrounding penumbrawhere neurons die more slowly by apoptosis This latter partsends death signals and starts a dual communication withthe neurogenic niches On one hand the penumbra sendsSOS signals to the SVZ which generates protective factorsand activates neurogenesis [30] On the other hand deathsignals released from the focus of degeneration form a bio-chemical barrier impeding the neuroblasts migrating fromSVZ to the damaged region This was also modelled in silicoby using cortexSVZstriatum cultures [16] In this paperwe identified extracellular ATP and microglia activation asfactors impeding neurogenesis and the interaction of SDF-1120572with its receptor CXCR4 as a key signalling pathway drivingneuroblasts migration

The effect of neuroinflammation on neurogenesis afterOGD has been largely studied in hippocampal organotypic

4 Stem Cells International

cultures In this culture OGD generates a selective damagein the CA1 region and microglia recruitment to the damagedzone Several papers demonstrated that anti-inflammatorytreatment in organotypic slices facilitated the neurogenesisfrom the SGZ through the inhibition of both the p38mitogen-activated protein kinase and metalloproteinases [3132] Metalloproteinases are involved in the activation of sev-eral neuroinflammatory events Also during OGD this classof enzyme can sustain neuroinflammation and modulateneurogenesis in the dentate gyrus In fact the modulation ofculturemicroenvironment afterOGD in hippocampal organ-otypic cultures can promote the proliferation of glioblastswith predominant generation of oligodendrocyte progenitors[32]

42 Parkinsonrsquos Disease Parkinsonrsquos disease (PD) is causedby the selective neurodegeneration of dopaminergic neuronsThemain affected areas are the substantia nigra (s nigra) andstriatum Nevertheless dopaminergic degeneration affectsalso other areas like cerebral cortex globus pallidus and tha-lamus The lack of dopamine and an unbalance of dopamin-ergic and glutamatergic signal cause the classical motorsymptoms of bradykinesia rigidity resting tremors and lossof postural reflexes The system ldquos nigrastriatumcortexrdquohas been reproduced in vitro with different organotypiccultures The first attempt was made by Plenz and Kitai [33]they cocultivated with the roller tube technique the threemain areas involved in PD degeneration cerebral cortexstriatum and s nigra After a few days the three regions wereconnected by new tyrosine hydroxylase positive fibers (TH+)Another coculture model to study dopaminergic degenera-tion was performed by combining the basal nucleus ofMeyn-ert ventral mesencephalon parietal cortex and dorsal stria-tum of postnatal 7ndash9 rat pups [34] Nevertheless the presenceof the SVZ in these organotypic slices is important besidethe interest in adult neurogenesis In fact the SVZ is directlyinnervated by TH fibers suggesting that these can sustainthe maintenance of the neurogenic niche We described anorganotypic model in which we maintained in one singleslice the connection between s nigrastriatumcortex andSVZ [15] In this culture dopaminergic degeneration canbe obtained either by mechanical lesion or by 6-OHDAinjection In both cases the loss of dopamine stimulated theTH expression and proliferation of SVZ cells suggesting theneurogenic niche can be activated following dopaminergiclesion Moreover while 6-OHDA generated more specificdopaminergic degeneration the mechanical lesion can beused to study glutamatergic pathways or GABA-ergic degen-eration which allows us to represent respectively early oradvanced PD model (see [35] for a review)

43 Alzheimerrsquos Disease Alzheimerrsquos disease (AD) is achronic neurodegenerative disorder characterized by a pro-gressive loss of memory and dementia The causes andaetiology of AD are still unknown and the brain regionsmoreaffected are the hippocampus and neocortex At molecularlevel brain patients are characterized by amyloid plaques(dense and insoluble deposits of the beta-amyloid peptide-A120573) and neurofibrillary tangles (intracellular aggregates of

the hyperphosphorylated microtubule-associated proteintau) One of the first pieces of evidence of neurodegenerationin AD is the neuronal loss of cholinergic fibers of the entorhi-nal cortex connecting the CA1 layer of hippocampus [36]AD ismimicked by different animalmodels from invertebrate[37] to rodents [38] Except for the mouse SAMP8 [39] alltransgenic models combine different mutations for amyloidprecursor protein tau and presenilin 1

The best organotypic model that represents cellular alter-ation observed in AD is the hippocampal slice Alberdiand colleagues [14] used an organotypic slice in which theymaintained the connection between entorhinal cortex andhippocampus This model could be particularly useful tostudy cholinergic degeneration and activation of neurogen-esis in the SGZ Induction of neurodegeneration is obtainedby treatment of slices with different A120573 peptides As forseveral animal models A120573 together with neuronal damagemay play a role in the regulation of adult neurogenesis [4]In particular low concentration and the small A120573

25ndash35 pep-tide result in an increase of mossy fibers density and stim-ulation of endogenous SGZ neurogenesis Nonetheless neu-roinflammation associated with A120573 toxicity can mediate theinhibition of SGZ neurogenesis through the release of variousproinflammatory cytokines

5 Other Organotypic Models forNeuroregeneration Studies

In addition to neurogenesis in the brain organotypic cultureswere employed also to study cell regeneration in spinal cordand PNS Organotypic slices of mice adult spinal cord canbe prepared with the liquid-air interface [40] Spinal cordcells incorporate BrdU and express nestin Oct34 andDppa in the inner mass and have been used for modellingregeneration in spinal cord lesion [41] Enteric neural stemcells (ENSC) have been isolated from adult intestine Theyexpress markers like Ret p75 and CD49b [42] and can dif-ferentiate primarily into glia [43] but also into neurons andmyofibroblasts If neurogenic potential of ENSC has beendemonstrated in vivo there is still discrepancy in resultsobtained in vitro Ex vivo organotypic cultures from longi-tudinal muscle and myenteric plexus tissue demonstrated 5-ethynyl-21015840-deoxyuridine incorporation in ENSC and poten-tial proliferation dependent on the PTENPI3KAkt pathway[44] To investigate the neurogenesis in auditory systemAburto and colleagues [45] used organotypic cultures ofexplanted chicken otic vesicles (OV) Neuroepithelial oticprogenitors transit through states of cell proliferation cell fatespecification cell cycle exit migration and differentiationshowing characteristics of neural stem cells In this caseautophagy covers a relevant role in maintaining clearanceof apoptotic cells and facilitating neuronal differentiation ofprogenitor cells [45] The inhibition of LC3B a gene markerof autophagy in organotypic cultures of OV provoked themisregulation of the cell cycle and impairing neurogenesis

Other factors like GDNF can contribute to the final neu-ritogenesis of NSC-derived PNS neurons as demonstratedin organotypic slices of mice spiral ganglia [46] Stimulationof GDNF-family receptor 120572-1 (GFR1205721) activated two parallel

Stem Cells International 5

pathways PI3KAkt and MEKErk especially during earlypostnatal days

6 Concluding Remarks

Organotypic cultures of the neurogenic niches represent avalid alternative method to study neurogenesis in the CNSwhich complements in vivomodels andneurosphere culturesMoreover these cultures represent a good method to modelbrain damage in diseases like AD PD or stroke and to studyhow to implement neurogenesis as a potential mechanism ofbrain repair in these disorders Organotypic slices have beenused especially for toxicity studies therefore in neurogenesisresearch they became valuable for testing drug effects onneurogenesis activation or improving cell fate specification

Competing Interests

The authors declare that they have no competing interests

References

[1] A Mewes H Franke and D Singer ldquoOrganotypic brain slicecultures of adult transgenic P301S micemdashamodel for tauopathystudiesrdquo PLoS ONE vol 7 no 9 Article ID e45017 2012

[2] F B Kleine Borgmann O Bracko and S Jessberger ldquoImagingneurite development of adult-born granule cellsrdquoDevelopmentvol 140 no 13 pp 2823ndash2827 2013

[3] H Kim E Kim M Park E Lee and K Namkoong ldquoOrgan-otypic hippocampal slice culture from the adult mouse braina versatile tool for translational neuropsychopharmacologyrdquoProgress in Neuro-Psychopharmacology and Biological Psychia-try vol 41 pp 36ndash43 2013

[4] L Z Rui Y LeTourneau S R Gregg et al ldquoNeuroblast divi-sion during migration toward the ischemic striatum a study ofdynamic migratory and proliferative characteristics of neurob-lasts from the subventricular zonerdquoThe Journal of Neurosciencevol 27 no 12 pp 3157ndash3162 2007

[5] B Lacar S Z Young J-C Platel and A Bordey ldquoImaging andrecording subventricular zone progenitor cells in live tissue ofpostnatalmicerdquo Frontiers inNeuroscience vol 4 article 43 2010

[6] D R Hochbaum Y Zhao S L Farhi et al ldquoAll-optical elec-trophysiology in mammalian neurons using engineered micro-bial rhodopsinsrdquo Nature Methods vol 11 no 8 pp 825ndash8332014

[7] M J Hogue ldquoHuman fetal brain cells in tissue cultures theiridentification and motilityrdquo Journal of Experimental Zoologyvol 106 no 1 pp 85ndash107 1947

[8] B H Gahwiler ldquoOrganotypic monolayer cultures of nervoustissuerdquo Journal of Neuroscience Methods vol 4 no 4 pp 329ndash342 1981

[9] T Knopfel L Rietschin and B H Gahwiler ldquoOrganotypic co-cultures of rat locus coeruleus and hippocampusrdquo EuropeanJournal of Neuroscience vol 1 no 6 pp 678ndash689 1989

[10] S M Crain and E R Peterson ldquoDevelopment of specific sen-sory-evoked synaptic networks in fetal mouse cord-brainstemculturesrdquo Science vol 188 no 4185 pp 275ndash278 1975

[11] U F Braschler A Iannone C Spenger J Streit and H-RLuscher ldquoAmodified roller tube technique for organotypic coc-ultures of embryonic rat spinal cord sensory ganglia and

skeletal musclerdquo Journal of Neuroscience Methods vol 29 no2 pp 121ndash129 1989

[12] RHAndres ADDucrayA Perez-Bouza et al ldquoCreatine sup-plementation improves dopaminergic cell survival and protectsagainst MPP+ toxicity in an organotypic tissue culture systemrdquoCell Transplantation vol 14 no 8 pp 537ndash550 2005

[13] L Stoppini P-A Buchs and D Muller ldquoA simple method fororganotypic cultures of nervous tissuerdquo Journal of NeuroscienceMethods vol 37 no 2 pp 173ndash182 1991

[14] E Alberdi M V Sanchez-Gomez F Cavaliere et al ldquoAmyloid120573 oligomers induce Ca2+ dysregulation and neuronal deaththrough activation of ionotropic glutamate receptorsrdquo CellCalcium vol 47 no 3 pp 264ndash272 2010

[15] F Cavaliere E S Vicente and C Matute ldquoAn organotypicculture model to study nigro-striatal degenerationrdquo Journal ofNeuroscience Methods vol 188 no 2 pp 205ndash212 2010

[16] D Vergni F Castiglione M Briani et al ldquoAmodel of ischemia-induced neuroblast activation in the adult subventricular zonerdquoPLoS ONE vol 4 no 4 Article ID e5278 2009

[17] P Codega V Silva-Vargas A Paul et al ldquoProspective identifica-tion and purification of quiescent adult neural stem cells fromtheir in vivo nicherdquo Neuron vol 82 no 3 pp 545ndash559 2014

[18] L Ruan B W-M Lau J Wang et al ldquoNeurogenesis in neu-rological and psychiatric diseases and brain injury from benchto bedsiderdquo Progress in Neurobiology vol 115 pp 116ndash137 2014

[19] A Alvarez-Buylla MTheelen and F Nottebohm ldquoMapping ofradial glia and of a new cell type in adult canary brainrdquo TheJournal of Neuroscience vol 8 no 8 pp 2707ndash2712 1988

[20] D A Lim and A Alvarez-Buylla ldquoAdult neural stem cells staketheir groundrdquo Trends in Neurosciences vol 37 no 10 pp 563ndash571 2014

[21] L Katsimpardi N K Litterman P A Schein et al ldquoVascularand neurogenic rejuvenation of the agingmouse brain by youngsystemic factorsrdquo Science vol 344 no 6184 pp 630ndash634 2014

[22] O Baron A Ratzka and C Grothe ldquoFibroblast growth factor2 regulates adequate nigrostriatal pathway formation in micerdquoJournal of Comparative Neurology vol 520 no 17 pp 3949ndash3961 2012

[23] J T Neumann J WThompson A P Raval C H Cohan K BKoronowski andMA Perez-Pinzon ldquoIncreasedBDNFproteinexpression after ischemic or PKC epsilon preconditioning pro-motes electrophysiologic changes that lead to neuroprotectionrdquoJournal of Cerebral Blood Flow andMetabolism vol 35 no 1 pp121ndash130 2015

[24] G Kempermann LWiskott and FHGage ldquoFunctional signif-icance of adult neurogenesisrdquo Current Opinion in Neurobiologyvol 14 no 2 pp 186ndash191 2004

[25] B Leuner E R Glasper and E Gould ldquoThymidine analogmethods for studies of adult neurogenesis are not equally sen-sitiverdquo Journal of Comparative Neurology vol 517 no 2 pp 123ndash133 2009

[26] D A Laplagne M S Esposito V C Piatti et al ldquoFunctionalconvergence of neurons generated in the developing and adulthippocampusrdquo PLoS Biology vol 4 no 12 article e409 2006

[27] J M Encinas and G Enikolopov ldquoIdentifying and quantitatingneural stem and progenitor cells in the adult brainrdquoMethods inCell Biology vol 85 pp 243ndash272 2008

[28] A Sierra S Martın-Suarez R Valcarcel-Martın et al ldquoNeu-ronal hyperactivity accelerates depletion of neural stem cellsand impairs hippocampal neurogenesisrdquo Cell Stem Cell vol 16no 5 pp 488ndash503 2015

6 Stem Cells International

[29] J Toslashnnesen C L Parish A T Soslashrensen et al ldquoFunctionalintegration of grafted neural stem cell-derived dopaminergicneurons monitored by optogenetics in an in vitro Parkinsonmodelrdquo PLoS ONE vol 6 no 3 Article ID e17560 2011

[30] F Cavaliere K Dinkel and K Reymann ldquoThe subventricularzone releases factors which can be protective in oxygenglucosedeprivation-induced cortical damage an organotypic studyrdquoExperimental Neurology vol 201 no 1 pp 66ndash74 2006

[31] O Chechneva K Dinkel F Cavaliere M Martinez-Sanchezand K G Reymann ldquoAnti-inflammatory treatment in oxygen-glucose-deprived hippocampal slice cultures is neuroprotectiveand associated with reduced cell proliferation and intact neu-rogenesisrdquo Neurobiology of Disease vol 23 no 2 pp 247ndash2592006

[32] M Ziemka-Nałecz L Stanaszek and T Zalewska ldquoOxygen-glucose deprivation promotes gliogenesis and microglia acti-vation in organotypic hippocampal slice culture Involvementof metalloproteinasesrdquoActa Neurobiologiae Experimentalis vol73 no 1 pp 130ndash142 2013

[33] D Plenz and S T Kitai ldquoOrganotypic cortex-striatum-mes-encephalon cultures the nigrostriatal pathwayrdquo NeuroscienceLetters vol 209 no 3 pp 177ndash180 1996

[34] C Ullrich and C Humpel ldquoRotenone induces cell death of cho-linergic neurons in an organotypic co-culture brain slicemodelrdquo Neurochemical Research vol 34 no 12 pp 2147ndash21532009

[35] N Daviaud E Garbayo P C Schiller M Perez-Pinzon andC N Montero-Menei ldquoOrganotypic cultures as tools for opti-mizing central nervous system cell therapiesrdquo ExperimentalNeurology vol 248 pp 429ndash440 2013

[36] M Gallagher and M T Koh ldquoEpisodic memory on the path toAlzheimerrsquos diseaserdquo Current Opinion in Neurobiology vol 21no 6 pp 929ndash934 2011

[37] K PruszligingAVoigt and J B Schulz ldquoDrosophilamelanogasteras a model organism for Alzheimerrsquos diseaserdquo Molecular Neu-rodegeneration vol 8 article 35 2013

[38] S J Webster A D Bachstetter P T Nelson F A Schmitt andL J Van Eldik ldquoUsing mice to model Alzheimerrsquos dementia anoverview of the clinical disease and the preclinical behavioralchanges in 10 mouse modelsrdquo Frontiers in Genetics vol 5 pp88ndash95 2014

[39] J F Flood and J E Morley ldquoLearning and memory in theSAMP8 mouserdquo Neuroscience and Biobehavioral Reviews vol22 no 1 pp 1ndash20 1998

[40] M V Glazova E S Pak and A K Murashov ldquoNeurogenicpotential of spinal cord organotypic culturerdquo NeuroscienceLetters vol 594 pp 60ndash65 2015

[41] M Heidemann J Streit and A Tscherter ldquoInvestigating func-tional regeneration in organotypic spinal cord co-culturesgrown on multi-electrode arraysrdquo Journal of Visualized Experi-ments vol 23 no 103 Article ID e53121 2015

[42] R Suarez-Rodrıguez and J Belkind-Gerson ldquoCultured nestin-positive cells from postnatal mouse small bowel differentiate exvivo into neurons glia and smooth musclerdquo STEM CELLS vol22 no 7 pp 1373ndash1385 2004

[43] N M Joseph S He E Quintana Y-G Kim G Nunez andS J Morrison ldquoEnteric glia are multipotent in culture but pri-marily form glia in the adult rodent gutrdquo Journal of ClinicalInvestigation vol 121 no 9 pp 3398ndash3411 2011

[44] L Becker J Peterson S Kulkarni and P J Pasricha ldquoEx vivoneurogenesis within enteric ganglia occurs in a PTEN depen-dent mannerrdquo PLoS ONE vol 8 no 3 Article ID e59452 2013

[45] M R Aburto H Sanchez-Calderon J M Hurle I Varela-Nieto and M Magarinos ldquoEarly otic development depends onautophagy for apoptotic cell clearance and neural differentia-tionrdquo Cell Death and Disease vol 3 article e394 2012

[46] S Euteneuer K H Yang E Chavez et al ldquoGlial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis inthe cochlear spiral ganglion via neural cell adhesion molecule(NCAM)rdquoMolecular and Cellular Neuroscience vol 54 pp 30ndash43 2013

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2 Stem Cells International

CortexHippocampusentorhinal cortex

HippocampusSVZstriatumcortex

Cerebellum

(a)

cp

lv

sn

cx

Substantia nigrastriatumSVZcortex

(b)

Figure 1 Different organotypic slices can be obtained from P2 to P7 postnatal forebrain or cerebellum by coronal sections of 350ndash400 120583m(a) In (b) the brain sectioning to obtain 45∘ transversal sections of s nigrastriatumSVZcortex (sn substantia nigra cp caudate putamenlv lateral ventricle cx cortex) used to model PD is shown

In the roller tube cultures the tissue is embedded in aplasma clot and attached on a glass coverslip The coverslipwith the embedded slice tissue is located in a tube that under-goes continuous slow rotation in a cell culture incubatorTheoxygenation is maintained by continuous exchange of liquid-gas interface generated by the slow rotation The techniquewas successively modified several times (eg [11 12]) butthe roller tube technique always yields very thin cultures(from an initial 400 120583m to about 50 120583m) with consequentialpreferential use for experiments that require optimal opticalconditions (eg electron microscopy or electrophysiology)

At the beginning of the 90s Stoppini and colleagues[13] published a new method to cultivate organotypic slicesIn this method brain slices were placed on a semiporousmembrane and cultivated at the air-liquid interface Theabsence of clot facilitates the studies of synaptic reorga-nization and became a useful tool to study plasticity andsprouting already during the first days of culture The realadvantage of this technique is that cultures are easily preparedand offer great advantages when a 3D structure is desired(from an initial 400120583m thickness slices are cultivated upto 100ndash150 120583m) As described more in detail below theair-liquid interface has become a key instrument to studyadult neurogenesis Organotypic slices can be obtained fromdifferent brain regions (as described in Figure 1) but forthe study of adult neurogenesis in normal and pathologicalconditions the hippocampal region containing the SGZ andthe slices containing the SVZ are most preferred Thus forexample neurogenesis in Alzheimer disease or Parkinsonrsquosdisease can be studied in hippocampusentorhinal cortexor s nigrastriatumSVZcortex slices [14 15] Organotypiccultures match the tridimensional space where neural pro-genitors migrate to reach maturation in vivo In the paperby Vergni and colleagues [16] we ideally represented theslice culture comprising subventricular zone as the spatialextension to elaborate amathematicalmodel to describe neu-roblasts activation and migration following oxygen and glu-cose deprivation

2 Adult Neurogenesis in CNS

Neurogenesis in the normal adult brain occurs mainly in thedentate gyrus from the subgranular zone of hippocampus(SGZ) and in the olfactory bulb from the subventricularzone of lateral ventricle (SVZ) In both cases the neurogenicniches the SGZ and SVZ host multipotent cells that give riseto neuroblasts or glioblasts throughout life The stem cells inthe neurogenic niches (type 1 in the SGZor B cells in the SVZ)give rise to the more proliferative transit-amplifying progeni-tors (type 2 or C cells) that in turn give rise to neuroblast (type3 or A cells) or glioblasts [17]The study of adult neurogenesisin the SGZ is relevant especially for the study of repairmecha-nism of neurodegeneration related to loss of memory or neu-ropsychiatric disorders (for a review see [18]) New neurob-lasts generated in the SGZmigrate to the granular zone of thedentate gyrus covering short distances whereas neuroblastsgenerated in the SVZ move through the rostral migratorystream (RMS) to the olfactory bulb (OB) where finally theymigrate radially and differentiate into new neurons

Adult neural stem cells (NSC) are a specialized formof glia deriving from the embryonic radial glia [19] Theypossess a regional pattern within the SVZ which is relatedto their embryonic origin and reflect the ability to generatedifferent types of OB interneurons (eg granule cells TH+or calbindin+ periglomerular cells see [20]) The neuro-genic moiety is guaranteed by different subpopulation ofmultipotent cells characterized by the expression of differentmarkers in different temporal stages as shown inTable 1 NSCcan be activated and neurogenesis can be induced underdifferent conditions A predominant role in vivo is coveredby the blood vessel and local vascular plexus that bringtrophic factors or stress molecule signals This was recentlydemonstrated by Katsimpardi et al [21] who potentiatedthe neurogenesis of old animals after transfusion of bloodfrom younger animals The absence of local circulation inthe organotypic cultures is fixed by using different trophicfactors in the medium that stimulate intrinsic signals mainly

Stem Cells International 3

Table 1 SVZ neural stem cell characterization by cellular marker expression

GFAP CD133 EGFR Nestin DCX 120573III tubulin NeuN PDGFR ProliferationB cells

qNSC + + + ++ minus minus minus minus minus minus minus

aNSC + + + ++ ++ + minus minus minus + +

C cellsTransit amplifying + minus + + + + minus minus minus ++ + + +

A cellsNeuroblast minus minus minus + + + + + minus minus ++

Immature neurons minus minus minus minus minus + + + + minus +

Glioblast + + + minus minus + minus minus minus + + + +

Proliferation is expressed by BrdU incorporation ability (Codega et al 2014) [17] Quiescent neural stem cells qNSC and activated neural stem cells aNSC

transcription factors for example sox2 olig2 or the bmpfamily [22 23]

3 Techniques Used to StudyNeurogenesis in OC

From a technical point of view the organotypic cultures rep-resent a versatile tool to study neurogenesis or cell regenera-tion In vivo neurogenesis is a multistep process that involvesproliferation migration and differentiation of neural stemcells as well as integration into preexisting network andfunctionality [24] Each of thementioned steps can be assayedin an organotypic slice The method more used for studyingthe cell proliferation is the labelling with cell duplicationmarkers The most used ones are the nucleotide analogue5-bromodeoxyuridine (BrdU) or 5-iododeoxyuridine (IdU)and the nuclear protein Ki67 BrdU and IdU incorporate intothe duplicating DNA (during the S phase) whereas Ki67protein is a nuclear protein expressed in all phases of cellduplication all of which are subsequently visualized byimmunofluorescence In addition the combination of BrdUand IdU can be used for time window experiments andcell characterization (for a review see [25]) Infection of flu-orescent proteins with retroviruses is often used for timelapse experiments Time set of different infections allowedthe researcher to perform connectivity and lineage studies onnewly generated cells directly in vitro [12 26]

To study cell differentiation BrdUIdU immunofluores-cence can be combined with antibodies for the differentmarkers of differentiation shown in Table 1 Differentiationcan be assayed also in organotypic cultures derived fromtransgenic animal expressing fluorescent reporter genesThisis the case of reporter mouse lines in which neural stem andprogenitor cells express various fluorescent proteins (GFPCFPnuc H2B-GFP DsRedTimer and mCherry) under thecontrol of the nestin promoter [27] With these animals onecan follow all processes of differentiation and proliferationand evaluate the changes induced by various neurogenic andantineurogenic stimuli [28] Using the fluorescent reportergenes is convenient also to verify the final integration of newlygenerated cells into a preexisting network by electrophysiol-ogy

Organotypic cultures can be used especially to study themechanisms of integration of new cells into preexisting cir-cuitries In the model proposed by Toslashnnesen et al [29] theytransplanted in vitro cultured GFP-TH neurospheres over-expressing embryonic Wnt5a into striatal organotypic slicesThey observed neuronal differentiation (expression of neu-ronal markers and spontaneous firing of action potentials)synapse formation and functional expression of dopamineD2 autoreceptors In the same work the authors could acti-vate or inactivate optogenetically grafted cells demonstratingbidirectional synaptic interactions between grafted cells andhost neurons and extensive synaptic connectivity within thegraft

4 Organotypic Cultures as a Model forCNS Neurodegeneration and Studieson Neurogenesis

41 Ischemia Cerebral ischemia is generated by the lossof oxygen and nutrient in the brain In vivo ischemia isgenerated in mice by occlusion of the middle cerebral arterythat generates the damage of specific areas (cortex striatumand hippocampus) degeneration of neurons and activa-tion of microglia In organotypic slices ischemic damage ismodelled by oxygen and glucose deprivation (OGD) Theneuronal damage generated is divided in a central corewhere neurons die by necrosis and a surrounding penumbrawhere neurons die more slowly by apoptosis This latter partsends death signals and starts a dual communication withthe neurogenic niches On one hand the penumbra sendsSOS signals to the SVZ which generates protective factorsand activates neurogenesis [30] On the other hand deathsignals released from the focus of degeneration form a bio-chemical barrier impeding the neuroblasts migrating fromSVZ to the damaged region This was also modelled in silicoby using cortexSVZstriatum cultures [16] In this paperwe identified extracellular ATP and microglia activation asfactors impeding neurogenesis and the interaction of SDF-1120572with its receptor CXCR4 as a key signalling pathway drivingneuroblasts migration

The effect of neuroinflammation on neurogenesis afterOGD has been largely studied in hippocampal organotypic

4 Stem Cells International

cultures In this culture OGD generates a selective damagein the CA1 region and microglia recruitment to the damagedzone Several papers demonstrated that anti-inflammatorytreatment in organotypic slices facilitated the neurogenesisfrom the SGZ through the inhibition of both the p38mitogen-activated protein kinase and metalloproteinases [3132] Metalloproteinases are involved in the activation of sev-eral neuroinflammatory events Also during OGD this classof enzyme can sustain neuroinflammation and modulateneurogenesis in the dentate gyrus In fact the modulation ofculturemicroenvironment afterOGD in hippocampal organ-otypic cultures can promote the proliferation of glioblastswith predominant generation of oligodendrocyte progenitors[32]

42 Parkinsonrsquos Disease Parkinsonrsquos disease (PD) is causedby the selective neurodegeneration of dopaminergic neuronsThemain affected areas are the substantia nigra (s nigra) andstriatum Nevertheless dopaminergic degeneration affectsalso other areas like cerebral cortex globus pallidus and tha-lamus The lack of dopamine and an unbalance of dopamin-ergic and glutamatergic signal cause the classical motorsymptoms of bradykinesia rigidity resting tremors and lossof postural reflexes The system ldquos nigrastriatumcortexrdquohas been reproduced in vitro with different organotypiccultures The first attempt was made by Plenz and Kitai [33]they cocultivated with the roller tube technique the threemain areas involved in PD degeneration cerebral cortexstriatum and s nigra After a few days the three regions wereconnected by new tyrosine hydroxylase positive fibers (TH+)Another coculture model to study dopaminergic degenera-tion was performed by combining the basal nucleus ofMeyn-ert ventral mesencephalon parietal cortex and dorsal stria-tum of postnatal 7ndash9 rat pups [34] Nevertheless the presenceof the SVZ in these organotypic slices is important besidethe interest in adult neurogenesis In fact the SVZ is directlyinnervated by TH fibers suggesting that these can sustainthe maintenance of the neurogenic niche We described anorganotypic model in which we maintained in one singleslice the connection between s nigrastriatumcortex andSVZ [15] In this culture dopaminergic degeneration canbe obtained either by mechanical lesion or by 6-OHDAinjection In both cases the loss of dopamine stimulated theTH expression and proliferation of SVZ cells suggesting theneurogenic niche can be activated following dopaminergiclesion Moreover while 6-OHDA generated more specificdopaminergic degeneration the mechanical lesion can beused to study glutamatergic pathways or GABA-ergic degen-eration which allows us to represent respectively early oradvanced PD model (see [35] for a review)

43 Alzheimerrsquos Disease Alzheimerrsquos disease (AD) is achronic neurodegenerative disorder characterized by a pro-gressive loss of memory and dementia The causes andaetiology of AD are still unknown and the brain regionsmoreaffected are the hippocampus and neocortex At molecularlevel brain patients are characterized by amyloid plaques(dense and insoluble deposits of the beta-amyloid peptide-A120573) and neurofibrillary tangles (intracellular aggregates of

the hyperphosphorylated microtubule-associated proteintau) One of the first pieces of evidence of neurodegenerationin AD is the neuronal loss of cholinergic fibers of the entorhi-nal cortex connecting the CA1 layer of hippocampus [36]AD ismimicked by different animalmodels from invertebrate[37] to rodents [38] Except for the mouse SAMP8 [39] alltransgenic models combine different mutations for amyloidprecursor protein tau and presenilin 1

The best organotypic model that represents cellular alter-ation observed in AD is the hippocampal slice Alberdiand colleagues [14] used an organotypic slice in which theymaintained the connection between entorhinal cortex andhippocampus This model could be particularly useful tostudy cholinergic degeneration and activation of neurogen-esis in the SGZ Induction of neurodegeneration is obtainedby treatment of slices with different A120573 peptides As forseveral animal models A120573 together with neuronal damagemay play a role in the regulation of adult neurogenesis [4]In particular low concentration and the small A120573

25ndash35 pep-tide result in an increase of mossy fibers density and stim-ulation of endogenous SGZ neurogenesis Nonetheless neu-roinflammation associated with A120573 toxicity can mediate theinhibition of SGZ neurogenesis through the release of variousproinflammatory cytokines

5 Other Organotypic Models forNeuroregeneration Studies

In addition to neurogenesis in the brain organotypic cultureswere employed also to study cell regeneration in spinal cordand PNS Organotypic slices of mice adult spinal cord canbe prepared with the liquid-air interface [40] Spinal cordcells incorporate BrdU and express nestin Oct34 andDppa in the inner mass and have been used for modellingregeneration in spinal cord lesion [41] Enteric neural stemcells (ENSC) have been isolated from adult intestine Theyexpress markers like Ret p75 and CD49b [42] and can dif-ferentiate primarily into glia [43] but also into neurons andmyofibroblasts If neurogenic potential of ENSC has beendemonstrated in vivo there is still discrepancy in resultsobtained in vitro Ex vivo organotypic cultures from longi-tudinal muscle and myenteric plexus tissue demonstrated 5-ethynyl-21015840-deoxyuridine incorporation in ENSC and poten-tial proliferation dependent on the PTENPI3KAkt pathway[44] To investigate the neurogenesis in auditory systemAburto and colleagues [45] used organotypic cultures ofexplanted chicken otic vesicles (OV) Neuroepithelial oticprogenitors transit through states of cell proliferation cell fatespecification cell cycle exit migration and differentiationshowing characteristics of neural stem cells In this caseautophagy covers a relevant role in maintaining clearanceof apoptotic cells and facilitating neuronal differentiation ofprogenitor cells [45] The inhibition of LC3B a gene markerof autophagy in organotypic cultures of OV provoked themisregulation of the cell cycle and impairing neurogenesis

Other factors like GDNF can contribute to the final neu-ritogenesis of NSC-derived PNS neurons as demonstratedin organotypic slices of mice spiral ganglia [46] Stimulationof GDNF-family receptor 120572-1 (GFR1205721) activated two parallel

Stem Cells International 5

pathways PI3KAkt and MEKErk especially during earlypostnatal days

6 Concluding Remarks

Organotypic cultures of the neurogenic niches represent avalid alternative method to study neurogenesis in the CNSwhich complements in vivomodels andneurosphere culturesMoreover these cultures represent a good method to modelbrain damage in diseases like AD PD or stroke and to studyhow to implement neurogenesis as a potential mechanism ofbrain repair in these disorders Organotypic slices have beenused especially for toxicity studies therefore in neurogenesisresearch they became valuable for testing drug effects onneurogenesis activation or improving cell fate specification

Competing Interests

The authors declare that they have no competing interests

References

[1] A Mewes H Franke and D Singer ldquoOrganotypic brain slicecultures of adult transgenic P301S micemdashamodel for tauopathystudiesrdquo PLoS ONE vol 7 no 9 Article ID e45017 2012

[2] F B Kleine Borgmann O Bracko and S Jessberger ldquoImagingneurite development of adult-born granule cellsrdquoDevelopmentvol 140 no 13 pp 2823ndash2827 2013

[3] H Kim E Kim M Park E Lee and K Namkoong ldquoOrgan-otypic hippocampal slice culture from the adult mouse braina versatile tool for translational neuropsychopharmacologyrdquoProgress in Neuro-Psychopharmacology and Biological Psychia-try vol 41 pp 36ndash43 2013

[4] L Z Rui Y LeTourneau S R Gregg et al ldquoNeuroblast divi-sion during migration toward the ischemic striatum a study ofdynamic migratory and proliferative characteristics of neurob-lasts from the subventricular zonerdquoThe Journal of Neurosciencevol 27 no 12 pp 3157ndash3162 2007

[5] B Lacar S Z Young J-C Platel and A Bordey ldquoImaging andrecording subventricular zone progenitor cells in live tissue ofpostnatalmicerdquo Frontiers inNeuroscience vol 4 article 43 2010

[6] D R Hochbaum Y Zhao S L Farhi et al ldquoAll-optical elec-trophysiology in mammalian neurons using engineered micro-bial rhodopsinsrdquo Nature Methods vol 11 no 8 pp 825ndash8332014

[7] M J Hogue ldquoHuman fetal brain cells in tissue cultures theiridentification and motilityrdquo Journal of Experimental Zoologyvol 106 no 1 pp 85ndash107 1947

[8] B H Gahwiler ldquoOrganotypic monolayer cultures of nervoustissuerdquo Journal of Neuroscience Methods vol 4 no 4 pp 329ndash342 1981

[9] T Knopfel L Rietschin and B H Gahwiler ldquoOrganotypic co-cultures of rat locus coeruleus and hippocampusrdquo EuropeanJournal of Neuroscience vol 1 no 6 pp 678ndash689 1989

[10] S M Crain and E R Peterson ldquoDevelopment of specific sen-sory-evoked synaptic networks in fetal mouse cord-brainstemculturesrdquo Science vol 188 no 4185 pp 275ndash278 1975

[11] U F Braschler A Iannone C Spenger J Streit and H-RLuscher ldquoAmodified roller tube technique for organotypic coc-ultures of embryonic rat spinal cord sensory ganglia and

skeletal musclerdquo Journal of Neuroscience Methods vol 29 no2 pp 121ndash129 1989

[12] RHAndres ADDucrayA Perez-Bouza et al ldquoCreatine sup-plementation improves dopaminergic cell survival and protectsagainst MPP+ toxicity in an organotypic tissue culture systemrdquoCell Transplantation vol 14 no 8 pp 537ndash550 2005

[13] L Stoppini P-A Buchs and D Muller ldquoA simple method fororganotypic cultures of nervous tissuerdquo Journal of NeuroscienceMethods vol 37 no 2 pp 173ndash182 1991

[14] E Alberdi M V Sanchez-Gomez F Cavaliere et al ldquoAmyloid120573 oligomers induce Ca2+ dysregulation and neuronal deaththrough activation of ionotropic glutamate receptorsrdquo CellCalcium vol 47 no 3 pp 264ndash272 2010

[15] F Cavaliere E S Vicente and C Matute ldquoAn organotypicculture model to study nigro-striatal degenerationrdquo Journal ofNeuroscience Methods vol 188 no 2 pp 205ndash212 2010

[16] D Vergni F Castiglione M Briani et al ldquoAmodel of ischemia-induced neuroblast activation in the adult subventricular zonerdquoPLoS ONE vol 4 no 4 Article ID e5278 2009

[17] P Codega V Silva-Vargas A Paul et al ldquoProspective identifica-tion and purification of quiescent adult neural stem cells fromtheir in vivo nicherdquo Neuron vol 82 no 3 pp 545ndash559 2014

[18] L Ruan B W-M Lau J Wang et al ldquoNeurogenesis in neu-rological and psychiatric diseases and brain injury from benchto bedsiderdquo Progress in Neurobiology vol 115 pp 116ndash137 2014

[19] A Alvarez-Buylla MTheelen and F Nottebohm ldquoMapping ofradial glia and of a new cell type in adult canary brainrdquo TheJournal of Neuroscience vol 8 no 8 pp 2707ndash2712 1988

[20] D A Lim and A Alvarez-Buylla ldquoAdult neural stem cells staketheir groundrdquo Trends in Neurosciences vol 37 no 10 pp 563ndash571 2014

[21] L Katsimpardi N K Litterman P A Schein et al ldquoVascularand neurogenic rejuvenation of the agingmouse brain by youngsystemic factorsrdquo Science vol 344 no 6184 pp 630ndash634 2014

[22] O Baron A Ratzka and C Grothe ldquoFibroblast growth factor2 regulates adequate nigrostriatal pathway formation in micerdquoJournal of Comparative Neurology vol 520 no 17 pp 3949ndash3961 2012

[23] J T Neumann J WThompson A P Raval C H Cohan K BKoronowski andMA Perez-Pinzon ldquoIncreasedBDNFproteinexpression after ischemic or PKC epsilon preconditioning pro-motes electrophysiologic changes that lead to neuroprotectionrdquoJournal of Cerebral Blood Flow andMetabolism vol 35 no 1 pp121ndash130 2015

[24] G Kempermann LWiskott and FHGage ldquoFunctional signif-icance of adult neurogenesisrdquo Current Opinion in Neurobiologyvol 14 no 2 pp 186ndash191 2004

[25] B Leuner E R Glasper and E Gould ldquoThymidine analogmethods for studies of adult neurogenesis are not equally sen-sitiverdquo Journal of Comparative Neurology vol 517 no 2 pp 123ndash133 2009

[26] D A Laplagne M S Esposito V C Piatti et al ldquoFunctionalconvergence of neurons generated in the developing and adulthippocampusrdquo PLoS Biology vol 4 no 12 article e409 2006

[27] J M Encinas and G Enikolopov ldquoIdentifying and quantitatingneural stem and progenitor cells in the adult brainrdquoMethods inCell Biology vol 85 pp 243ndash272 2008

[28] A Sierra S Martın-Suarez R Valcarcel-Martın et al ldquoNeu-ronal hyperactivity accelerates depletion of neural stem cellsand impairs hippocampal neurogenesisrdquo Cell Stem Cell vol 16no 5 pp 488ndash503 2015

6 Stem Cells International

[29] J Toslashnnesen C L Parish A T Soslashrensen et al ldquoFunctionalintegration of grafted neural stem cell-derived dopaminergicneurons monitored by optogenetics in an in vitro Parkinsonmodelrdquo PLoS ONE vol 6 no 3 Article ID e17560 2011

[30] F Cavaliere K Dinkel and K Reymann ldquoThe subventricularzone releases factors which can be protective in oxygenglucosedeprivation-induced cortical damage an organotypic studyrdquoExperimental Neurology vol 201 no 1 pp 66ndash74 2006

[31] O Chechneva K Dinkel F Cavaliere M Martinez-Sanchezand K G Reymann ldquoAnti-inflammatory treatment in oxygen-glucose-deprived hippocampal slice cultures is neuroprotectiveand associated with reduced cell proliferation and intact neu-rogenesisrdquo Neurobiology of Disease vol 23 no 2 pp 247ndash2592006

[32] M Ziemka-Nałecz L Stanaszek and T Zalewska ldquoOxygen-glucose deprivation promotes gliogenesis and microglia acti-vation in organotypic hippocampal slice culture Involvementof metalloproteinasesrdquoActa Neurobiologiae Experimentalis vol73 no 1 pp 130ndash142 2013

[33] D Plenz and S T Kitai ldquoOrganotypic cortex-striatum-mes-encephalon cultures the nigrostriatal pathwayrdquo NeuroscienceLetters vol 209 no 3 pp 177ndash180 1996

[34] C Ullrich and C Humpel ldquoRotenone induces cell death of cho-linergic neurons in an organotypic co-culture brain slicemodelrdquo Neurochemical Research vol 34 no 12 pp 2147ndash21532009

[35] N Daviaud E Garbayo P C Schiller M Perez-Pinzon andC N Montero-Menei ldquoOrganotypic cultures as tools for opti-mizing central nervous system cell therapiesrdquo ExperimentalNeurology vol 248 pp 429ndash440 2013

[36] M Gallagher and M T Koh ldquoEpisodic memory on the path toAlzheimerrsquos diseaserdquo Current Opinion in Neurobiology vol 21no 6 pp 929ndash934 2011

[37] K PruszligingAVoigt and J B Schulz ldquoDrosophilamelanogasteras a model organism for Alzheimerrsquos diseaserdquo Molecular Neu-rodegeneration vol 8 article 35 2013

[38] S J Webster A D Bachstetter P T Nelson F A Schmitt andL J Van Eldik ldquoUsing mice to model Alzheimerrsquos dementia anoverview of the clinical disease and the preclinical behavioralchanges in 10 mouse modelsrdquo Frontiers in Genetics vol 5 pp88ndash95 2014

[39] J F Flood and J E Morley ldquoLearning and memory in theSAMP8 mouserdquo Neuroscience and Biobehavioral Reviews vol22 no 1 pp 1ndash20 1998

[40] M V Glazova E S Pak and A K Murashov ldquoNeurogenicpotential of spinal cord organotypic culturerdquo NeuroscienceLetters vol 594 pp 60ndash65 2015

[41] M Heidemann J Streit and A Tscherter ldquoInvestigating func-tional regeneration in organotypic spinal cord co-culturesgrown on multi-electrode arraysrdquo Journal of Visualized Experi-ments vol 23 no 103 Article ID e53121 2015

[42] R Suarez-Rodrıguez and J Belkind-Gerson ldquoCultured nestin-positive cells from postnatal mouse small bowel differentiate exvivo into neurons glia and smooth musclerdquo STEM CELLS vol22 no 7 pp 1373ndash1385 2004

[43] N M Joseph S He E Quintana Y-G Kim G Nunez andS J Morrison ldquoEnteric glia are multipotent in culture but pri-marily form glia in the adult rodent gutrdquo Journal of ClinicalInvestigation vol 121 no 9 pp 3398ndash3411 2011

[44] L Becker J Peterson S Kulkarni and P J Pasricha ldquoEx vivoneurogenesis within enteric ganglia occurs in a PTEN depen-dent mannerrdquo PLoS ONE vol 8 no 3 Article ID e59452 2013

[45] M R Aburto H Sanchez-Calderon J M Hurle I Varela-Nieto and M Magarinos ldquoEarly otic development depends onautophagy for apoptotic cell clearance and neural differentia-tionrdquo Cell Death and Disease vol 3 article e394 2012

[46] S Euteneuer K H Yang E Chavez et al ldquoGlial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis inthe cochlear spiral ganglion via neural cell adhesion molecule(NCAM)rdquoMolecular and Cellular Neuroscience vol 54 pp 30ndash43 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 3

Table 1 SVZ neural stem cell characterization by cellular marker expression

GFAP CD133 EGFR Nestin DCX 120573III tubulin NeuN PDGFR ProliferationB cells

qNSC + + + ++ minus minus minus minus minus minus minus

aNSC + + + ++ ++ + minus minus minus + +

C cellsTransit amplifying + minus + + + + minus minus minus ++ + + +

A cellsNeuroblast minus minus minus + + + + + minus minus ++

Immature neurons minus minus minus minus minus + + + + minus +

Glioblast + + + minus minus + minus minus minus + + + +

Proliferation is expressed by BrdU incorporation ability (Codega et al 2014) [17] Quiescent neural stem cells qNSC and activated neural stem cells aNSC

transcription factors for example sox2 olig2 or the bmpfamily [22 23]

3 Techniques Used to StudyNeurogenesis in OC

From a technical point of view the organotypic cultures rep-resent a versatile tool to study neurogenesis or cell regenera-tion In vivo neurogenesis is a multistep process that involvesproliferation migration and differentiation of neural stemcells as well as integration into preexisting network andfunctionality [24] Each of thementioned steps can be assayedin an organotypic slice The method more used for studyingthe cell proliferation is the labelling with cell duplicationmarkers The most used ones are the nucleotide analogue5-bromodeoxyuridine (BrdU) or 5-iododeoxyuridine (IdU)and the nuclear protein Ki67 BrdU and IdU incorporate intothe duplicating DNA (during the S phase) whereas Ki67protein is a nuclear protein expressed in all phases of cellduplication all of which are subsequently visualized byimmunofluorescence In addition the combination of BrdUand IdU can be used for time window experiments andcell characterization (for a review see [25]) Infection of flu-orescent proteins with retroviruses is often used for timelapse experiments Time set of different infections allowedthe researcher to perform connectivity and lineage studies onnewly generated cells directly in vitro [12 26]

To study cell differentiation BrdUIdU immunofluores-cence can be combined with antibodies for the differentmarkers of differentiation shown in Table 1 Differentiationcan be assayed also in organotypic cultures derived fromtransgenic animal expressing fluorescent reporter genesThisis the case of reporter mouse lines in which neural stem andprogenitor cells express various fluorescent proteins (GFPCFPnuc H2B-GFP DsRedTimer and mCherry) under thecontrol of the nestin promoter [27] With these animals onecan follow all processes of differentiation and proliferationand evaluate the changes induced by various neurogenic andantineurogenic stimuli [28] Using the fluorescent reportergenes is convenient also to verify the final integration of newlygenerated cells into a preexisting network by electrophysiol-ogy

Organotypic cultures can be used especially to study themechanisms of integration of new cells into preexisting cir-cuitries In the model proposed by Toslashnnesen et al [29] theytransplanted in vitro cultured GFP-TH neurospheres over-expressing embryonic Wnt5a into striatal organotypic slicesThey observed neuronal differentiation (expression of neu-ronal markers and spontaneous firing of action potentials)synapse formation and functional expression of dopamineD2 autoreceptors In the same work the authors could acti-vate or inactivate optogenetically grafted cells demonstratingbidirectional synaptic interactions between grafted cells andhost neurons and extensive synaptic connectivity within thegraft

4 Organotypic Cultures as a Model forCNS Neurodegeneration and Studieson Neurogenesis

41 Ischemia Cerebral ischemia is generated by the lossof oxygen and nutrient in the brain In vivo ischemia isgenerated in mice by occlusion of the middle cerebral arterythat generates the damage of specific areas (cortex striatumand hippocampus) degeneration of neurons and activa-tion of microglia In organotypic slices ischemic damage ismodelled by oxygen and glucose deprivation (OGD) Theneuronal damage generated is divided in a central corewhere neurons die by necrosis and a surrounding penumbrawhere neurons die more slowly by apoptosis This latter partsends death signals and starts a dual communication withthe neurogenic niches On one hand the penumbra sendsSOS signals to the SVZ which generates protective factorsand activates neurogenesis [30] On the other hand deathsignals released from the focus of degeneration form a bio-chemical barrier impeding the neuroblasts migrating fromSVZ to the damaged region This was also modelled in silicoby using cortexSVZstriatum cultures [16] In this paperwe identified extracellular ATP and microglia activation asfactors impeding neurogenesis and the interaction of SDF-1120572with its receptor CXCR4 as a key signalling pathway drivingneuroblasts migration

The effect of neuroinflammation on neurogenesis afterOGD has been largely studied in hippocampal organotypic

4 Stem Cells International

cultures In this culture OGD generates a selective damagein the CA1 region and microglia recruitment to the damagedzone Several papers demonstrated that anti-inflammatorytreatment in organotypic slices facilitated the neurogenesisfrom the SGZ through the inhibition of both the p38mitogen-activated protein kinase and metalloproteinases [3132] Metalloproteinases are involved in the activation of sev-eral neuroinflammatory events Also during OGD this classof enzyme can sustain neuroinflammation and modulateneurogenesis in the dentate gyrus In fact the modulation ofculturemicroenvironment afterOGD in hippocampal organ-otypic cultures can promote the proliferation of glioblastswith predominant generation of oligodendrocyte progenitors[32]

42 Parkinsonrsquos Disease Parkinsonrsquos disease (PD) is causedby the selective neurodegeneration of dopaminergic neuronsThemain affected areas are the substantia nigra (s nigra) andstriatum Nevertheless dopaminergic degeneration affectsalso other areas like cerebral cortex globus pallidus and tha-lamus The lack of dopamine and an unbalance of dopamin-ergic and glutamatergic signal cause the classical motorsymptoms of bradykinesia rigidity resting tremors and lossof postural reflexes The system ldquos nigrastriatumcortexrdquohas been reproduced in vitro with different organotypiccultures The first attempt was made by Plenz and Kitai [33]they cocultivated with the roller tube technique the threemain areas involved in PD degeneration cerebral cortexstriatum and s nigra After a few days the three regions wereconnected by new tyrosine hydroxylase positive fibers (TH+)Another coculture model to study dopaminergic degenera-tion was performed by combining the basal nucleus ofMeyn-ert ventral mesencephalon parietal cortex and dorsal stria-tum of postnatal 7ndash9 rat pups [34] Nevertheless the presenceof the SVZ in these organotypic slices is important besidethe interest in adult neurogenesis In fact the SVZ is directlyinnervated by TH fibers suggesting that these can sustainthe maintenance of the neurogenic niche We described anorganotypic model in which we maintained in one singleslice the connection between s nigrastriatumcortex andSVZ [15] In this culture dopaminergic degeneration canbe obtained either by mechanical lesion or by 6-OHDAinjection In both cases the loss of dopamine stimulated theTH expression and proliferation of SVZ cells suggesting theneurogenic niche can be activated following dopaminergiclesion Moreover while 6-OHDA generated more specificdopaminergic degeneration the mechanical lesion can beused to study glutamatergic pathways or GABA-ergic degen-eration which allows us to represent respectively early oradvanced PD model (see [35] for a review)

43 Alzheimerrsquos Disease Alzheimerrsquos disease (AD) is achronic neurodegenerative disorder characterized by a pro-gressive loss of memory and dementia The causes andaetiology of AD are still unknown and the brain regionsmoreaffected are the hippocampus and neocortex At molecularlevel brain patients are characterized by amyloid plaques(dense and insoluble deposits of the beta-amyloid peptide-A120573) and neurofibrillary tangles (intracellular aggregates of

the hyperphosphorylated microtubule-associated proteintau) One of the first pieces of evidence of neurodegenerationin AD is the neuronal loss of cholinergic fibers of the entorhi-nal cortex connecting the CA1 layer of hippocampus [36]AD ismimicked by different animalmodels from invertebrate[37] to rodents [38] Except for the mouse SAMP8 [39] alltransgenic models combine different mutations for amyloidprecursor protein tau and presenilin 1

The best organotypic model that represents cellular alter-ation observed in AD is the hippocampal slice Alberdiand colleagues [14] used an organotypic slice in which theymaintained the connection between entorhinal cortex andhippocampus This model could be particularly useful tostudy cholinergic degeneration and activation of neurogen-esis in the SGZ Induction of neurodegeneration is obtainedby treatment of slices with different A120573 peptides As forseveral animal models A120573 together with neuronal damagemay play a role in the regulation of adult neurogenesis [4]In particular low concentration and the small A120573

25ndash35 pep-tide result in an increase of mossy fibers density and stim-ulation of endogenous SGZ neurogenesis Nonetheless neu-roinflammation associated with A120573 toxicity can mediate theinhibition of SGZ neurogenesis through the release of variousproinflammatory cytokines

5 Other Organotypic Models forNeuroregeneration Studies

In addition to neurogenesis in the brain organotypic cultureswere employed also to study cell regeneration in spinal cordand PNS Organotypic slices of mice adult spinal cord canbe prepared with the liquid-air interface [40] Spinal cordcells incorporate BrdU and express nestin Oct34 andDppa in the inner mass and have been used for modellingregeneration in spinal cord lesion [41] Enteric neural stemcells (ENSC) have been isolated from adult intestine Theyexpress markers like Ret p75 and CD49b [42] and can dif-ferentiate primarily into glia [43] but also into neurons andmyofibroblasts If neurogenic potential of ENSC has beendemonstrated in vivo there is still discrepancy in resultsobtained in vitro Ex vivo organotypic cultures from longi-tudinal muscle and myenteric plexus tissue demonstrated 5-ethynyl-21015840-deoxyuridine incorporation in ENSC and poten-tial proliferation dependent on the PTENPI3KAkt pathway[44] To investigate the neurogenesis in auditory systemAburto and colleagues [45] used organotypic cultures ofexplanted chicken otic vesicles (OV) Neuroepithelial oticprogenitors transit through states of cell proliferation cell fatespecification cell cycle exit migration and differentiationshowing characteristics of neural stem cells In this caseautophagy covers a relevant role in maintaining clearanceof apoptotic cells and facilitating neuronal differentiation ofprogenitor cells [45] The inhibition of LC3B a gene markerof autophagy in organotypic cultures of OV provoked themisregulation of the cell cycle and impairing neurogenesis

Other factors like GDNF can contribute to the final neu-ritogenesis of NSC-derived PNS neurons as demonstratedin organotypic slices of mice spiral ganglia [46] Stimulationof GDNF-family receptor 120572-1 (GFR1205721) activated two parallel

Stem Cells International 5

pathways PI3KAkt and MEKErk especially during earlypostnatal days

6 Concluding Remarks

Organotypic cultures of the neurogenic niches represent avalid alternative method to study neurogenesis in the CNSwhich complements in vivomodels andneurosphere culturesMoreover these cultures represent a good method to modelbrain damage in diseases like AD PD or stroke and to studyhow to implement neurogenesis as a potential mechanism ofbrain repair in these disorders Organotypic slices have beenused especially for toxicity studies therefore in neurogenesisresearch they became valuable for testing drug effects onneurogenesis activation or improving cell fate specification

Competing Interests

The authors declare that they have no competing interests

References

[1] A Mewes H Franke and D Singer ldquoOrganotypic brain slicecultures of adult transgenic P301S micemdashamodel for tauopathystudiesrdquo PLoS ONE vol 7 no 9 Article ID e45017 2012

[2] F B Kleine Borgmann O Bracko and S Jessberger ldquoImagingneurite development of adult-born granule cellsrdquoDevelopmentvol 140 no 13 pp 2823ndash2827 2013

[3] H Kim E Kim M Park E Lee and K Namkoong ldquoOrgan-otypic hippocampal slice culture from the adult mouse braina versatile tool for translational neuropsychopharmacologyrdquoProgress in Neuro-Psychopharmacology and Biological Psychia-try vol 41 pp 36ndash43 2013

[4] L Z Rui Y LeTourneau S R Gregg et al ldquoNeuroblast divi-sion during migration toward the ischemic striatum a study ofdynamic migratory and proliferative characteristics of neurob-lasts from the subventricular zonerdquoThe Journal of Neurosciencevol 27 no 12 pp 3157ndash3162 2007

[5] B Lacar S Z Young J-C Platel and A Bordey ldquoImaging andrecording subventricular zone progenitor cells in live tissue ofpostnatalmicerdquo Frontiers inNeuroscience vol 4 article 43 2010

[6] D R Hochbaum Y Zhao S L Farhi et al ldquoAll-optical elec-trophysiology in mammalian neurons using engineered micro-bial rhodopsinsrdquo Nature Methods vol 11 no 8 pp 825ndash8332014

[7] M J Hogue ldquoHuman fetal brain cells in tissue cultures theiridentification and motilityrdquo Journal of Experimental Zoologyvol 106 no 1 pp 85ndash107 1947

[8] B H Gahwiler ldquoOrganotypic monolayer cultures of nervoustissuerdquo Journal of Neuroscience Methods vol 4 no 4 pp 329ndash342 1981

[9] T Knopfel L Rietschin and B H Gahwiler ldquoOrganotypic co-cultures of rat locus coeruleus and hippocampusrdquo EuropeanJournal of Neuroscience vol 1 no 6 pp 678ndash689 1989

[10] S M Crain and E R Peterson ldquoDevelopment of specific sen-sory-evoked synaptic networks in fetal mouse cord-brainstemculturesrdquo Science vol 188 no 4185 pp 275ndash278 1975

[11] U F Braschler A Iannone C Spenger J Streit and H-RLuscher ldquoAmodified roller tube technique for organotypic coc-ultures of embryonic rat spinal cord sensory ganglia and

skeletal musclerdquo Journal of Neuroscience Methods vol 29 no2 pp 121ndash129 1989

[12] RHAndres ADDucrayA Perez-Bouza et al ldquoCreatine sup-plementation improves dopaminergic cell survival and protectsagainst MPP+ toxicity in an organotypic tissue culture systemrdquoCell Transplantation vol 14 no 8 pp 537ndash550 2005

[13] L Stoppini P-A Buchs and D Muller ldquoA simple method fororganotypic cultures of nervous tissuerdquo Journal of NeuroscienceMethods vol 37 no 2 pp 173ndash182 1991

[14] E Alberdi M V Sanchez-Gomez F Cavaliere et al ldquoAmyloid120573 oligomers induce Ca2+ dysregulation and neuronal deaththrough activation of ionotropic glutamate receptorsrdquo CellCalcium vol 47 no 3 pp 264ndash272 2010

[15] F Cavaliere E S Vicente and C Matute ldquoAn organotypicculture model to study nigro-striatal degenerationrdquo Journal ofNeuroscience Methods vol 188 no 2 pp 205ndash212 2010

[16] D Vergni F Castiglione M Briani et al ldquoAmodel of ischemia-induced neuroblast activation in the adult subventricular zonerdquoPLoS ONE vol 4 no 4 Article ID e5278 2009

[17] P Codega V Silva-Vargas A Paul et al ldquoProspective identifica-tion and purification of quiescent adult neural stem cells fromtheir in vivo nicherdquo Neuron vol 82 no 3 pp 545ndash559 2014

[18] L Ruan B W-M Lau J Wang et al ldquoNeurogenesis in neu-rological and psychiatric diseases and brain injury from benchto bedsiderdquo Progress in Neurobiology vol 115 pp 116ndash137 2014

[19] A Alvarez-Buylla MTheelen and F Nottebohm ldquoMapping ofradial glia and of a new cell type in adult canary brainrdquo TheJournal of Neuroscience vol 8 no 8 pp 2707ndash2712 1988

[20] D A Lim and A Alvarez-Buylla ldquoAdult neural stem cells staketheir groundrdquo Trends in Neurosciences vol 37 no 10 pp 563ndash571 2014

[21] L Katsimpardi N K Litterman P A Schein et al ldquoVascularand neurogenic rejuvenation of the agingmouse brain by youngsystemic factorsrdquo Science vol 344 no 6184 pp 630ndash634 2014

[22] O Baron A Ratzka and C Grothe ldquoFibroblast growth factor2 regulates adequate nigrostriatal pathway formation in micerdquoJournal of Comparative Neurology vol 520 no 17 pp 3949ndash3961 2012

[23] J T Neumann J WThompson A P Raval C H Cohan K BKoronowski andMA Perez-Pinzon ldquoIncreasedBDNFproteinexpression after ischemic or PKC epsilon preconditioning pro-motes electrophysiologic changes that lead to neuroprotectionrdquoJournal of Cerebral Blood Flow andMetabolism vol 35 no 1 pp121ndash130 2015

[24] G Kempermann LWiskott and FHGage ldquoFunctional signif-icance of adult neurogenesisrdquo Current Opinion in Neurobiologyvol 14 no 2 pp 186ndash191 2004

[25] B Leuner E R Glasper and E Gould ldquoThymidine analogmethods for studies of adult neurogenesis are not equally sen-sitiverdquo Journal of Comparative Neurology vol 517 no 2 pp 123ndash133 2009

[26] D A Laplagne M S Esposito V C Piatti et al ldquoFunctionalconvergence of neurons generated in the developing and adulthippocampusrdquo PLoS Biology vol 4 no 12 article e409 2006

[27] J M Encinas and G Enikolopov ldquoIdentifying and quantitatingneural stem and progenitor cells in the adult brainrdquoMethods inCell Biology vol 85 pp 243ndash272 2008

[28] A Sierra S Martın-Suarez R Valcarcel-Martın et al ldquoNeu-ronal hyperactivity accelerates depletion of neural stem cellsand impairs hippocampal neurogenesisrdquo Cell Stem Cell vol 16no 5 pp 488ndash503 2015

6 Stem Cells International

[29] J Toslashnnesen C L Parish A T Soslashrensen et al ldquoFunctionalintegration of grafted neural stem cell-derived dopaminergicneurons monitored by optogenetics in an in vitro Parkinsonmodelrdquo PLoS ONE vol 6 no 3 Article ID e17560 2011

[30] F Cavaliere K Dinkel and K Reymann ldquoThe subventricularzone releases factors which can be protective in oxygenglucosedeprivation-induced cortical damage an organotypic studyrdquoExperimental Neurology vol 201 no 1 pp 66ndash74 2006

[31] O Chechneva K Dinkel F Cavaliere M Martinez-Sanchezand K G Reymann ldquoAnti-inflammatory treatment in oxygen-glucose-deprived hippocampal slice cultures is neuroprotectiveand associated with reduced cell proliferation and intact neu-rogenesisrdquo Neurobiology of Disease vol 23 no 2 pp 247ndash2592006

[32] M Ziemka-Nałecz L Stanaszek and T Zalewska ldquoOxygen-glucose deprivation promotes gliogenesis and microglia acti-vation in organotypic hippocampal slice culture Involvementof metalloproteinasesrdquoActa Neurobiologiae Experimentalis vol73 no 1 pp 130ndash142 2013

[33] D Plenz and S T Kitai ldquoOrganotypic cortex-striatum-mes-encephalon cultures the nigrostriatal pathwayrdquo NeuroscienceLetters vol 209 no 3 pp 177ndash180 1996

[34] C Ullrich and C Humpel ldquoRotenone induces cell death of cho-linergic neurons in an organotypic co-culture brain slicemodelrdquo Neurochemical Research vol 34 no 12 pp 2147ndash21532009

[35] N Daviaud E Garbayo P C Schiller M Perez-Pinzon andC N Montero-Menei ldquoOrganotypic cultures as tools for opti-mizing central nervous system cell therapiesrdquo ExperimentalNeurology vol 248 pp 429ndash440 2013

[36] M Gallagher and M T Koh ldquoEpisodic memory on the path toAlzheimerrsquos diseaserdquo Current Opinion in Neurobiology vol 21no 6 pp 929ndash934 2011

[37] K PruszligingAVoigt and J B Schulz ldquoDrosophilamelanogasteras a model organism for Alzheimerrsquos diseaserdquo Molecular Neu-rodegeneration vol 8 article 35 2013

[38] S J Webster A D Bachstetter P T Nelson F A Schmitt andL J Van Eldik ldquoUsing mice to model Alzheimerrsquos dementia anoverview of the clinical disease and the preclinical behavioralchanges in 10 mouse modelsrdquo Frontiers in Genetics vol 5 pp88ndash95 2014

[39] J F Flood and J E Morley ldquoLearning and memory in theSAMP8 mouserdquo Neuroscience and Biobehavioral Reviews vol22 no 1 pp 1ndash20 1998

[40] M V Glazova E S Pak and A K Murashov ldquoNeurogenicpotential of spinal cord organotypic culturerdquo NeuroscienceLetters vol 594 pp 60ndash65 2015

[41] M Heidemann J Streit and A Tscherter ldquoInvestigating func-tional regeneration in organotypic spinal cord co-culturesgrown on multi-electrode arraysrdquo Journal of Visualized Experi-ments vol 23 no 103 Article ID e53121 2015

[42] R Suarez-Rodrıguez and J Belkind-Gerson ldquoCultured nestin-positive cells from postnatal mouse small bowel differentiate exvivo into neurons glia and smooth musclerdquo STEM CELLS vol22 no 7 pp 1373ndash1385 2004

[43] N M Joseph S He E Quintana Y-G Kim G Nunez andS J Morrison ldquoEnteric glia are multipotent in culture but pri-marily form glia in the adult rodent gutrdquo Journal of ClinicalInvestigation vol 121 no 9 pp 3398ndash3411 2011

[44] L Becker J Peterson S Kulkarni and P J Pasricha ldquoEx vivoneurogenesis within enteric ganglia occurs in a PTEN depen-dent mannerrdquo PLoS ONE vol 8 no 3 Article ID e59452 2013

[45] M R Aburto H Sanchez-Calderon J M Hurle I Varela-Nieto and M Magarinos ldquoEarly otic development depends onautophagy for apoptotic cell clearance and neural differentia-tionrdquo Cell Death and Disease vol 3 article e394 2012

[46] S Euteneuer K H Yang E Chavez et al ldquoGlial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis inthe cochlear spiral ganglion via neural cell adhesion molecule(NCAM)rdquoMolecular and Cellular Neuroscience vol 54 pp 30ndash43 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Stem Cells International

cultures In this culture OGD generates a selective damagein the CA1 region and microglia recruitment to the damagedzone Several papers demonstrated that anti-inflammatorytreatment in organotypic slices facilitated the neurogenesisfrom the SGZ through the inhibition of both the p38mitogen-activated protein kinase and metalloproteinases [3132] Metalloproteinases are involved in the activation of sev-eral neuroinflammatory events Also during OGD this classof enzyme can sustain neuroinflammation and modulateneurogenesis in the dentate gyrus In fact the modulation ofculturemicroenvironment afterOGD in hippocampal organ-otypic cultures can promote the proliferation of glioblastswith predominant generation of oligodendrocyte progenitors[32]

42 Parkinsonrsquos Disease Parkinsonrsquos disease (PD) is causedby the selective neurodegeneration of dopaminergic neuronsThemain affected areas are the substantia nigra (s nigra) andstriatum Nevertheless dopaminergic degeneration affectsalso other areas like cerebral cortex globus pallidus and tha-lamus The lack of dopamine and an unbalance of dopamin-ergic and glutamatergic signal cause the classical motorsymptoms of bradykinesia rigidity resting tremors and lossof postural reflexes The system ldquos nigrastriatumcortexrdquohas been reproduced in vitro with different organotypiccultures The first attempt was made by Plenz and Kitai [33]they cocultivated with the roller tube technique the threemain areas involved in PD degeneration cerebral cortexstriatum and s nigra After a few days the three regions wereconnected by new tyrosine hydroxylase positive fibers (TH+)Another coculture model to study dopaminergic degenera-tion was performed by combining the basal nucleus ofMeyn-ert ventral mesencephalon parietal cortex and dorsal stria-tum of postnatal 7ndash9 rat pups [34] Nevertheless the presenceof the SVZ in these organotypic slices is important besidethe interest in adult neurogenesis In fact the SVZ is directlyinnervated by TH fibers suggesting that these can sustainthe maintenance of the neurogenic niche We described anorganotypic model in which we maintained in one singleslice the connection between s nigrastriatumcortex andSVZ [15] In this culture dopaminergic degeneration canbe obtained either by mechanical lesion or by 6-OHDAinjection In both cases the loss of dopamine stimulated theTH expression and proliferation of SVZ cells suggesting theneurogenic niche can be activated following dopaminergiclesion Moreover while 6-OHDA generated more specificdopaminergic degeneration the mechanical lesion can beused to study glutamatergic pathways or GABA-ergic degen-eration which allows us to represent respectively early oradvanced PD model (see [35] for a review)

43 Alzheimerrsquos Disease Alzheimerrsquos disease (AD) is achronic neurodegenerative disorder characterized by a pro-gressive loss of memory and dementia The causes andaetiology of AD are still unknown and the brain regionsmoreaffected are the hippocampus and neocortex At molecularlevel brain patients are characterized by amyloid plaques(dense and insoluble deposits of the beta-amyloid peptide-A120573) and neurofibrillary tangles (intracellular aggregates of

the hyperphosphorylated microtubule-associated proteintau) One of the first pieces of evidence of neurodegenerationin AD is the neuronal loss of cholinergic fibers of the entorhi-nal cortex connecting the CA1 layer of hippocampus [36]AD ismimicked by different animalmodels from invertebrate[37] to rodents [38] Except for the mouse SAMP8 [39] alltransgenic models combine different mutations for amyloidprecursor protein tau and presenilin 1

The best organotypic model that represents cellular alter-ation observed in AD is the hippocampal slice Alberdiand colleagues [14] used an organotypic slice in which theymaintained the connection between entorhinal cortex andhippocampus This model could be particularly useful tostudy cholinergic degeneration and activation of neurogen-esis in the SGZ Induction of neurodegeneration is obtainedby treatment of slices with different A120573 peptides As forseveral animal models A120573 together with neuronal damagemay play a role in the regulation of adult neurogenesis [4]In particular low concentration and the small A120573

25ndash35 pep-tide result in an increase of mossy fibers density and stim-ulation of endogenous SGZ neurogenesis Nonetheless neu-roinflammation associated with A120573 toxicity can mediate theinhibition of SGZ neurogenesis through the release of variousproinflammatory cytokines

5 Other Organotypic Models forNeuroregeneration Studies

In addition to neurogenesis in the brain organotypic cultureswere employed also to study cell regeneration in spinal cordand PNS Organotypic slices of mice adult spinal cord canbe prepared with the liquid-air interface [40] Spinal cordcells incorporate BrdU and express nestin Oct34 andDppa in the inner mass and have been used for modellingregeneration in spinal cord lesion [41] Enteric neural stemcells (ENSC) have been isolated from adult intestine Theyexpress markers like Ret p75 and CD49b [42] and can dif-ferentiate primarily into glia [43] but also into neurons andmyofibroblasts If neurogenic potential of ENSC has beendemonstrated in vivo there is still discrepancy in resultsobtained in vitro Ex vivo organotypic cultures from longi-tudinal muscle and myenteric plexus tissue demonstrated 5-ethynyl-21015840-deoxyuridine incorporation in ENSC and poten-tial proliferation dependent on the PTENPI3KAkt pathway[44] To investigate the neurogenesis in auditory systemAburto and colleagues [45] used organotypic cultures ofexplanted chicken otic vesicles (OV) Neuroepithelial oticprogenitors transit through states of cell proliferation cell fatespecification cell cycle exit migration and differentiationshowing characteristics of neural stem cells In this caseautophagy covers a relevant role in maintaining clearanceof apoptotic cells and facilitating neuronal differentiation ofprogenitor cells [45] The inhibition of LC3B a gene markerof autophagy in organotypic cultures of OV provoked themisregulation of the cell cycle and impairing neurogenesis

Other factors like GDNF can contribute to the final neu-ritogenesis of NSC-derived PNS neurons as demonstratedin organotypic slices of mice spiral ganglia [46] Stimulationof GDNF-family receptor 120572-1 (GFR1205721) activated two parallel

Stem Cells International 5

pathways PI3KAkt and MEKErk especially during earlypostnatal days

6 Concluding Remarks

Organotypic cultures of the neurogenic niches represent avalid alternative method to study neurogenesis in the CNSwhich complements in vivomodels andneurosphere culturesMoreover these cultures represent a good method to modelbrain damage in diseases like AD PD or stroke and to studyhow to implement neurogenesis as a potential mechanism ofbrain repair in these disorders Organotypic slices have beenused especially for toxicity studies therefore in neurogenesisresearch they became valuable for testing drug effects onneurogenesis activation or improving cell fate specification

Competing Interests

The authors declare that they have no competing interests

References

[1] A Mewes H Franke and D Singer ldquoOrganotypic brain slicecultures of adult transgenic P301S micemdashamodel for tauopathystudiesrdquo PLoS ONE vol 7 no 9 Article ID e45017 2012

[2] F B Kleine Borgmann O Bracko and S Jessberger ldquoImagingneurite development of adult-born granule cellsrdquoDevelopmentvol 140 no 13 pp 2823ndash2827 2013

[3] H Kim E Kim M Park E Lee and K Namkoong ldquoOrgan-otypic hippocampal slice culture from the adult mouse braina versatile tool for translational neuropsychopharmacologyrdquoProgress in Neuro-Psychopharmacology and Biological Psychia-try vol 41 pp 36ndash43 2013

[4] L Z Rui Y LeTourneau S R Gregg et al ldquoNeuroblast divi-sion during migration toward the ischemic striatum a study ofdynamic migratory and proliferative characteristics of neurob-lasts from the subventricular zonerdquoThe Journal of Neurosciencevol 27 no 12 pp 3157ndash3162 2007

[5] B Lacar S Z Young J-C Platel and A Bordey ldquoImaging andrecording subventricular zone progenitor cells in live tissue ofpostnatalmicerdquo Frontiers inNeuroscience vol 4 article 43 2010

[6] D R Hochbaum Y Zhao S L Farhi et al ldquoAll-optical elec-trophysiology in mammalian neurons using engineered micro-bial rhodopsinsrdquo Nature Methods vol 11 no 8 pp 825ndash8332014

[7] M J Hogue ldquoHuman fetal brain cells in tissue cultures theiridentification and motilityrdquo Journal of Experimental Zoologyvol 106 no 1 pp 85ndash107 1947

[8] B H Gahwiler ldquoOrganotypic monolayer cultures of nervoustissuerdquo Journal of Neuroscience Methods vol 4 no 4 pp 329ndash342 1981

[9] T Knopfel L Rietschin and B H Gahwiler ldquoOrganotypic co-cultures of rat locus coeruleus and hippocampusrdquo EuropeanJournal of Neuroscience vol 1 no 6 pp 678ndash689 1989

[10] S M Crain and E R Peterson ldquoDevelopment of specific sen-sory-evoked synaptic networks in fetal mouse cord-brainstemculturesrdquo Science vol 188 no 4185 pp 275ndash278 1975

[11] U F Braschler A Iannone C Spenger J Streit and H-RLuscher ldquoAmodified roller tube technique for organotypic coc-ultures of embryonic rat spinal cord sensory ganglia and

skeletal musclerdquo Journal of Neuroscience Methods vol 29 no2 pp 121ndash129 1989

[12] RHAndres ADDucrayA Perez-Bouza et al ldquoCreatine sup-plementation improves dopaminergic cell survival and protectsagainst MPP+ toxicity in an organotypic tissue culture systemrdquoCell Transplantation vol 14 no 8 pp 537ndash550 2005

[13] L Stoppini P-A Buchs and D Muller ldquoA simple method fororganotypic cultures of nervous tissuerdquo Journal of NeuroscienceMethods vol 37 no 2 pp 173ndash182 1991

[14] E Alberdi M V Sanchez-Gomez F Cavaliere et al ldquoAmyloid120573 oligomers induce Ca2+ dysregulation and neuronal deaththrough activation of ionotropic glutamate receptorsrdquo CellCalcium vol 47 no 3 pp 264ndash272 2010

[15] F Cavaliere E S Vicente and C Matute ldquoAn organotypicculture model to study nigro-striatal degenerationrdquo Journal ofNeuroscience Methods vol 188 no 2 pp 205ndash212 2010

[16] D Vergni F Castiglione M Briani et al ldquoAmodel of ischemia-induced neuroblast activation in the adult subventricular zonerdquoPLoS ONE vol 4 no 4 Article ID e5278 2009

[17] P Codega V Silva-Vargas A Paul et al ldquoProspective identifica-tion and purification of quiescent adult neural stem cells fromtheir in vivo nicherdquo Neuron vol 82 no 3 pp 545ndash559 2014

[18] L Ruan B W-M Lau J Wang et al ldquoNeurogenesis in neu-rological and psychiatric diseases and brain injury from benchto bedsiderdquo Progress in Neurobiology vol 115 pp 116ndash137 2014

[19] A Alvarez-Buylla MTheelen and F Nottebohm ldquoMapping ofradial glia and of a new cell type in adult canary brainrdquo TheJournal of Neuroscience vol 8 no 8 pp 2707ndash2712 1988

[20] D A Lim and A Alvarez-Buylla ldquoAdult neural stem cells staketheir groundrdquo Trends in Neurosciences vol 37 no 10 pp 563ndash571 2014

[21] L Katsimpardi N K Litterman P A Schein et al ldquoVascularand neurogenic rejuvenation of the agingmouse brain by youngsystemic factorsrdquo Science vol 344 no 6184 pp 630ndash634 2014

[22] O Baron A Ratzka and C Grothe ldquoFibroblast growth factor2 regulates adequate nigrostriatal pathway formation in micerdquoJournal of Comparative Neurology vol 520 no 17 pp 3949ndash3961 2012

[23] J T Neumann J WThompson A P Raval C H Cohan K BKoronowski andMA Perez-Pinzon ldquoIncreasedBDNFproteinexpression after ischemic or PKC epsilon preconditioning pro-motes electrophysiologic changes that lead to neuroprotectionrdquoJournal of Cerebral Blood Flow andMetabolism vol 35 no 1 pp121ndash130 2015

[24] G Kempermann LWiskott and FHGage ldquoFunctional signif-icance of adult neurogenesisrdquo Current Opinion in Neurobiologyvol 14 no 2 pp 186ndash191 2004

[25] B Leuner E R Glasper and E Gould ldquoThymidine analogmethods for studies of adult neurogenesis are not equally sen-sitiverdquo Journal of Comparative Neurology vol 517 no 2 pp 123ndash133 2009

[26] D A Laplagne M S Esposito V C Piatti et al ldquoFunctionalconvergence of neurons generated in the developing and adulthippocampusrdquo PLoS Biology vol 4 no 12 article e409 2006

[27] J M Encinas and G Enikolopov ldquoIdentifying and quantitatingneural stem and progenitor cells in the adult brainrdquoMethods inCell Biology vol 85 pp 243ndash272 2008

[28] A Sierra S Martın-Suarez R Valcarcel-Martın et al ldquoNeu-ronal hyperactivity accelerates depletion of neural stem cellsand impairs hippocampal neurogenesisrdquo Cell Stem Cell vol 16no 5 pp 488ndash503 2015

6 Stem Cells International

[29] J Toslashnnesen C L Parish A T Soslashrensen et al ldquoFunctionalintegration of grafted neural stem cell-derived dopaminergicneurons monitored by optogenetics in an in vitro Parkinsonmodelrdquo PLoS ONE vol 6 no 3 Article ID e17560 2011

[30] F Cavaliere K Dinkel and K Reymann ldquoThe subventricularzone releases factors which can be protective in oxygenglucosedeprivation-induced cortical damage an organotypic studyrdquoExperimental Neurology vol 201 no 1 pp 66ndash74 2006

[31] O Chechneva K Dinkel F Cavaliere M Martinez-Sanchezand K G Reymann ldquoAnti-inflammatory treatment in oxygen-glucose-deprived hippocampal slice cultures is neuroprotectiveand associated with reduced cell proliferation and intact neu-rogenesisrdquo Neurobiology of Disease vol 23 no 2 pp 247ndash2592006

[32] M Ziemka-Nałecz L Stanaszek and T Zalewska ldquoOxygen-glucose deprivation promotes gliogenesis and microglia acti-vation in organotypic hippocampal slice culture Involvementof metalloproteinasesrdquoActa Neurobiologiae Experimentalis vol73 no 1 pp 130ndash142 2013

[33] D Plenz and S T Kitai ldquoOrganotypic cortex-striatum-mes-encephalon cultures the nigrostriatal pathwayrdquo NeuroscienceLetters vol 209 no 3 pp 177ndash180 1996

[34] C Ullrich and C Humpel ldquoRotenone induces cell death of cho-linergic neurons in an organotypic co-culture brain slicemodelrdquo Neurochemical Research vol 34 no 12 pp 2147ndash21532009

[35] N Daviaud E Garbayo P C Schiller M Perez-Pinzon andC N Montero-Menei ldquoOrganotypic cultures as tools for opti-mizing central nervous system cell therapiesrdquo ExperimentalNeurology vol 248 pp 429ndash440 2013

[36] M Gallagher and M T Koh ldquoEpisodic memory on the path toAlzheimerrsquos diseaserdquo Current Opinion in Neurobiology vol 21no 6 pp 929ndash934 2011

[37] K PruszligingAVoigt and J B Schulz ldquoDrosophilamelanogasteras a model organism for Alzheimerrsquos diseaserdquo Molecular Neu-rodegeneration vol 8 article 35 2013

[38] S J Webster A D Bachstetter P T Nelson F A Schmitt andL J Van Eldik ldquoUsing mice to model Alzheimerrsquos dementia anoverview of the clinical disease and the preclinical behavioralchanges in 10 mouse modelsrdquo Frontiers in Genetics vol 5 pp88ndash95 2014

[39] J F Flood and J E Morley ldquoLearning and memory in theSAMP8 mouserdquo Neuroscience and Biobehavioral Reviews vol22 no 1 pp 1ndash20 1998

[40] M V Glazova E S Pak and A K Murashov ldquoNeurogenicpotential of spinal cord organotypic culturerdquo NeuroscienceLetters vol 594 pp 60ndash65 2015

[41] M Heidemann J Streit and A Tscherter ldquoInvestigating func-tional regeneration in organotypic spinal cord co-culturesgrown on multi-electrode arraysrdquo Journal of Visualized Experi-ments vol 23 no 103 Article ID e53121 2015

[42] R Suarez-Rodrıguez and J Belkind-Gerson ldquoCultured nestin-positive cells from postnatal mouse small bowel differentiate exvivo into neurons glia and smooth musclerdquo STEM CELLS vol22 no 7 pp 1373ndash1385 2004

[43] N M Joseph S He E Quintana Y-G Kim G Nunez andS J Morrison ldquoEnteric glia are multipotent in culture but pri-marily form glia in the adult rodent gutrdquo Journal of ClinicalInvestigation vol 121 no 9 pp 3398ndash3411 2011

[44] L Becker J Peterson S Kulkarni and P J Pasricha ldquoEx vivoneurogenesis within enteric ganglia occurs in a PTEN depen-dent mannerrdquo PLoS ONE vol 8 no 3 Article ID e59452 2013

[45] M R Aburto H Sanchez-Calderon J M Hurle I Varela-Nieto and M Magarinos ldquoEarly otic development depends onautophagy for apoptotic cell clearance and neural differentia-tionrdquo Cell Death and Disease vol 3 article e394 2012

[46] S Euteneuer K H Yang E Chavez et al ldquoGlial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis inthe cochlear spiral ganglion via neural cell adhesion molecule(NCAM)rdquoMolecular and Cellular Neuroscience vol 54 pp 30ndash43 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Stem Cells International 5

pathways PI3KAkt and MEKErk especially during earlypostnatal days

6 Concluding Remarks

Organotypic cultures of the neurogenic niches represent avalid alternative method to study neurogenesis in the CNSwhich complements in vivomodels andneurosphere culturesMoreover these cultures represent a good method to modelbrain damage in diseases like AD PD or stroke and to studyhow to implement neurogenesis as a potential mechanism ofbrain repair in these disorders Organotypic slices have beenused especially for toxicity studies therefore in neurogenesisresearch they became valuable for testing drug effects onneurogenesis activation or improving cell fate specification

Competing Interests

The authors declare that they have no competing interests

References

[1] A Mewes H Franke and D Singer ldquoOrganotypic brain slicecultures of adult transgenic P301S micemdashamodel for tauopathystudiesrdquo PLoS ONE vol 7 no 9 Article ID e45017 2012

[2] F B Kleine Borgmann O Bracko and S Jessberger ldquoImagingneurite development of adult-born granule cellsrdquoDevelopmentvol 140 no 13 pp 2823ndash2827 2013

[3] H Kim E Kim M Park E Lee and K Namkoong ldquoOrgan-otypic hippocampal slice culture from the adult mouse braina versatile tool for translational neuropsychopharmacologyrdquoProgress in Neuro-Psychopharmacology and Biological Psychia-try vol 41 pp 36ndash43 2013

[4] L Z Rui Y LeTourneau S R Gregg et al ldquoNeuroblast divi-sion during migration toward the ischemic striatum a study ofdynamic migratory and proliferative characteristics of neurob-lasts from the subventricular zonerdquoThe Journal of Neurosciencevol 27 no 12 pp 3157ndash3162 2007

[5] B Lacar S Z Young J-C Platel and A Bordey ldquoImaging andrecording subventricular zone progenitor cells in live tissue ofpostnatalmicerdquo Frontiers inNeuroscience vol 4 article 43 2010

[6] D R Hochbaum Y Zhao S L Farhi et al ldquoAll-optical elec-trophysiology in mammalian neurons using engineered micro-bial rhodopsinsrdquo Nature Methods vol 11 no 8 pp 825ndash8332014

[7] M J Hogue ldquoHuman fetal brain cells in tissue cultures theiridentification and motilityrdquo Journal of Experimental Zoologyvol 106 no 1 pp 85ndash107 1947

[8] B H Gahwiler ldquoOrganotypic monolayer cultures of nervoustissuerdquo Journal of Neuroscience Methods vol 4 no 4 pp 329ndash342 1981

[9] T Knopfel L Rietschin and B H Gahwiler ldquoOrganotypic co-cultures of rat locus coeruleus and hippocampusrdquo EuropeanJournal of Neuroscience vol 1 no 6 pp 678ndash689 1989

[10] S M Crain and E R Peterson ldquoDevelopment of specific sen-sory-evoked synaptic networks in fetal mouse cord-brainstemculturesrdquo Science vol 188 no 4185 pp 275ndash278 1975

[11] U F Braschler A Iannone C Spenger J Streit and H-RLuscher ldquoAmodified roller tube technique for organotypic coc-ultures of embryonic rat spinal cord sensory ganglia and

skeletal musclerdquo Journal of Neuroscience Methods vol 29 no2 pp 121ndash129 1989

[12] RHAndres ADDucrayA Perez-Bouza et al ldquoCreatine sup-plementation improves dopaminergic cell survival and protectsagainst MPP+ toxicity in an organotypic tissue culture systemrdquoCell Transplantation vol 14 no 8 pp 537ndash550 2005

[13] L Stoppini P-A Buchs and D Muller ldquoA simple method fororganotypic cultures of nervous tissuerdquo Journal of NeuroscienceMethods vol 37 no 2 pp 173ndash182 1991

[14] E Alberdi M V Sanchez-Gomez F Cavaliere et al ldquoAmyloid120573 oligomers induce Ca2+ dysregulation and neuronal deaththrough activation of ionotropic glutamate receptorsrdquo CellCalcium vol 47 no 3 pp 264ndash272 2010

[15] F Cavaliere E S Vicente and C Matute ldquoAn organotypicculture model to study nigro-striatal degenerationrdquo Journal ofNeuroscience Methods vol 188 no 2 pp 205ndash212 2010

[16] D Vergni F Castiglione M Briani et al ldquoAmodel of ischemia-induced neuroblast activation in the adult subventricular zonerdquoPLoS ONE vol 4 no 4 Article ID e5278 2009

[17] P Codega V Silva-Vargas A Paul et al ldquoProspective identifica-tion and purification of quiescent adult neural stem cells fromtheir in vivo nicherdquo Neuron vol 82 no 3 pp 545ndash559 2014

[18] L Ruan B W-M Lau J Wang et al ldquoNeurogenesis in neu-rological and psychiatric diseases and brain injury from benchto bedsiderdquo Progress in Neurobiology vol 115 pp 116ndash137 2014

[19] A Alvarez-Buylla MTheelen and F Nottebohm ldquoMapping ofradial glia and of a new cell type in adult canary brainrdquo TheJournal of Neuroscience vol 8 no 8 pp 2707ndash2712 1988

[20] D A Lim and A Alvarez-Buylla ldquoAdult neural stem cells staketheir groundrdquo Trends in Neurosciences vol 37 no 10 pp 563ndash571 2014

[21] L Katsimpardi N K Litterman P A Schein et al ldquoVascularand neurogenic rejuvenation of the agingmouse brain by youngsystemic factorsrdquo Science vol 344 no 6184 pp 630ndash634 2014

[22] O Baron A Ratzka and C Grothe ldquoFibroblast growth factor2 regulates adequate nigrostriatal pathway formation in micerdquoJournal of Comparative Neurology vol 520 no 17 pp 3949ndash3961 2012

[23] J T Neumann J WThompson A P Raval C H Cohan K BKoronowski andMA Perez-Pinzon ldquoIncreasedBDNFproteinexpression after ischemic or PKC epsilon preconditioning pro-motes electrophysiologic changes that lead to neuroprotectionrdquoJournal of Cerebral Blood Flow andMetabolism vol 35 no 1 pp121ndash130 2015

[24] G Kempermann LWiskott and FHGage ldquoFunctional signif-icance of adult neurogenesisrdquo Current Opinion in Neurobiologyvol 14 no 2 pp 186ndash191 2004

[25] B Leuner E R Glasper and E Gould ldquoThymidine analogmethods for studies of adult neurogenesis are not equally sen-sitiverdquo Journal of Comparative Neurology vol 517 no 2 pp 123ndash133 2009

[26] D A Laplagne M S Esposito V C Piatti et al ldquoFunctionalconvergence of neurons generated in the developing and adulthippocampusrdquo PLoS Biology vol 4 no 12 article e409 2006

[27] J M Encinas and G Enikolopov ldquoIdentifying and quantitatingneural stem and progenitor cells in the adult brainrdquoMethods inCell Biology vol 85 pp 243ndash272 2008

[28] A Sierra S Martın-Suarez R Valcarcel-Martın et al ldquoNeu-ronal hyperactivity accelerates depletion of neural stem cellsand impairs hippocampal neurogenesisrdquo Cell Stem Cell vol 16no 5 pp 488ndash503 2015

6 Stem Cells International

[29] J Toslashnnesen C L Parish A T Soslashrensen et al ldquoFunctionalintegration of grafted neural stem cell-derived dopaminergicneurons monitored by optogenetics in an in vitro Parkinsonmodelrdquo PLoS ONE vol 6 no 3 Article ID e17560 2011

[30] F Cavaliere K Dinkel and K Reymann ldquoThe subventricularzone releases factors which can be protective in oxygenglucosedeprivation-induced cortical damage an organotypic studyrdquoExperimental Neurology vol 201 no 1 pp 66ndash74 2006

[31] O Chechneva K Dinkel F Cavaliere M Martinez-Sanchezand K G Reymann ldquoAnti-inflammatory treatment in oxygen-glucose-deprived hippocampal slice cultures is neuroprotectiveand associated with reduced cell proliferation and intact neu-rogenesisrdquo Neurobiology of Disease vol 23 no 2 pp 247ndash2592006

[32] M Ziemka-Nałecz L Stanaszek and T Zalewska ldquoOxygen-glucose deprivation promotes gliogenesis and microglia acti-vation in organotypic hippocampal slice culture Involvementof metalloproteinasesrdquoActa Neurobiologiae Experimentalis vol73 no 1 pp 130ndash142 2013

[33] D Plenz and S T Kitai ldquoOrganotypic cortex-striatum-mes-encephalon cultures the nigrostriatal pathwayrdquo NeuroscienceLetters vol 209 no 3 pp 177ndash180 1996

[34] C Ullrich and C Humpel ldquoRotenone induces cell death of cho-linergic neurons in an organotypic co-culture brain slicemodelrdquo Neurochemical Research vol 34 no 12 pp 2147ndash21532009

[35] N Daviaud E Garbayo P C Schiller M Perez-Pinzon andC N Montero-Menei ldquoOrganotypic cultures as tools for opti-mizing central nervous system cell therapiesrdquo ExperimentalNeurology vol 248 pp 429ndash440 2013

[36] M Gallagher and M T Koh ldquoEpisodic memory on the path toAlzheimerrsquos diseaserdquo Current Opinion in Neurobiology vol 21no 6 pp 929ndash934 2011

[37] K PruszligingAVoigt and J B Schulz ldquoDrosophilamelanogasteras a model organism for Alzheimerrsquos diseaserdquo Molecular Neu-rodegeneration vol 8 article 35 2013

[38] S J Webster A D Bachstetter P T Nelson F A Schmitt andL J Van Eldik ldquoUsing mice to model Alzheimerrsquos dementia anoverview of the clinical disease and the preclinical behavioralchanges in 10 mouse modelsrdquo Frontiers in Genetics vol 5 pp88ndash95 2014

[39] J F Flood and J E Morley ldquoLearning and memory in theSAMP8 mouserdquo Neuroscience and Biobehavioral Reviews vol22 no 1 pp 1ndash20 1998

[40] M V Glazova E S Pak and A K Murashov ldquoNeurogenicpotential of spinal cord organotypic culturerdquo NeuroscienceLetters vol 594 pp 60ndash65 2015

[41] M Heidemann J Streit and A Tscherter ldquoInvestigating func-tional regeneration in organotypic spinal cord co-culturesgrown on multi-electrode arraysrdquo Journal of Visualized Experi-ments vol 23 no 103 Article ID e53121 2015

[42] R Suarez-Rodrıguez and J Belkind-Gerson ldquoCultured nestin-positive cells from postnatal mouse small bowel differentiate exvivo into neurons glia and smooth musclerdquo STEM CELLS vol22 no 7 pp 1373ndash1385 2004

[43] N M Joseph S He E Quintana Y-G Kim G Nunez andS J Morrison ldquoEnteric glia are multipotent in culture but pri-marily form glia in the adult rodent gutrdquo Journal of ClinicalInvestigation vol 121 no 9 pp 3398ndash3411 2011

[44] L Becker J Peterson S Kulkarni and P J Pasricha ldquoEx vivoneurogenesis within enteric ganglia occurs in a PTEN depen-dent mannerrdquo PLoS ONE vol 8 no 3 Article ID e59452 2013

[45] M R Aburto H Sanchez-Calderon J M Hurle I Varela-Nieto and M Magarinos ldquoEarly otic development depends onautophagy for apoptotic cell clearance and neural differentia-tionrdquo Cell Death and Disease vol 3 article e394 2012

[46] S Euteneuer K H Yang E Chavez et al ldquoGlial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis inthe cochlear spiral ganglion via neural cell adhesion molecule(NCAM)rdquoMolecular and Cellular Neuroscience vol 54 pp 30ndash43 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Stem Cells International

[29] J Toslashnnesen C L Parish A T Soslashrensen et al ldquoFunctionalintegration of grafted neural stem cell-derived dopaminergicneurons monitored by optogenetics in an in vitro Parkinsonmodelrdquo PLoS ONE vol 6 no 3 Article ID e17560 2011

[30] F Cavaliere K Dinkel and K Reymann ldquoThe subventricularzone releases factors which can be protective in oxygenglucosedeprivation-induced cortical damage an organotypic studyrdquoExperimental Neurology vol 201 no 1 pp 66ndash74 2006

[31] O Chechneva K Dinkel F Cavaliere M Martinez-Sanchezand K G Reymann ldquoAnti-inflammatory treatment in oxygen-glucose-deprived hippocampal slice cultures is neuroprotectiveand associated with reduced cell proliferation and intact neu-rogenesisrdquo Neurobiology of Disease vol 23 no 2 pp 247ndash2592006

[32] M Ziemka-Nałecz L Stanaszek and T Zalewska ldquoOxygen-glucose deprivation promotes gliogenesis and microglia acti-vation in organotypic hippocampal slice culture Involvementof metalloproteinasesrdquoActa Neurobiologiae Experimentalis vol73 no 1 pp 130ndash142 2013

[33] D Plenz and S T Kitai ldquoOrganotypic cortex-striatum-mes-encephalon cultures the nigrostriatal pathwayrdquo NeuroscienceLetters vol 209 no 3 pp 177ndash180 1996

[34] C Ullrich and C Humpel ldquoRotenone induces cell death of cho-linergic neurons in an organotypic co-culture brain slicemodelrdquo Neurochemical Research vol 34 no 12 pp 2147ndash21532009

[35] N Daviaud E Garbayo P C Schiller M Perez-Pinzon andC N Montero-Menei ldquoOrganotypic cultures as tools for opti-mizing central nervous system cell therapiesrdquo ExperimentalNeurology vol 248 pp 429ndash440 2013

[36] M Gallagher and M T Koh ldquoEpisodic memory on the path toAlzheimerrsquos diseaserdquo Current Opinion in Neurobiology vol 21no 6 pp 929ndash934 2011

[37] K PruszligingAVoigt and J B Schulz ldquoDrosophilamelanogasteras a model organism for Alzheimerrsquos diseaserdquo Molecular Neu-rodegeneration vol 8 article 35 2013

[38] S J Webster A D Bachstetter P T Nelson F A Schmitt andL J Van Eldik ldquoUsing mice to model Alzheimerrsquos dementia anoverview of the clinical disease and the preclinical behavioralchanges in 10 mouse modelsrdquo Frontiers in Genetics vol 5 pp88ndash95 2014

[39] J F Flood and J E Morley ldquoLearning and memory in theSAMP8 mouserdquo Neuroscience and Biobehavioral Reviews vol22 no 1 pp 1ndash20 1998

[40] M V Glazova E S Pak and A K Murashov ldquoNeurogenicpotential of spinal cord organotypic culturerdquo NeuroscienceLetters vol 594 pp 60ndash65 2015

[41] M Heidemann J Streit and A Tscherter ldquoInvestigating func-tional regeneration in organotypic spinal cord co-culturesgrown on multi-electrode arraysrdquo Journal of Visualized Experi-ments vol 23 no 103 Article ID e53121 2015

[42] R Suarez-Rodrıguez and J Belkind-Gerson ldquoCultured nestin-positive cells from postnatal mouse small bowel differentiate exvivo into neurons glia and smooth musclerdquo STEM CELLS vol22 no 7 pp 1373ndash1385 2004

[43] N M Joseph S He E Quintana Y-G Kim G Nunez andS J Morrison ldquoEnteric glia are multipotent in culture but pri-marily form glia in the adult rodent gutrdquo Journal of ClinicalInvestigation vol 121 no 9 pp 3398ndash3411 2011

[44] L Becker J Peterson S Kulkarni and P J Pasricha ldquoEx vivoneurogenesis within enteric ganglia occurs in a PTEN depen-dent mannerrdquo PLoS ONE vol 8 no 3 Article ID e59452 2013

[45] M R Aburto H Sanchez-Calderon J M Hurle I Varela-Nieto and M Magarinos ldquoEarly otic development depends onautophagy for apoptotic cell clearance and neural differentia-tionrdquo Cell Death and Disease vol 3 article e394 2012

[46] S Euteneuer K H Yang E Chavez et al ldquoGlial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis inthe cochlear spiral ganglion via neural cell adhesion molecule(NCAM)rdquoMolecular and Cellular Neuroscience vol 54 pp 30ndash43 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology