Vol. 6 Issue 2

www.natureprotocols.com

❱ Subtype-specific neurons from postnatal astroglia ❱ Spike-in SILAC ❱ Circular polymerase extension cloning ❱ Imaging fluorescent zebrafish ❱ Assaying stem cell mechanobiology

nature publishing group © 2011 Nature America, Inc. All rights reserved. ( genes neural pro the overexpression retroviralof that foundstudy, wethis in astroglia the acquisition aof true neuronal identity, as reflected by the ability of Subsequently, it was shown that the effect of Pax6 encompasses the ronal morphology and the gradual expression of neuronal markers toward a neuronal identity, as revealed by the acquisition aof neu astroglia cortical mouse cultured in switch fate a induce can tors ( boxed6determinant pairedgene fate neurogenic the of expressionforced that studies previous in acquisition of a stable astroglial cell fate not give rise to neurons without manipulation, thus indicating the proliferating, astroglial cells remain within the glial lineage and do underwhich conditions theyproliferate monolayer.aas Although can be maintained in primary culture in serum Astroglia obtained from the early postnatal cerebral cortex (P5–P7) telencephalic neurogenic fate determinant neurogenin and aldehyde dehydrogenase 1 L1, and they are devoid of the dorsal transporter (GLAST), glutamine GFAP synthetase, express not glutamate do transporterFurthermore, mouse astroglia express S100 1, the in which glia, radial genic protein (GFAP), which clearly distinguishes these cells from neuro­ astroglia postnatal genesis and glutamatergic of GABAergic functional neurons from the instruct selectively to protocol robust and simple a provide neurons become to determinants fate neurogenic by astroglia isolated at early postnatal stages (P5–P7) can genes be instructed neurogenic of ing intermediate progenitors through or transformation direct by either astroglia, to rise give onic neurogenesis (~E18), they lose their neurogenic potential and a as source for serve neurons and neurogenic glia progenitors, but radial at late mouse, embry the in corticogenesis early During I conversion from non-neuronal cells, with potential for brain regenerative medicine. thus offering an alternative to the use of embryonic or neural stem cells. Moreover, it can be a useful model for studies of lineage neurogenic transcription factor provides a stringent experimental system to study the ofspecification a selective neuronal subtype, neurons, respectively (7–21 d for different degrees of maturity). as complexachaete-scute homolog 1 or conversion induced by persistent and strong retroviral expression of synapse-forming neurons. a robust protocol for the efficient Instructing glial cells to generate neurons may prove to be a strategy to replace neurons that have degenerated. Here, we describe Published online February 3 2011; doi:10.1038/nprot.2010.188 Research Center for Environment and Health, Neuherberg, Germany. Correspondence should be addressed to B.B. ([email protected] 1 Tatiana Simon-Ebert Christophe Heinrich postnatal astroglia of the mouse cerebral cortex Generation of subtype-specific neurons from 214 Department of Physiological Genomics,of Department Institute Physiology,of Ludwig Ascl1 NTRO protocol Astroglia are defined by the expression of glial fibrillary acidic fibrillary glial of expression the by defined are Astroglia

| VOL.6 NO.2VOL.6 , referred to hereafter as D UCT - I derived neurons to fire action potentialsactionfire derivedneuronsto ON Neurog2 | 2011 in in vitro | r amla achaete mammalian or

natureprotocols 1 1 1 2 , Gascón Sergio , Timm Schroeder , concomitant with an epigenetic silenc . Recent work has shown that cortical cortical that shown has work Recent . . T his his protocol involves two steps: (i) expansion of astroglial cells (7 d) and (ii) astroglia-to-neuron Mash1 Pax6 in in vitro β ) is even more effective to drive ,the 2,4 ) driven by retroviralvec bydriven ) . However, we have shown Ascl1 l conversion of postnatal astroglia from the mouse cerebral cortex into functional, - 1 glutamate/ , 2 - , Giacomo Masserdotti containing medium, ) ) and/or distal-less homeobox 2 ( 2 - , Magdalena Götz ct hmlg 1 homolog scute - 3–5 2 (refs. 3–5). 5 . Moreover,. l . Here,we . - aspartate - Maximilians Maximilians University Munich, Munich, Germany. 6,7 3 - ­ ­ ­ ­ . .

O 1 ur ur protocol of astroglia-to-neuron conversion by a single , 2 may provide an alternative experimental model to embryonic system stem our specification, subtype neuronal studying researchers (ref. (~70% astroglia with compared (ref. (~20% fibroblasts in elicited process reprogramming distance mesoderm with compared neurons glia single factor is sufficient for neuronal conversion of postnatal astro of neurons toward full functionality required three factors, while a tors for introducing the transcription factors. Moreover, maturationthis procedure differs from our protocol in the use of lentiviral vec factor 1 scription factor 2, also referred to as forced coexpression of embryonic and perinatal fibroblasts into glutamatergic neurons by recently an alternative procedure has also been described to convert Dlx2 glutamatergic (using encephalon,developedwe efficient an procedure togenerate both ing the genesis of different neuronal subtypes in the developing tel respective transcription factor on basis of its known role in instruct ­synapse constructs retroviral neurogenic fate determinant using silencing allydiminished. Indeed, stronger andprolonged expression the of prohence,silencing; pCLIG Failure to complete neurogenesis may be because of the fact that the thus suggesting that astroglia as well as the lack of spontaneous or evoked synaptic transmission, reflected by the absence of synaptic clustering of presynapticrecordings proteins able excitelectrical of generationtowardthe astroglia cortical mouse Neurog2 & Berninger Benedikt For researchers interested in studying lineage reprogramming, lineage studying in interested researchers For 1 4 , Lepier Alexandra

, possibly due to the closer lineage relationship of astroglia and ) neurons from the same population of cortical astroglia neurons, as revealed by electrophysiologypatchrevealedneurons,byusing as - - - based retroviral vectorsretroviral based 1 like protein ( omn neurons forming 1 (encoding neurogenin-2) or may also contribute to the rather low efficiency of the the of efficiency low rather the to contribute also may 5 . However, no functional synaptogenesis was observed, as Dlx2 ) ) for generation of glutamatergic or G 9 Myt11 - Neurog2 allowed for the genesis of fully functional, fully of genesis the for allowed neural gene expression had become gradu become had expression gene neural Mash1 1 , Rodrigo Sanchez 4 Ipraty b te eeto o the of selection the by Importantly, . ) 1 - 1 , to 2 0 ) or GABAergic (using ). Besides the different cellular source, , 2 - Institute for Stem Cell Research, National Pou3f2 neuron conversion was incomplete 8 used in this study were prone to pronewere study this in used Brn-2 - derived fibroblasts. Lineage Lineage fibroblasts. derived (POU domain class 3, tran 4 Mash1 )). On the other hand, for hand, other the On )). ) and myelin transcription - resistant pCAG - muenchen.de). (also referred to 1 ,

Mash1 A B A and/or - ergic - clamp 4 based . 1 0 )),

5 ­ ­ ­ ­ ­ ­ ­ .

© 2011 Nature America, Inc. All rights reserved. combining more than one factor ( glutamatergic neurons by by induced version astroglia following neurons GABAergic of yield neurons functional toward induce a neuronal identity and to promote a complete maturation respective factors used transcription with regard to their ability to largely unknown and require further characterization. version of quiescent astroglia into sphere efficiency astroglia adult presented here can principally also be adapted for cells derived from rise to self isolated when and proliferation, resume tissue following injury to the cerebral cortex, astroglia within the lesioned astroglia, which is unfavorable to large This is likely to be due to the limited proliferative potential of adult astroglia. mouse adult culture routinelyprotocolsto available are limited yield of astroglial cells following culturing, and currently no postnatal or even adult stages is hampered in first place by the experimental animals.theof Conversion cells astroglial at later of rather tors used. One major limitation of the protocol is related to the age tergic or GABAergic neurons, depending on the fac transcription astroglial cells from the early postnatal cerebral cortex into glutamaIn its present form, the protocol warrants an efficient conversion of the protocolof Limitations that convert non from the human brain in culture tions, the protocol presented here may be extendable to cells derivedsion of neurogenic fate determinants. Finally, with some modifica of the neuronal phenotypes that can be obtained by forced expres brain areas other than the cerebral cortex may expand the diversity gic or spinal motor neurons. Furthermore, culturing astroglia from or GABAergic neurons, such as, for instance, midbrain dopaminer the possibility to generate other types of neurons than glutamatergic fication. Further developments of the protocol should also examine underlying glia ble to biochemical analysis to decipher the molecular mechanisms sented here, this experimental system should be in principle amena converted with high efficiency and into scale neurons large using at the cultured protocol be precan astroglia postnatal that Given Potential the protocolof applications targeting, which can be helpful in analyzing lineage decisions gene to amenable easily Moreover,arethis. preclude cells fact ES undergoing neuronal conversion typically exit the cell cycle may in tion of distinct glutamatergic subtypes. The fact that astroglial cells genera sequential such undergoing of capable are determinants notclear underwhetherastroglia theneurogenicinfluence of fate icking the behavior of cultured cortical progenitors subtypes glutamatergic distinct of generation tial the presence of a sonic hedgehog inhibitor recapitulate the sequen neurons from mouse ES cells allow for the selective generation glutamatergicof and GABAergic neural stem cells types cell pluripotent other cells, (ES) approach consists in the expansion of the astroglial cells under under cells astroglial the of expansion the in consists approach In addition, we observed marked differences in the potency of the 4 - . However, the molecular processes the underlying con renewing and multipotent neurospheres - - derived neurosphere cells and works with similar similar with works and cells neurosphere derived to 13,14 - neurogenic cells into neurons. - neuron conversion and neuronal subtype speci . Specifically, protocols have been developed that Mash1 Neurog2 or or 4 . Indeed, we always observed a lower a observed always we Indeed, . 15–20 Dlx2 2 4 . Interestingly, ES cells cultured in to screen for transcription factors Mash1 (ref. , compared with the genesis of of genesis the with ,compared - 4 1 scale expansion. However, ). This can be overcome by 2 - and or even more restricted restricted more even or forming astroglia are still Dlx2 in vitro in - 2 ). An to 2 1 2 2 ,mim therein 5 . It is currently . The protocol - neuron con neuron

alternative , can give can , 2 3 . ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­

tions; ref.see dayspostnatal 14–16 (C.H., M.G. and B.B., observa unpublished Box 1 neurosphere conditions before astroglia five forced expression of distinct neurogenic fate determinants using a postnatal astroglia isolated from the mouse cerebral cortex, by the vitro in Overview of the procedure. design Experimental rons that demonstrates such are restrictions not insurmountable. can give rise not only to glutamatergic but also to GABAergic neu gic neurons. However, the fact that the very same cortical astroglia which may, hence, show a bias toward the generation of glutamater protocol has been established for astroglia from the cerebral cortex, the of astroglia molecular basis for this difference is not yet well understood ( due in part to the higher expression SRYof The higher susceptibility of astroglia sphere conditions greatly enhanced neurogenesis induced by demonstrate that cell division is not required for astrogliaments in Berninger retroviraltransductionchoice methodthe wasof formostexperi experimentalthe addressed.questionbe to Forinstance, although will mainly depend on the costs, availability of biosafety astrogliafacilities toward and full functional neurons. The choice of gene delivery well as plasmid transfection allow for conversion Choiceof postnatal of thecortical gene delivery method. cerebral cortex rat postnatal the from astroglia conversion the of to applied fully the cerebral cortex of C57BL/6J mice glia The protocol described here has been optimized for the successful • • • • • Sox2 and/or neuronglutamatergic identity, forced expression of into astroglia. Whereas forced neurogenic fate of thegene determinantsfollowing transfer theastroglia until neurons process isagradual thatrequires from 7dto 4weeks Astroglia-to-neuron conversion the corresponding retroviral vector plasmids. Mash1 vectors encoding neurogenic fate determinants such as At 2–4 h after seeding, the cells are transduced retroviral with Retroviral transduction or plasmid transfection of astroglial cells poly are removed andseeded onto from theflask trypsinization by cells Passaging astroglial of andseeding factorgrowth andbasicfibroblastgrowth factor. FBS, 5%(vol/vol) horse serum, B27supplement, epidermal plastic flasks for expansion in medium containing 10% (vol/vol) Primary sociated to asingle obtain mice (P5–P7) isdissectedfrom andmechanically dis thebrain thecerebral of cortex Dissection Finally, another parameter likely to contribute to the outcome the to contribute to likely parameter Finally,another - - to step procedure, as outlined below ( - ) by astroglia under ) neurosphereby astroglia conditions, but precisethe ). Astroglial cells can indeed give rise to neurosphere cells until d - generation of glutamatergic or GABAergic neurons from neurons GABAergic or glutamatergic of generation neuron conversion of early postnatal astroglia derived from or - Dlx2 lysine (PDL)

culture of astroglial cells culture astroglial of Dlx2 induces aGABAergic fate. 26). Prior expansion of astroglial cells 26).under neuro astroglial Prior expansion of 2 - 7 . Alternatively, astroglia can be transfected with to . - et al. - neuron conversion is the origin. astroglial This derived neurons reach functionality, full - coated cover glass slips. 5 and Heinrich natureprotocols - The protocol presented here allows the cell suspension. : Conversion into astroglia of Neurog2 : cerebral The postnatal cortex of : are Cells placed into uncoated

- Both retroviral transduction as 3–5 to et al. - , and has also been success - : After 7d, cultured cells Fig. neuron conversion may be to expression induces a 4 - , transfection was used to

- neuron conversion (see | VOL.6 NO.2VOL.6 box containing gene 2 1 ): protocol Mash1 | - 2011 to Neurog2 - neuron

4 Dlx2

| .

215 : ­ , ­ ­ ­ ­ ­ ­ . © 2011 Nature America, Inc. All rights reserved. sion may increase as a result of their prolonged proliferation. relative proportion of astroglia failing to undergo neuronal conver leave the cell cycle or continue proliferating and, thus, over time,faithfullyprogeny, transmittedalltheto whetherindependentcells of retroviralgenome,thetransgeneis genomictheintegration of on liferating and thus lose episomal plasmids more rapidly. In contrast, to respond to the neurogenic fate determinants often continue pro over time, suggesting persistent expression, whereas astroglia failing division, become postmitotic and, therefore, keeping neuronalhigh reporterconversion levelsmaximally undergo one round of cell cycle may be slightly overestimated. Indeed, astroglia(i.e., successfully percentage undergo of neurons per reporter titer is determined. In addition, the relative efficacyhigher of degreeglia conversionof variability than retroviral conversion.transduction, Of oncenote, thegene viraldelivery through transfection is subject to a 216 Mash1 protocol Box 1 (starting from Step17). Thereafter, follow the protocol described inthe PROCEDUREsection for retroviral transduction and cultivation of transduced cells neurosphere medium in24-welltissueculture platesand incubate at37°Cwith5%CO 24. Seed50,000–60,000cellsonto poly- 23. Remove the supernatant and resuspend the pelletin500 22. Centrifuge at250 21. Add 750 20. Dissociate the neurospheres byslowly pipetting the mixture upand down witha200- 19. Gently triturate the mixture upand down witha200- ­mixture at37°Cfor 2min. 18. Carefully remove the supernatant and add 750 17. Centrifuge at120 16. Collectthe medium containing the astroglia-derived neurospheres ina15-mlconical tube.  obtain neurospheres withinthistime frame. 15. Transfer the cellsuspension toa25-cm adding prewarmed (37°C),CO 14. Count the cellsinahemocytometer, using Trypan bluetoexclude dead cells, aspreviously described inref. CO 13. Carefully remove the supernatant and resuspend the cellpelletinanappropriate volume (~1ml)of prewarmed (37°C), 12. Centrifuge at250 11. Gently apply 2 ml of cell suspension obtained in step 9 to the top of the 10. Fillanew12 15-mlconical tubewith12mlof ice-coldml neurosphere of solutionice-cold III. neurosphere solution III prepared in step 10. 9. Carefully remove the supernatant and resuspend the cellsin2mlof ice-cold neurosphere solution III. 8. Centrifuge at450 7. Remove the supernatant and resuspend the cellsin10mlof ice-cold neurosphere solution II. 6. Centrifuge at200 15-ml conical tube. 5. Pass the obtained cellularsuspension through a70- disposable pipette. 4. Add 5mlof ice-cold neurosphere solution IIIto5mldissociation medium to blocktrypsinactivity. Gently mixwitha5-ml  3. Incubate the solution containing now partially dissociated cellsand remaining tissuepieces at37°Cfor anadditional 15min. 2. Gently triturate the solution containing tissuepieces afewtimes (~10times) upand down witha5-mldisposablepipette. of prewarmed (37°C)dissociation medium. Incubate at37°Cfor 15min. 1. Mince the dissectedcortices, withtwoblades, into small tissuepieces and transfer them into a15-mlconical tubecontaining 5ml PROCEDURE). Thisprotocol ismainly adapted from reference The firststeps dealing withcortical tissuedissection are identical tothose described inthe PROCEDUREsection (seeSteps1–4 of P We have used different retroviral constructs to drive drive to constructs retroviral different used Wehave

O 2 | CR CR VOL.6 NO.2VOL.6 -equilibrated neurosphere medium. STNATAL C and and I I T T I I CAL CAL Dlx2 |

PREPARATI µ STEP STEP l of neurosphere solution IIIand gently mix. | 2011 expression. By using retroviral backbones with with backbones retroviral using By expression. The totalincubation time inthe dissociation solution should not exceed 30min. Ifneurospheres are allowedtogrow for toolong time, partial differentiation may occur. g g | (2,000r.p.m.) for 10minat4°C. (1,300r.p.m.) for 5minat4°C. g g g

O (1,500r.p.m.) for 1minatroom temperature. (1,000r.p.m.) for 5minatroom temperature. (1,500r.p.m.) for 7minat4°C. natureprotocols RTE 2 X -equilibrated neurosphere medium toobtain 10cellsper O

● N -

positive cells) by transfection T O IMI d F NEUR -lysine-coated glasscoverslips in500 2 (or75-cm N G 5–7D µ l of 0.05%(wt/vol)trypsin/EDTA tothe neurosphere pelletand incubate the O 2 µ ) tissueculture flaskand incubate at37°Cwith5%CO SPHERES FR m cellstrainer toremove the tissuedebris and collectthe cellsinto afresh Neurog2 µ l filtertipand incubate for 2minat37°C. 37. µ l of fresh neurosphere medium. Count the cells. ­ ­ ­ , self by driven was determinants fate neurogenic of expression forced fluorescence over time following transduction promoter such as pCLIG, reporter we decrease noted of a gradual native long terminal repeat–driven expression without an internal blasts fibro­ perinatal and embryonic of reprogramming successful the ­astroglia toxicity or differentiation rant aber of signs observe not did we development, during exit cycle of transcription factors that are typically switched off following cell latter retroviral vectors. note,Of despite the continued expression astroglia efficient more obtained scription factor (C.H. and B.B., unpublished data). Accordingly, we decrease in reporter the pCAG retroviral vector ( that had been developed for a persistent expression - inactivating vectors, due to the deletion of the 3 1 O 0 , lentiviral gene delivery may prove to be a viable M ASTR - to µ - l of fresh, prewarmed (37°C),CO neuron conversion by lentiviral vectors. However, given 2 . µ l of medium (i.e., clonal density). µ - l filtertip. fluorescence O GLIA FR Fig. 4 . Finally, we have not yet assessed assessed yet not have Finally,we . 2 - 4 or nuclear staining for the tran to ) 9,29 - O neuronconversion the using , we did not detect any overt M THE 2 for 5–7d. We usually 35. Dilutethe cellsby 2 -equilibrated 5,14 . In contrast, when ′ U3 enhancer in in vivo

O

USE

alternative

such as

2 8 ­ ­ , © 2011 Nature America, Inc. All rights reserved. • astroglia Cultures cortical postnatal of • animals Experimental REAGENTS M targeted by the retrovirus coexpress GFAP 1 d post (pCAG tures with a retroviral vector encoding the expression of DsRed only converted into neurons. To this aim, wecul astroglial transduced important to ascertain the astroglial identity of the cells that will be Controls. prerequisite for astroglia a be to not found was proliferation note, Of culture. the within vectorslentiviral would also allow targeting no to glutamatergic or GABAergic neurons. protocol for direct conversion of postnatal astroglia into synapse-forming Figure 1 cat. no. 14065 Hanks’ balanced CaCl saltsolution with animals for research purposes. relevant governmental andinstitutional regulations regarding theuseof ! preparedastroglia from thecerebral cortex day between postnatal 5and7. developed for andneuronal theefficient culturing conversion of C57BL/6J wild cerebral cortexof Primary cultureof Dissection ofthe Trypsinization of (7 dto4weeks) ATER

Glia-to-neuron P5–P7 mouse astroglial cell astroglial cell astroglial cell retroviral delivery for neuronal conversion of astroglia. Moreover, CAUT Transduction/ transfectio conversion Seeding of (15 min) (1–2 h) (5–7 d) (24 h) (2 h) I I -

ON | IRES ALS This schematic diagram illustrates the major steps of the

n inaccordance performed shouldbe experiments All all with s s s The protocol involves two types of controls. First, it is is controls. it First, of involvesprotocoltypes twoThe

- - - 049) DsRed type mice day atpostnatal 5–7 type Glutamatergi Neurog2 neuron Astro mediu Astro mediu Dorsal view ). Immunocytochemistry revealed that cells cells that revealed Immunocytochemistry ). c Mash1 m m GABAergic - to neuron - transfectio 4 hplasmid neuron conversion B27 differentiatio Astro mediu medium n 2 andMgCl 50,0000–60,000 transduction Retrovira cells perwell m  n l Glutamatergi

CR Neurog2 neuron 2 I (10×HBSS; Invitrogen, T I n 4 CAL . proliferating proliferating cells

infection (d.p.i.), c The protocol The is Dlx2 GABAergi neuron

Ventral view Dlx 2 c / Mash1

­

thus confirming their astroglial identity astroglial their confirming thus • • • • • • of of immunocytochemical and genetic tools to ascertain the astroglial into another. In our previous studies, we employed a combination can provide definitive evidence for the conversion one of cell type mapping fate converted, be togenetic cells of majority the of tity immunocytochemical analysis allows for the assessment of the iden tion of the cellular identity of the lineage any lineage reprogramming study involves the precise characteriza cellular identity.of characterization Genetic and properties did not firepotentials action cells transduced with a control retrovirus exhibited passive electric for negative be to found were pCAG with transduced and cortex cerebral P5–P7 non their nature, with neurogenic Consistent d.p.i.). 32 and 11 (7, transduction as such markers formed immunocytochemistry to assess the expression pCAGof neuronal with cultures astroglial transduced we aim, Tothis potential. neurogenic intrinsic with few,any, contains culture if cells astroglial the that ensure to sary neces is it below.Second, described as mapping, fate genetic by provided be can cells converted the of identity astroglial the for of the reporter locus when neurogenesis is completed is neurogenesis when locus reporter the of However, it is important to keep in mind to activate recombination of an astroglia which an inducible Cre recombinase is expressed under the control labeled by this fate neurons are generated expression of neurogenic fate determinants, no NG2 98.7 ± 0.7% of GFAP pups through the mother’s milk, reporter induced between P2 and P7 by of administration tamoxifen to the Z/EG reporter mouse line astroglia the of control tamoxifen a GLAST used we Toaim, this astroglia undergoing cells the of nature of the of time course the of glia microscopyvideo allows for the direct and visualization inference fate genetically of tracking mapping should be induced at postnatal stages. Finally, single cortex cerebral the in observed are neurons positive when recombination is induced by E18, very few ( HEPES buffer solution (1M; Invitrogen, cat. no. 15630 Invitrogen, cat. no. 14200 Dulbecco’s phosphate Invitrogen, cat. no. 11058 Reduced cat. no. 14155048) Earle’s balanced CaCl saltsolution without l Dulbecco’s Eagle’s modified medium: MixtureNutrient Ham’s F cat. no. 24020 Hanks’ balanced CaCl saltsolution with - glutamine (DMEM/Ham’sglutamine F - positive cells. Of crucial importance, in the absence of forced - serum medium with medium with serum - - inducible form of the Cre recombinase under the the under recombinase Cre the of form inducible 091) - specific promoter may be employed to the same aim. β - III tubulin at different time periods following following periods time different at tubulin III mapping mapping strategy. Other fate - buffered CaCl salinewithout

> - positive astroglia and to a limited number of 4

98% of astroglial cells prepared from the the from prepared cells astroglial of 98% - - , indicating the gliogenic nature of the cells - 067) 021) specific GLAST promoter, crossed with a promoter, GLAST with crossed specific natureprotocols 3 1 . We showed that when recombination is - - l 12; PAA, cat. no. E15 mapped astroglial cells by time by cells astroglial mapped - - glutamine andHEPESglutamine (Opti  to β CreERT2/Z/EG mice CreERT2/Z/EG - - IRES III tubulin III neuron conversion 2 andMgCl - 2 andMgCl DsRed - - converted cells. Although - to 5 . An additional control control additional An . positive cells give rise to

| - 4 VOL.6 NO.2VOL.6 5 . Moreover, . astroglial neuron conversion neuron 2 . ervrs n per and retrovirus 2 - (1×HBSS; Invitrogen, andMgCl β 012)

2 III tubulin A central aspect of A central of aspect (EBSS; Invitrogen, - - 056) mapping mapping tools in protocol

<

4 4%) 4%) reporter . - 3 3 - 0 IRES 0 MEM; , expressing expressing , 2 . Thus, fate fate Thus, . | (10×PBS; 2011 3 - 0 12 without 12 without . Indeed, . - positive - DsRed

- lapse lapse | -

217 cell cell 4,5 - ­ ­ ­ ­ - .

© 2011 Nature America, Inc. All rights reserved. • Electrophysiological recordings • • • • Immunocytochemistry • • • • • • • • • • • • • • • • • • • • • SfiI, PmeI and NotI are restriction sites of the restriction enzymes. post-transcriptional regulatory element; SIN, self-inactivating ( ribosomal entry site; PPT, polypurine tract; WPRE, woodchuck hepatitis virus synthetic intron 29; the minimum enhancer sequence of CMV, chicken extended retroviral packaging signal; CAG, compound promoter containing of the cytomegalovirus (CMV) promoter; LTR, long-terminal repeat; Neurog2 Figure 2 218 • of neurons;of for practicability, we recommend B27, theuseof but incaseof B27for long of inthequality noticed some variability Store inappropriate aliquots at B27 serum aliquots andstore at Inactivate by theserum incubationat56°Cfor 30min; prepare appropriate Heat currently no serum the important technical caveat that, in contrast to neuronal cultures, there is of unsuccessful attempts of astroglia promote astroglial differentiation, it is important to test different sera in case may contain unknown or variable concentrations of growth factors that aliquots and store at Inactivate by theserum incubationat56°Cfor 30min; prepare appropriate Heat d d Penicillin/streptomycin (Invitrogen, cat. no. 15140 protocol Potassium chloride (KCl; Merck, cat. no. 104938) chlorideSodium (NaCl; Sigma (See Antibodies Triton X underand handle alaminarflow hood. formaldehyde Paraformaldehyde (PFA; Sigma BSA (Sigma Trypan blue stain(Invitrogen, cat. no. 15250 buffers andcoverof slipcleaning andrinsing) theaqueousUltrapure solutions, water for (used thepreparation of dilution Poly Ethanol ( Acetone (Carl Roth, cat. no. 9372.4) Hydrochloric (1M; acid AppliChem, cat. no. A1434) 2000 (Invitrogen,Lipofectamine cat. no. 11668 Albumin from (BSA; serum Sigma bovine Hyaluronidase from testes bovine (Sigma Trypsin from pancreas bovine (Sigma Trypsin/EDTA (0.25%(wt/vol); Invitrogen, cat. no. 25200 manufacturer’s recommendations at and store factors thegrowth all inadequate aliquots according to the Calbiochem, cat. no. 203702; REAGENT see SETUP) Recombinant human brain recommendations at factors thegrowth all inadequate aliquots according to themanufacturer’s cat. no. 13256 Recombinant human basicfibroblastgrowth factor (bFGF; Invitrogen, recommendations at factorsgrowth inadequate aliquots according to themanufacturer’s PHG0311; REAGENT see SETUP) Recombinant human factor epidermalgrowth (EGF; Invitrogen, cat. no.  GlutaMAX may indicated be inconsistent results, analternative theuseof defined supplement medium RV - -

( ( CR

CMV + + | - VOL.6 NO.2VOL.6 - -

d I ) ) inactivated horse (Invitrogen, serum cat. no. 16050 inactivated FBS(Invitrogen, cat. no. 10106 T - - - -IRES- IE saccharose (Carl Roth, cat. no. 4621) glucose solution (45%inH

I lysine hydrobromide (PDL; Sigma | CAL - Schematic structure of the recombinant retroviral vector of pCAG- 5 ≥ 100 (Sigma ′ LTR - 99.8%; Carl Roth, cat. no. 9065.4) - free supplement (Invitrogen, cat. no. 17504 - I supplement (200mM; Invitrogen, cat. no. 35050 Store inappropriate aliquots at Aldrich, cat. no. A2153) DsRed - - 029; REAGENT see SETUP) releasing agent, PFA carcinogen. isasuspected notinhale Do Ψ Table + 3 | 2 2011 . CAG - . RV, retrovirus; CMV Neurog2 free defined medium available for thegrowth of astroglia.

− -

−

− − Aldrich, cat. no. T9284)

20 °C for up to 6 months. 1

20 °Cfor upto 6months.

20 °Cfor upto 6months. 20 °Cfor upto 6months. ) Sfil |

EcoRI natureprotocols - , cDNA encoding neurogenin-2; IRES, internal derived neurotrophic factor (BDNF; Neurog2 - - Aldrich, cat. no. S3014)

− Aldrich, cat. no. P6148)

20 °Cfor upto 1year 2  O; Sigma - EcoRI to

− - CR - Aldrich, cat. no. T9201)

IE 20 °Cfor upto 6months. neuron conversion. This point raises Pmel , immediate early enhancer element I - T - IRES - Aldrich, cat. no. H3884) I Aldrich, cat. no. P0899) Aldrich, cat. no. A2153) CAL  -

- − 061) Aldrich, cat. no. G8769)

CR

20 °Cfor upto 6months. - DsRe Dilute andstore the all 019)  - β I 169) T -actin promoter and a

- CR I 122) d CAL NotI I  - PPT T  term maintenance - I

044) Dilute andstore  CAL CR

! - CR

122). -

CAUT WPR CR I 056) I T As serum T I  I I CAL - T ∆ E CAL 038)

 I CR I U3). EcoRI, CAL ON

Dilute CR SIN I ψ

T

As a

+ I We

I

, CAL

3 T ′ I LT

CAL R

• • • • • Microdissection instruments EQUIPMENT • • vectorsRetroviral • • • pH adjustment • • • • • • • • • • TA • • • • • CultureCell • vGluT1 vGaT Tbr2 Tbr1 DsRed/RFP protein (RFP) fluorescent DsRed/red MAP2 GFP GFAP CamKII β A Extra Surgical scissors (8.5cm; FineScience Tools, cat. no. 14084 Dumont no. 5SFforceps (FineScience Tools, cat. no. 11252 Dumont no. 5forceps (FineScience Tools, cat. no. 11251 ! pSKSP shuttle vector (Stratagene) REAGENT SETUP) Retroviral vectors expressing theneurogenic fate determinants (see HCl, 1N(Merck, cat. no. 1090571000) KOH, 0.1N(Merck, cat. no. 1099210001) NaOH, 1N(Merck, cat. no. 1091371000) Dimethyl sulfoxide (DMSO; Sigma cat. no. 0190) 6 ( Amphotericin B(Calbiochem; cat. no. 171375) Sigma Ethylene glycol Potassium d HEPES (Sigma Magnesium chloride, hexahydrate (MgCl Calcium chloride, dihydrate (CaCl Cover slips (diameter 12 mm; Menzel, cat. no. CB00120RA1) Tissue culture plates (24well; Scientific, Orange cat. no. 5530305) Tissue culture flasks(75cm Tissue culture flasks(25cm Tissue culture dishes(60mm; Greiner Bio blades (Schreiber surgical Disposable Instrumente, cat. no. SM11

- + - B III III tubulin ntibody CAUT Cyano (

LE )

+ - Bicuculline (Tocris Bioscience, cat. no. 0130)

- ) 1 - fine spring scissors (8.5cm;fine spring FineScience Tools, cat. no. 15003 - I Aldrich, cat. no. E3889) Glucose (Sigma ON α - | 7

- List List of primary antibodies for immunocytochemistry. D are instruments All sterilized by autoclaving nitroquinoxaline - gluconate (Sigma - - Aldrich, cat. no. H3375) bis Rabbit Guinea pig Rabbit Rabbit Rat Rabbit Mouse (IgG1) Chicken Rabbit Mouse (IgG1) (IgG2b) Mouse Host (2 - aminoethylether) - Aldrich, cat. no. G8270) 2 2 , 250ml; Greiner Bio , Greiner Bio - 2,3 - Aldrich, cat. no. G4500) - dion (CNQX; Tocris Biosciences, 2 - , 2H Aldrich, cat. no. D5879) 1:1,000 1:200 1:500 1:1,000 1:500 1:500 1:200 1:1,000 1:2,000 1:200 1:500 Dilution 2 - O; Merck, cat. no. 208291) 2 N - , 6H One, cat. no. 690175) - , N One, cat. no. 628160) , 2 N O; AppliChem, cat. no. A3618) ´, N - One, cat. no. 658175) ´ - 135302 Synaptic Systems, 131004 Synaptic Systems, Millipore, AB9618 Millipore, AB9616 Chromotek, 5F8 Chemicon, AB3216 M4403 Sigma-Aldrich, Aves Labs, GFP-1020 Z0334 Dako Cytomation, Abcam, ab2725 T8660 Sigma-Aldrich, S tetraacetic acid (EGTA; acid tetraacetic upplier, cat.no. - 20) - - 08) 00) - 0020

- 08) - 22) © 2011 Nature America, Inc. All rights reserved. 1 ml of B27 supplement with GlutaMAX B27supplement 2mM with atafinal 1 mlof concentration of B27 differentiation medium prepared before use. 50of ml with basic medium. and GlutaMAX at a final concentration 2of mM; bring to a final volume concentration 10of ng ml serum (final concentration, 5%), 1 ml B27of supplement, EGF at a final inactivated FBS (final concentration, 10%), 2.5 ml heat of Astro medium the basicmedium at4°Cfor upto 2weeks. penicillin/streptomycinand 5mlof DMEM/Ham’s to 500mlof F Basic medium prepared fresh before shortly use. neurosphere solution below). I(see powder hyaluronidase and3.5mgof powder trypsin 3.4 mgof in5ml solution Dissociation medium at 4 °C for up to 2 weeks. (1 M) (final concentration, 10 mM) to 500 ml of HBSS 1×. Store the dissection medium Dissection REAGENT SETUP • • pH adjustment • • Immunocytochemistry • • • • • • • • • • • • • • • • • Electrophysiological recordings • • • • • • • • • • • • • • • medium should be freshlymedium shouldbe prepared before use. with basic medium. to 50ml and bring afinal volume of pH Werkstätten) pHmeter InoLab pH720(WTW,Laboratory Wissenschaftlich Confocal microscope LSM710(Zeiss) Epifluorescence microscope BX61(Olympus) Clampfit 10software (MolecularDevices) (Molecular Devices) pCLAMP electrophysiology dataacquisition andanalysis software Minipuls 3Peristaltic Pump (Gilson) PP 1.5 ±0.05mm; GlassCompany, Garner cat. no. KG33) Glass micropipettes (Inner diameter 1.0±0.05mm; outer diameter Filter 43HECy3 set shiftfree (Zeiss, cat. no. 489043 immersion objectives (Zeiss) ×4(Achroplan with nation equipped 440020)and×40(Achroplan 440090) Axioskop microscope bright 2upright with Remote control for sixaxes (Luigs &Neumann, cat. no. 200 Headstage holder for Axopatch (Luigs & Neumann, cat. cat.no. no. 210 100 LN Display for controller (6×Luigs &Neumann, cat. no. 200 SM Headstage CV203BU (Molecular Devices) Axon 1322A(Molecular Digidata Devices) Axon Axopatch 200B(Molecular Devices) TMC 65 Custom Stereomicroscope (MZ6, Leica) ×10and×40objectives (Axiovert with equipped 40CFL, Zeiss) Inverted tissueculture microscope phase contrast andepifluorescence with Laminar flow hood Water bathat37°C Microcentrifuge (Eppendorf, cat. no. 5452 Humidified cell culture incubator (set at 37Humidified °C, 10% cell CO culture incubator (set at 37 °C, 5% CO (Universal50 mltubes 320R; Hettich LabTechnology, cat. no. 1406) Refrigerated andswing centrifuge Hecht Improved Neubauer hemocytometer (0.1 Bottle filtersSyringe (0.2 cat. no. 612 Cotton (70 strainers Cell Polypropylene conical (50ml; tubes Greiner Bio Polypropylene conical (15ml; tubes Greiner Bio - - - - 830 GlassMicroelectrode Puller (Narishige) Mini 25manipulator block XYZ (MRE)(Luigs &Neumann, sensitve 61(WTW) Sentix electrode 5 Controller for sixaxes (Luigs &Neumann, cat. no. 200 - - top filter systems (0.22 Assistent, cat. no. 191844272) - - plugged glass Pasteurplugged glass pipettes (230mm; VWR International, - built Faraday front cage with side open 560 anti - - 100 0000010) 1702; sterilized by autoclaving)

To prepare basicmedium, 45% 5mlof add To prepare 50 ml astro of medium, add 5 ml heat of - µ - vibration table (Technicalvibration Manufacturing Corporation) m; Falcon, cat. no. 352350) µ

To prepare the dissection medium, add 5 ml of HEPES HEPES of ml 5 add medium, dissection To the prepare m pore size; Starstedt, cat. no. 831826001)

To solution, prepare dissociation 5mlof dissolve

−

1 , bFGF at a final concentration 10of ng ml 

- µ To prepare B27medium, 50mlof combine

CR m pore, 500 ml; Millipore, cat. no. SCGPT05RE) I T -  I out rotor adapters for with 15mland CAL

CR This medium should be freshly I T - I - mm depth, 0.0025mm CAL 000 - field and epifluoresencefield illumi - This solution This must be 018) - - One, cat. no. 277261) One, cat. no. 188271) - 2 2 9901 ; Binder, cat. no. CB210) ; Binder, cat. no. CB210) -  inactivated horse - -

d CR 000) 100 9100030) - - 100 9009010) - - ( I 350 100 9002621)

T + - I Technische

) CAL - - -

12. Store 2 glucose - 20004 ;

This This

−

1

-

AP)

­ streptomycin, 20ngml EGFatafinal concentration of HEPES (1M), penicillin/ 0.4mlof B27supplement 0.5mlof with of Neurosphere medium aliquots andstore at (1N) andalabpHmeter. Filter with EBSS. tobring 500a mlfinal volume of Adjustwith the pH to 7.5 NaOH rosphere solution III, HEPES BSA; (1M)and4%(wt/vol) combine 10mlof Neurosphere solution III(BSA-EBSS-HEPES) to 6months. sterilize thesolution, prepare appropriate aliquots andstore at pure water. Adjust NaOH thepHto 7.5with (1N)andalabpHmeter. Filter with 0.9M(154g); of 500ml final to afinalbring concentration volumeof sphere solution II, HBSS10×, combine 25mlof and Neurosphere solution II(saccharose-HBSS) tion, prepare appropriate aliquots and store at the pH to NaOH 7.5 with (1N) and a lab pH meter. Filter HEPES (1 M);of to bring a finalwith pure 500 ml volume water. of Adjust solution I, mix 50 ml HBSSof 10× with 6 ml 45%of Neurosphere solution I (HBSS-glucose) theEGFis10 tion of lyophilized EGF(100 before briefly opening the to bring contents bottom.to the Reconstitute the factor growth Epidermal Ham’s F 20ngml concentration of 1 week. 10 mOsm. and a lab pH meter. the The solution final osmolarity of shouldbe 310 ± volume 1 liter of pure with water. Adjust the pH to 7.4 NaOH with (1N) (1 M), HEPES 10 ml (1 of M) and 5 ml of 150 ml of NaCl (1 M) with 3 ml of KCl (1 M), 3 ml of CaCl Extracellular solution out under aflow carried hood.should be Do notinhalePFA fumes. to 6months. (1N) andalabpHmeter. Prepare 10 PBS(1×).1 liter with Filter thesolution andadjust thepHto 7.4using HCl becomes clear. Cool down thesolution on ice. to Bring afinal volume of thesolution.(1×) andheatto stirring 60°Cwhile Wait thesolution until Paraformaldehyde (4%(wt/vol)) cover glass theplated cells on dried isobtained fully slips. of cover slipsat4°Cfor nolonger than1week. pure water, thecover andlet inalaminarairflow. slipsdry Store glass coated at least2hor overnight at37°C. Wash thoroughly three or fourwith times culture plate, each wellcontaining cover aglass slip. Incubate theplate for towel under thelaminarairflow. 1 h. Finally, on a paper themsolution in100%ethanol them andlet dry rinse cover slipsina70%(vol/vol) solution ethanol andshake (~50r.p.m.) for bathandthen shakein anultrasonic (~50r.p.m.) for 1h. the Then transfer 1 h. Afterward, transfer them into acetone, place the beaker for the first 20 min slips inabeaker, cover HCl (0.1M)andshake asolution for of them with toneed cleaned be according to procedure. the following Put cover theglass cover theglass of PDL coating slips sterile PBS, filter PDL working solution aliquots andstore them at 1mgml centration of PDL stock solution them at BDNFis 10 final concentration of Reconstitute thelyophilized BDNF(10 before briefly opening the BDNF vial to bring contents bottom.to the Brain-derived neurotrophic factor at thebFGFis10 of tration the lyophilized bFGF(10 before briefly opening the vial to bring contents bottom.to the Reconstitute factorgrowth Basic fibroblast −

20 °Cfor upto 6months. Avoid freeze/thaw cycles.

Add 500 −

20 °Cfor upto 6months. Avoid freeze/thaw cycles. -

− 12.

20 °Cfor upto 6months. Avoid freeze/thaw cycles.  µ 

l of PDLworking a24 solution to eachl of wellof ! CR

CAUT CR I - T sterilize thesolution andstore itat4°Cfor upto 2weeks. I I T CAL

I I

CAL Dissolve 50 mg of PDLpowderDissolve 50mgof inpure water atacon − ON µ µ

−

g ml

20 °Cfor upto 6months.

1 g) with 10 ml of basicmedium. 10mlof final The concentrag) with Store the freshly prepared solution at 4 °C for up to , andthen filter To prepare neurosphere 50mlof medium, mix1ml To prepare 1 liter extracellular of solution, mix Add 1 ml of thePDLstock Add solution to 1mlof 50mlof Because of thetoxic PFA, of Because nature of thisprocedure This medium should be freshly medium shouldbe This prepared before use. µ µ

−

g ml Centrifuge therecombinantCentrifuge human EGFvial g) with 1 ml of basicmedium. 1mlof final The conceng) with −

− 1 ; bring to a final volume of 50 ml with DMEM/ ; to 50ml bring afinal volume of

1

20 °Cfor upto 6months. natureprotocols . Prepare 100

-

− sterilize thesolution, prepare appropriate Centrifuge therecombinantCentrifuge human bFGF

1 . Prepare 100

µ Add 40 g of PFAAdd 40gof powder PBS to 500mlof

g ml

- Centrifuge therecombinantCentrifuge human ml aliquots andstore at Before coating, cover theglass slips - µ sterilize thesolution. Prepare 1

−

g) in 1 ml of basicmedium.g) in1mlof The

1 To prepare neurosphere 500 ml of - D . Prepare 50 µ - l aliquots andstore them at glucose (1 M); bring to a final 

- To prepare neuro 500mlof

− µ

CR

To prepare neu 500mlof l aliquots andstore them 20 °C for up to 6 months.

I | T VOL.6 NO.2VOL.6 d I d CAL - - - (

− µ (

+

1

2 + l aliquots andstore , andbFGFatafinal (1 M), 2 ml of MgCl -

The best adherence best The ) protocol sterilize the solu

) - - - glucose and 7.5 ml well tissue saccharose ata

−

−

20 °Cfor up |

20 °Cfor up 2011

-

ml |

219

­ ­

­ ­ ­ ­ -

2

© 2011 Nature America, Inc. All rights reserved. 7|  medium. to dissociate the tissuemechanically and achieve asingle-cell suspension, and then add 13mlof ice-cold dissection 6| that canbeusedasanalternative procedure atthis step). containing 2mlof ice-cold dissection medium (seealso 5| tissue to ascertain the purity of the culture.  first myelin sheets). diencephalon, hippocampalformation and the developing whitematter (containing myelinating oligodendrocytes and the 4| culture by progenitor cells residing in the subependymal zone.  brain along the longitudinal fissure toseparate itinto twoequal hemispheres. equal partsatthe levelof the optic chiasm and discard the anterior part(asdepicted in 3| 2| tional regulations regarding the useof animals for research purposes. ! whole brain. 1| P PROCE and subcloned into thepSKSPshuttle theEcoRI vector site of (Stratagene). The site (IRES)to allow for simultaneous reporter gene expression ( DsRed or GFP(asreporter proteins) followed by entry aninternal ribosomal aninternal control compoundregulatory CAG of promoter previously as described stomatitis virus vectorsRetroviral light at 50mgml a freshly prepared or thawed amphotericin Bstock solution aliquot (2 of Amphotericin Bworking solution photericin B is light sensitive; therefore, use light The final concentration of the stock solution is 50 mg ml (5 min) and aliquot at 2 DMSO, vortex thoroughly (5 min), sonicate (5 min), vortex once more Amphotericin B stock solution for up to 6 months. When thawed, an aliquot can be used for up to 1 week. 300 ± 10 mOsm. (0.1 N) and a lab pH meter. The final the osmolarity solution of should be to a final volume 100with ml pureof water. Adjust the pH to 7.4 using KOH MgCl of potassiumof gluconate 583with Intracellular solution 220 cells with6ml of prewarmed (37°C)and CO protocol

reparation of primaryculture of adherent astroglia from themousepostnatalcortex CAUT

CR CR CR

Neurog2 - | protected microtubes. VOL.6 NO.2VOL.6 Centrifuge the cellsuspension at120 Pipette the mixture upand down afewtimes (approximately tentimes) withafire-polished glass Pasteur pipette Using twoblades, mince the dissectedcortices into small tissuepieces and transfer them into a15-mlconical tube Carefully remove the meninges using fine forceps. Separate the cortical gray matter tissue of both hemispheres from the Remove the cerebellum withablade and discard it (as depicted in Transfer the brain into a60-mmtissueculture dishcontaining 10mlof ice-cold dissection medium. Decapitate one (orseveral) mouse (mice) atthe age of postnatal days 5–7(P5–P7).Isolate the head and extract the I I I T T T 2 D (2 M), 100 I I I I ON URE CAL CAL CAL

− -

1 coding cDNA wasisolated from thepCLIG ). Handling of experimental animals must beperformed inaccordance withallrelevant governmental and institu -

 STEP STEP STEP glycoprotein) encoding



CR |

2011 Retroviruses pseudotyped with with RetrovirusesVSV pseudotyped CR µ I

l of HEPESl of (1 M) and 100 T Avoid generating air bubbles when triturating the tissue, as this will reduce the viability of the cells. Carefully remove the potentially remaining developing white matter from the dissected cortical gray matter We discard the anterior part of the brain to eliminate any risk of possible contamination of the astroglia I To prepare 100 ml of intracellular solution, mix 3.1975 g 4 I T . µ CAL I Neurog2 l volume. Store aliquots at CAL |

natureprotocols sensitive;Amphotericin Bislight therefore, use Store the solution in 15

Dissolve 4 mg of amphotericin B in 80 µ , Dlx2 l of KCll of (3 M), 300

Add 500 or Neurog2 Mash1 µ l of intracellular solution intracellular to l of - protected microtubes. , µ are expressed under the Dlx2 l of EGTAl of (0.2 M); bring

−

- g 20 °C for up to 1 month. µ ml aliquots at or (1,000r.p.m.) for 5minat4 °C, aspirate the supernatant and resuspend the l of NaCll of (3 M), 50

− -

2 G (vesicular 1 - . -equilibrated astro medium bygently triturating the cellularpellet. Transfer the Neurog2 Mash1  2 9

CR together with together with Fig. I were used T I (ref. CAL 2

− ).

20 °C µ Am

8 l Box 1 ) µ l ­ µ l

for the preparation of neurospheres from cortical astroglia the institution andunder S2/BL2conditions. retroviruses must done be inaccordance guidelines of biosafety thelocal with ! suspension andstore at promoter. the neurogenic fate determinants driven by thesilencing astroglia of reference see and titration gag tor containing neurogenic fate determinants andtheplasmidsencoding HEK293T cells co canbe vsv-g derived cells stably express the cellderivative theretroviral line293GPG packaging of promoter (pCAG encoding generate pCAG pMXIG Following thesamestrategy, cloned from pCLIG as pCAG thepCAGsites within retroviral vector. resulting The isreferred construct to From there, itwasthen placed between the5

CAUT To generate the VSV - pol under atet/VP16 thecontrol (Tet transactivator of I genes and - ON Dlx2 - Fig. Neurog2 DsRed  Production, concentration of andhandling VSV - to

CR (ref. - 1 neuron conversion strong andpersistent with expression of - I behind theIRESdriven behind by thesamecompound CAG Dlx2 , blackline). Cutthe brain coronally into two T - I - vsv-g IRES 3 CAL IRES - 3 Mash1 - ) and inserted into) andinserted thepCAG retroviral vector to IRES - Prepare appropriate theconcentrated aliquotsviral of . For protocol adetailed for retrovirus preparation - G -

DsRed − DsRed - - transfected with therecombinant vec with transfected viral

80 °C until use.80 °Cuntil Avoid repeated freeze/thaw cycles. pseudotyped viral particles, we aclonal used viral pseudotyped 29. - (ref. DsRed Dlx2  ). gag Fig. . Likewise, the

8 CR ● - ) to generate pCAG . For control experiments, we useavirus - coding cDNA wassubcloned from the pol I

T T 1 I genes of murine leukemia and murine virus genes of IMI CAL , red line). Cutthe rest of the We efficiency thebest obtained N ′ G - Mash1 SfiI and3 ~7d - coding cDNA wassub - Mash1 3 4 ′ . HEK293 These - - - off). Alternatively,off). PmeI restriction PmeI restriction resistant pCAG - G - IRES - pseudotyped pseudotyped - DsRed

­

- - .

­ © 2011 Nature America, Inc. All rights reserved. 17| R additional ~24hbefore performing the immunostaining) ( for GFAP ( and CO 16| the well outside the glass cover slip. Avoid generating air bubbles when seeding the cells.  culture plate(seeREAGENT SETUP),and incubate for 1hat37°Cwith5%CO 15| and CO ~1.0- to1.5×10 14|  a fewtimes using a5-mldisposablepipette, until the cellsuspension lookshomogenous. flask or~3ml for aconfluent 75-cm 13| 12| to a15-mlconical tube. Add 5mlof fresh, prewarmed (37°C)and CO 11| to time todislodge allcells. Tilt the flasktodistributethe trypsinoverthe cells. Incubate for 5minat37°C.Gently tapthe flaskon its side from time 10| monolayer. Remove these cells by brusquely shaking the culture flask several times.  monolayer with5mlof PBS. 9| P astroglial cellswith70–80%confluence within5–7dinculture ( basic medium. Add 6mlof fresh, prewarmed (37°C)and CO 8| incubate at37°Cwith5%CO cell suspension into a25-cm as revealed by maintenance of GFAP expression (green). Immunostaining was performed at 11 d post infection (d.p.i.). ( isolated from the cerebral cortex of P6 mice shown in cultured cells are GFAP-positive astroglia. Cells DAPI (blue) reveals that the vast majority of the ( culture flask (after 7 d at the end of the expansion phase in the tissue the cerebral cortex of postnatal day 6 (P6) mice imaged, adherent astroglial cells isolated from ( cells from the mouse postnatal cerebral cortex. Figure 3 plasmids using option B. c b a assaging assaging and plating of astroglial cells etroviral transduction or plasmid transfection of the astroglial cells ) Astroglia from the cerebral cortex of postnatal day 5 (P5) mice transduced with a control retrovirus (pCAG-IRES- ) Bright-field (BF) micrograph depicting live- ) Double-immunostaining for GFAP (green) and

CR CR CR At thispoint, the cellscaneither betransduced withretroviral vectorsusing option Aortransfected withretroviral After1h,when the vastmajority of the cellshaveattached tothe coverslips, add 400 Seed100 Count cellsinahemocytometer, using Trypan bluetoexclude dead cells, aspreviously described Remove the supernatant and resuspend the cellsinanappropriate volume (~1mlfor aconfluent 25-cm Centrifuge at120 Once allthe cellshavedetached from the flask,collect the cellsinthe trypsinsolution and transfer the cellsuspension Remove the PBSand the floating cellsand discard them. Add enough (0.05%(wt/vol))trypsin/EDTA tocoverthe cells. When the cellsbecome confluent (generally after5–7d), remove the astro medium from the flask and wash the cell After 3d, remove the medium and add 6mlof fresh basic medium. Gently shake the tissueculture flaskand remove the I I I T T T

2 2 I | I I -equilibrated astro medium toeachwell.Incubate the cellsat37°Cwith5%CO -equilibrated astro medium toobtain50,000–60,000cellsper100 Primary culture of adherent astroglial CAL CAL CAL T able

STEP STEP STEP µ l of cellsuspension directly onto eachof the air-dried, PDL-coatedglasscoverslipsina24-welltissue 1 6 Avoid generating air bubbles when triturating the cell suspension, as this will reduce the viability of the cells. ) may beperformed toensure the purityof the astroglial cultures, asdescribed in Make sure that the cell suspension stays on the cover slips, and no cells are wasted by direct adherence to A few contaminating oligodendrocyte precursor cells may have grown typically on top of the astroglia cellsfrom one confluent 25cm in vitro g (1,000r.p.m.) for 5minatroom temperature (20–25°C). (d.i.v.)). 2 (or75-cm 2 for 5–7d.

2 tissueculture flask) of fresh astro medium byslowlypipetting the mixture upand down 2 , inwhich caseresuspend the cellswith12mlof medium) tissueculture flaskand a ● were passaged, seeded onto glass cover slips and stained at day 6

a 7 d.i.v. P6 cerebralcortex, T IMI 2 N tissueculture flask.Dilutethe cellsbyadding fresh, prewarmed (37°C) G ~2 h Fig. 3b 2 -equilibrated astro medium. We usually achieve amonolayer of Fig. 3 – c ; seealsoref. 2 -equilibrated astro medium and mixbygently tapping. a b 6 d.i.v. P6 cerebralcortex, ). µ l of medium. 2 . 5). natureprotocols 2 for 2–4h.Immunocytochemistry DsRed µ l of fresh, prewarmed (37°C) , red) remain in the astroglial lineage, in vitro c 11 d.p.i. pCAG-IRES- P5 cerebralcortex, Box 2 3 5 (d.i.v.) after seeding. . Usually, weobtain

| VOL.6 NO.2VOL.6 (waitfor an DsRed 2 tissueculture protocol | 2011

|

221 © 2011 Nature America, Inc. All rights reserved. (B) ( 222 (viii) protocol A (vii) Box 2 9. Mount the glasscoverslipsonto microscope glassslides withanti-fading mounting medium. 8. Wash the cellsthree times withPBS(5mineachtime). temperature. 7. Add the appropriate species- orsubclass-specificsecondary antibodies dilutedinPBSand incubate for 2hinthe dark at room 6. Remove the primary antibody solution byaspiration and discard it.Wash the cellsthree times withPBS(5mineachtime). Day 2 incubate overnight at4°C. 5. Add primary antibodies (see 5. Incubate the cellswith50–80 4. Pretreat the cellswith0.5%(vol/vol)Triton X-100in50–80 3. Wash the cellsthree times with1mlof PBS. 2. Fixthe cellswith500 1. Remove the medium from the cells (from Step 16, 18 or 21) by aspiration and discard it. Wash the cells three times with 1 ml of PBS. Day 1 30 MIN (iii) 10. Analyze the coverslipsusing anepifluorescence microscope (BX61,Olympus)oraconfocal microscope (LSM710, Zeiss). (ix) (vi) (iv)

(ii) (ii) ) (v) (i) (i)

| T T VOL.6 NO.2VOL.6 ransfection ransfection of the astroglial cells with expression plasmids ransduction of theastroglial cellswithretroviral particles ? details)—and mixgently bytapping. IRES- incubate for 5minatroom temperature. ? ? medium collectedinStep17B(v)plus250 handle the virusunder S2/BL2conditions. ! retroviral pseudotyped particles (  sequence asacontrol (pCAG-IRES- viral particles pleaserefer toref. tissue culture platetoensure the homogenous distribution of the viral particles. Fordetails of titration of the retro ranging from: 1×10 Neurog2 ? distribution of the DNA/Lipofectamine 2000complexes. dropwise onto the 300 prepared inStep17B(iii),mixgently bytapping and incubate for 20minatroom temperature. Incubate the cellsfor 20hat37°Cwith5%CO Remove the transfection medium and add 250 Incubate for 4hat37°Cwith5%CO After 20minincubation, add the DNA/Lipofectamine 2000complexes prepared inStep17B(iv)(totalvolume 100 Remove the astro medium from the cellsfrom Step16and collectitasconditioned medium for laterreuse. Add 300 Add 50 Transfer 50 Transfer 0.5 Transfer 50 Incubate the cellsfor 24hat37°Cwith5%CO At 2–4hafterseeding the cellsonthe coverslips, add 1 of fresh and prewarmed (37°C)Opti-MEMtothe cells. : :

CAUT TROU

TROU TROU TROU CR DsRed I | T IMMUN I I O B B B B µ -IRES- ON CAL | LES LES LES LES 2011 l of Lipofectamine 2000–Opti-MEM mixture prepared inStep17B(ii)dropwise tothe plasmid-containing tube N DAY 2 When working withviral particles, youshould refer tothe localbiosafety guidelines of yourinstitution and , pCAG- µ µ

µ H H H H STEP l of Opti-MEMinto a2-mltube, add 0.5 l of Opti-MEMinto a2-mltube. l of Lipofectamine 2000directly into the Opti-MEM prepared inStep17B(i),mixgently bytapping and OOT OOT OOT DsRed OOT |

natureprotocols The bestrate of transduction of astroglial cellsistypically obtained withhigh-titer (concentrated) µ I I I I Dlx2 N N N O N l of 4%(wt/vol)paraformaldehyde inPBSfor 15minatroom temperature. , pCAG- G G G 6 G CYT to1×10

-IRES- µ T able l of Opti-MEMcovering the cellsand gently move the tissueculture platefor homogeneous µ O l PBScontaining 2%(wt/vol)BSA and 0.5%(vol/vol)Triton X-100for 30min. Dlx2 CHEMISTRY DsRed 1 ) dilutedin50–80 8 -IRES- colony-forming units(c.f.u.) ml

> 29. Transduce astroglial cellswithvectorsencoding only DsRed , pCAG-

1 ×10 2 DsRed . ). 7

Mash1 c.f.u. ml µ orpCAG- l of prewarmed (37°C)and CO µ ● µ 2 2 -IRES- l of the prewarmed (37°C) and CO . . l PBScontaining 2%(wt/vol)BSA and 0.5%(vol/vol)Triton X-100and

T

−

1 IMI µ Mash1 ). µ l PBSfor 30min. DsRed

g of the appropriate retroviral plasmid DNA(pCAG- N µ -IRES- l of the appropriate concentrated retroviral vector(pCAG- G orpCAG-IRES-

● 1H30MIN

●

T

− T IMI DsRed

1 IMI ) to500 N N G ~24h —see REAGENT SETUPfor further details; titer G G ~24 h 2 DsRed µ -equilibrated fresh astro medium. l of astro medium and gently shake the —see REAGENT SETUPfor further O 2 N DAY 1AND2H -equilibrated, conditioned astro DsRed behind anIRES Neurog2

- µ ­ l) µ l © 2011 Nature America, Inc. All rights reserved. Troubleshooting advice canbefound in ? ? retrovirus remain inthe glial lineage, show an astroglial morphology and express GFAP ( glutamatergic orGABAergic autapsesorsynapses ( recordings (see and vesicular GABA transporter (vGaT) for the GABAergic sublineage ( glutamate transporter 1(vGluT1), Ca protein 2(MAP2)( of astroglial markers such asGFAP; the acquisition of neuronal markers such as 21| formation. following transduction ortransfection) inorder topromote the maturation of the astroglia-derived neurons and synapse 20| BDNF (see below). change of medium throughout the course of astroglia-to-neuron conversion and neuronal differentiation, apart from adding maturation and synapse formation by incubating the cells in a 10% CO  become functional neurons after3–4weeks. retrovirus encoding the expression of neurogenic fate determinants. They willfurther develop overtime inculture and derived neurons. Immature astroglia-derived neurons canusually beobserved5–7days postinfection (d.p.i.), withthe 19| DsRed and GFAP (see ­experimental group canbefixed after48htoallow expression of the reporter gene and immunostained for the expression of ­transfection efficiency, one tothree coverslipscontaining astroglial cultures transduced ortransfected inparallel tothe and add 1mlof fresh, prewarmed (37°C)and CO 18| A in bar Scale in as the indicated micrographs.in ( bars fluorescence Scale from ref. permission with adapted panel been has This cells. among positive reporter respectively. The represent error bars the s.e.m. of the percent of neurons (CAG), a with control vector at ± ± 7.3 9.8 and 1.0, 3.1 analyzed ± 10.7 d 2.0 transductionfollowing (dark gray or bar) DsRed encoding retroviruses with among infection transducedall following cells ( neurons forced following expression of into the are transduced cells converted astroglial for (red) and DsRed extending one ( or long two processes. show an neuron immature cells morphology of the reporter-positive encoding ( DsRed a with control encoding retroviral construct ( neurons on forced positive expression of neurogenic fate determinants. 4 Figure d b a

stroglia-to-neuron conversion of astroglial cells ) The micrograph depicts astroglial cells 6 d post infection (d.p.i.) (d.p.i.) ) 6 The d infection cells micrograph post depicts astroglial ) ) The graph shows the percentage of ) Representative example of astroglial cells 6 d.p.i. with a 6 example with ofretrovirus d.p.i. cells astroglial ) Representative TROU TROU

CR Characterize the cellsfor their astroglia-to-neuron conversion byimmunocytochemistry (see Add BDNFatafinal concentration of 20 ng ml Maintain the cellsinculture inB27differentiation medium at37°Cwith10%CO At 24haftercompletion of retroviral transduction orplasmid transfection, remove the astro medium from the cells only (CAG; white bar), bar), (CAG; white only morphology. astroglial typical ). Note the exhibit that cells I T

I B B | 4. Astroglia were isolated between postnatal days 6–7 (P6–P7) days (P6–P7) 6–7 postnatal between 4. Astroglia were isolated Neurog2 CAL Postnatal cortical astroglia differentiate into differentiate astroglia Postnatal cortical LES LES c , , 60

H H STEP OOT OOT (pCAG- µ Mash1 m. Box 3 β III tubulin (green) reveals that the majority ofthe that majority reveals (green) tubulin III We obtained the best efficiency of astroglia-to-neuron conversion, as well as the most advanced neuronal I I Fig. N N and Neurog2 G G Box 2 ) allowassessment of the proper acquisition of electrical neuronal properties and the formation of Neurog2 4 Neurog2 Dlx2 ); and the expression of T-box brain 1protein (Tbr1),T-box brain 2protein (Tbr2),vesicular -IRES- together (black bar). Cultures were Cultures bar). (black together ). Count the number of DsRed-positivecellsamong allGFAP-positive cells. and (red bar), (red bar), c Neurog2 β DsRed ) Double immunocytochemistry immunocytochemistry ) Double III tubulin-positive neurons tubulin-positive III Dlx2 DsRed 2 ). Note that the vast majority ). Note the majority that vast -

+ Mash1 , , as shown 7 d.p.i.

calmodulin-dependent kinase IIalpha(CamKII Dlx2 β T only (pCAG-IRES- only able III tubulin-positive tubulin-positive III (gray bar), (gray bar), - Mash1 β 2 III tubulin- III . / a 2 Dlx2 , -equilibrated B27-differentiation medium. To estimate transduction or b Fig. 5e

− ), 120 ), 120

1 Mash1 everyfourth day during the differentiation period (starting onday 5 ● ,

T

IMI µ

– m; m;

h N ; seealsorefs. 4,5).Astroglial cellstransduced withcontrol G ~7 d to 4 weeks c a 7 d.p.i. pCAG- P6 cerebralcortex, 6 d.p.i. pCAG-IRES- P7 cerebralcortex, T able 2 Neurog2 atmosphere. We had the best experience with no DsRed 1 -IRES- ) ( Fig. 5a β DsRed III tubulinormicrotubule-associated DsRed α – 2 until the emergence of astroglia- d natureprotocols ) for the glutamatergic sublineage β Figs. 3c III tubulin , f DsRed ). In addition, electrophysiological d b 6 d.p.i. pCAG- P7 cerebralcortex,

and Percentage of Box 2 βIII tubulin-positive 4a

neurons/transduced cells Neurog2 100 20 40 60 80 0 ) tomonitor the loss ).

|

VOL.6 NO.2VOL.6 CAG

Neurog2 -IRES- protocol DsRed

Dlx2 |

2011 Mash1

Mash1 DsRed

|

Dlx2 223

© 2011 Nature America, Inc. All rights reserved. with the transcription factor used. Forinstance, wefound that neuronal conversion typically requires high expression levelsof the neurogenic fate determinants. Thisdependence varies degree of astroglia-to-neuron conversion isahigh transduction ortransfection efficiency (seeStep18).Second, successful The overall proportion of astroglial cellsconverted into neurons depends onvarious parameters. First,paramount to a high E toward full functionality. are important differences in the efficiency of the respective factors to induce a neuronal fate and to promote differentiation tion factor expression Neurog2 factors. Whereas forced astroglia can be converted into different neuronal subtypes by selective expression of distinct neurogenic transcription neurogenic fate determinants toward the generation of functional neurons establishing synapses after 3–4 weeks. Importantly, With this protocol, postnatal astroglia isolated from the mouse cerebral cortex can be directed ANT Steps 18–21,astroglia-to-neuron conversion of astroglial cells:7dto4weeks Step 17,retroviral transduction orplasmid transfection of astroglial cells:24h Steps 9–16,passaging and plating of astroglial cells: 2h Steps 1–8,preparation of primary culture of adherent astroglia from the mouse postnatal cortex: 7d ● TA 224 viral vector(pMXIG; compared withthe pCAG-vector) resulted innearly negligible neurogenesis incontrast to 21 21 17B(ix) 17B(viii) 17A(ii) 17A(i) S protocol fficiency of glia-to-neuron conversion

tep B T

LE IMI | I VOL.6 NO.2VOL.6 C 2 I PATE N | G

Troubleshooting table. D RESULTS | efficiency Low transfection transduction Cell death after retrovirus efficiency Low transduction P overexpression after No synapse formation reprogramming No neuronal transfection Cell death after plasmid 2011 roblem Neurog2 | natureprotocols Neurog2

→

expression induces a glutamatergic neuron identity, encompassing the cascade of transcrip Tbr2 → Tbr1, forced expression of Partial reprogramming Non-optimal culture conditions determinant Low expression of neurogenic fate cells cell death of the reprogrammed cell death of the transduced cells; Non-optimal culture conditions; cells Poor survival of the transfected Endotoxin-related toxicity 2000 complex formation Non-optimal DNA/Lipofectamine Low quality of the plasmids Endotoxin-related toxicity Low-titer retrovirus P ossible reason

Dlx2

Mash1 expression driven from aweakand silencing-prone retro

and/or following transfection mately double) the serum proportion in astro medium Reduce Lipofectamine 2000 amount. Increase (approxi preparations for transfection Use endotoxin-free or cesium chloride plasmid DNA Lipofectamine 2000 decreasing the amount of DNA compared with that of Optimize the ratio of DNA/Lipofectamine 2000 by aliquots to prevent DNA nicking and degradation fication). Avoid repetitive freeze/thaw cycles of plasmid Use high-quality plasmids (endotoxin-free plasmid puri suspension by several washing steps and centrifugation preparations for retrovirus production; clean the viral Use endotoxin-free or cesium chloride plasmid DNA cycles of viral vector aliquots Use high-titer retrovirus; avoid repeated freeze/thaw S using silencing-resistant vectors Increase the expression of neurogenic fate determinants alternative supplement medium) vival of neurons (for instance, change the B27 lot or try Ensure that culture conditions allow the long-term sur using silencing-resistant vectors Increase the expression of neurogenic fate determinants tive supplement medium) neurons (for instance, change the B27 lot or try alterna Ensure that culture conditions allow for the survival of olution Dlx2 induces a GABAergic neuron fate. There in vitro

by overexpression of

Dlx2 encoded

------

© 2011 Nature America, Inc. All rights reserved. on their glutamatergic orGABAergic subtypeidentity. Indeed, neurons derived from astroglia following forced expression ment ( from a multipolar to a mono- or bipolar stage, resembling the morphological transitions of cortical precursors during develop shows that astroglia subject to astroglia-to-neuron conversion often undergo a sequence of distinct morphological transitions and an immature neuronal morphology istypically acquired between5–7daftertransduction ortransfection ( terminants, asassessedby reporter expression ( astroglia-to-neuron conversion isnot much delayed compared withthe onset of forced expression of the respective fate de distinct maturational stepscanbedistinguished As conversion of postnatal astroglia into neurons byforced expression of neurogenic fate determinants isagradual process, Morphological changes from acatastrophic conflict of cell fate signals. For details onthe single-cell tracking method see reference during the process of astroglia-to-neuron conversion, anot yetquantified level of cell death occurs, which islikely to result over time inculture. Finally, single-cell tracking withtime-lapse video microscopy (see continue proliferating. Therefore, the relative proportion of astroglia failing toundergo neuronal conversion may increase Third, incontrast toastroglia undergoing neuronal conversion vectors. Yet, the degree of functionality of the neurons obtained byusing silencing-resistant vectorsismarkedly higher. as similarnumbers of astroglia-derived neurons canbeobtained using silencing-prone (pCLIG) orsilencing-resistant (pCAG) by astrong and silencing-resistant vector(pCAG; fluorescence micrographs. Scale bar ( postnatal days 5–7 (P5–P7) as indicated in the injection, 100 pA. Astroglia were isolated between subclass of GABAergic interneurons. Step-current little frequency adaptation, characteristic for a firing of action potentials at high frequency withencoding retrovirus. The trace shows repetitive astroglia-derived neuron at 17 d.p.i. with Step-current injection, 100 pA. ( consistent with a pyramidal neuron-like identity.moderate frequency with frequency adaptation, depicts repetitive firing of action potentials at following forced ( been adapted with permission from ref. their MAP2-positive processes. This panel has vGluT1-positive puncta outlining their soma and expression of that astroglia-derived neurons, following forced (red), MAP2 (blue) and vGluT1 (green) reveals black trace). ( (6-cyano-7-nitroquinoxaline-2,3-dion; 10 propionate)/kainate receptor antagonist CNQX AMPA ( (red trace) that is abolished in the presence of step-depolarizationthe evokes an autaptic response forced astroglia-derived neurons at 27 d.p.i. following (arrowhead in (white) reveals the up-regulation of Tbr1 at 7 d.p.i.Neurog2 regulation of (white) by 4 d.p.i. (arrowheads in is barely detectable. ( 4 d post infection (d.p.i.) sequential upregulation of Tbr2 and Tbr1. ( neuron conversion induced by expression of to distinct types of neurons following forced Figure 5 of g ) A live-imaged astroglia-derived neuron Neurog2 S Neurog2 α upplementary Video1 expression. (

S -amino-3-hydroxyl-5-methyl-4-isoxazole- | Postnatal cortical astroglia give rise upplementary Video1 β Neurog2 Neurog2 f exhibit a large soma size with more than three primary dendrites ( c III tubulin 7 d.p.i. following forced expression. The inset shows that ) Triple immunostaining for DsRed and Neurog2 d d b ). ( or , show a dense labeling of ) Immunostaining for Tbr1 ) Upregulation of Tbr2 expression. The trace e Dlx2 ) A cluster of live-imaged, β III tubulin expression . ( Neurog2 a h – ) A live-imaged d a ) Astroglia-to- and a ; seealsoref. – involves the d 4. b ), 20 ). At laterstages, astroglia-derived neurons acquire amorphology thatisdifferent, depending ). ( Dlx2 µ

a M, ) At c -

) Up- µ m.

4). Incaseof the h 27 d.p.i. pCAG- P5 cerebralcortex a g e S 2 17 d.p.i. pCAG- P7 cerebralcortex, 26 d.p.i. pCAG- P5 cerebralcortex, 7 upplementary Video1 Fig. . Direct observation bysingle-cell tracking Neurog2 DsRed Dlx2 Neurog2 2 ). However, inthe caseof -IRES- -IRES-

β , III tubulin -IRES- 4 d.p.i. DsRed DsRed Neurog2 4,5 DsRed , astroglia thatdo not respond toneurogenic cues often b -induced glia-to-neuron conversion, single-cell tracking ). Once astroglia-to-neuron conversion commenced, CNQX 10 15 ms Fig. 5e 100 pA 4 d.p.i. Tbr µ M Neurog2 2 , g 27 d.p.i. pCAG P5 cerebralcortex, c f ), whereas neurons derived from natureprotocols S upplementary Video1 - Neurog , the dependence isless obvious, DsRed 3 6 suggests thatthe onset of 2 -IRES-

β III tubuli 100 pA 7 d.p.i. DsRe n d

| d VOL.6 NO.2VOL.6 Figs. 4b 36. DsRe protocol 250 ms 250 ms ) reveals that d

vGluT1 , c | 2011 25 mV 25 mV and

7 d.p.i. MAP2 Tbr1 5c |

225 -

­

© 2011 Nature America, Inc. All rights reserved. positive terminals often originate from the same neuron the terminals impinge onto. Finally, consistentderived neurons. with their Moreover,glutamatergic neurons tend to form autaptic connections ever, as all neurons in these cultures are derived from astroglia, vGluT1-positive terminals must alsofrom originate the neuron from astroglia- covered by the puncta. Thus, they cannot serve to characterize the neurotransmitter importantidentity tobeaware that vGluT1-immunopositive puncta labelpresynaptic of terminals and dothe not necessarily originatecell. How of the ( neurons derived from cells express to asubpopulation of cells. At 10dposttransduction, 70.2±6.3%(at9.8 3.1 d.p.i.) of the nor Tbr1are detected in all transduction ( β to-neuron conversion in20.7 ±1.9%of the DsRed-positivecellsby4d.p.i., preceding the expression of the neuronal marker astroglia-to-neuron conversion, we observed a transient up-regulation of Tbr2 as one ofAstroglia-to-neuron the earliest conversion isaccompanied bythe rapid lossof GFAPsigns expression. Inthe caseof of successful astroglia- Immunocytochemical changes astroglia exhibit spine-like structures covering their dendritic processes by4weeksaftertransduction As revealed byimmun astroglia following forced expression of 226 identity, neurons derived from Fig. 5 protocol III tubulin( Box 3 CNQX (6-cyano-7-nitroquinoxaline-2,3-dion, 10 nist bicuculline methiodide (5–10 11. Ifautaptic responses canbedetected, assesswhether they canbeabolished inthe presence of either the GABA frequency of 0.05Hz. 10. To assessthe presence of autaptic connections holding potential of 9. To assesswhether agivencelliscapableof (repetitive) action potential firing, stimulate the cellunder current clampcondition ata 8. EstablishaG current induced byasquare voltage pulse(5mV). 7. Placethe micropipette slowlyindirect contact withthe plasma membrane of the cell,which canbemonitored byareduction of the 6. Move filledmicropipette closetoaselectedcellunder bright-field illumination using micromanipulators. 5. Placethe filledmicropipette onto the recording headstage, coupledtoanAxopatch 200Bpatch-clamp amplifier. containing anadditional 200 10 mMHEPESand 0.2mMEGTA (pH7.4)atanosmolarity of 300mOsmand back-fillthe micropipette withintracellular solution 4. Tip-fillthe micropipette withanintracellular solution consisting of 136.5mM K-gluconate, 17.5mM KCl, 9mM NaCl, 1mMMgCl the micropipettes should haveresistances of 2–2.5M 3. Prepare micropipettes from borosilicate glasscapillaries using amicroelectrode glasspuller. When placedinto extracellular solution, 2. Select transduced cells for patch-clamp recording on basis of their DsRed-fluorescence using a ×40 immersion objective ( 10 mMHEPESand 5mMglucose (pH7.4)atanosmolarity of 310mOsm. perfused atroom temperature at0.5mlmin an Axioskop 2microscope placedonananti-vibration tablesurrounded byaFaraday cage. The perfusion chambershould beconstantly 1. Transfer coverslipscontaining cellsfollowing astroglia-to-neuron conversion (from Step21)into aperfusion chambermounted on clamp techniqueseeLippiat patch-clamp amplifiers, seeCummins Clampfit 10software. Fora more detailed protocol for voltage-clamp and current-clamp patch-clamp recordings using Axopatch 200B Axopatch 200Bpatch-clampamplifierscoupledtoDIGIDATA 1322Ausing pCLAMP10software. Dataare subsequently analyzed with must beadapted for the simultaneous recording of asecond cell.Electrical signals are sampledat10kHzand filtered at5kHzwith the assessment of synaptic connections betweentwoneurons following astroglia-to-neuron conversion bydual recording, the setup action potential firing aswellthe presence of autaptic connections (i.e., synaptic connections of agiven neuron onto itself).For astroglia-to-neuron conversion. Recording of single neurons allowsthe assessment of neuronal electrical properties such asrepetitive The stepsdelineated here follow standard procedures for whole-cell perforated patch-clamprecordings of cultured neurons

| VOL.6 NO.2VOL.6 Neurog2 f ). Along withthe establishment of functional glutamatergic connections, by4weeksaftertransduction, 85.4±5.0% | ELECTR β Fig. 5a, Fig. 5c, III tubulin,indicating the acquisition of aneuronal identity ( -transduced cellsexpress the vesicular glutamate transporter 1(vGluT1;at26.3±2.2d.p.i., | 2011 Ω sealonto the selectedneuron bygentle aspiration. Wait for 5minfor the perforation tooccur.

− |

b

o Neurog2 d natureprotocols 70 mVbydepolarizing step-current injection (lasting for 1,000ms). ). Subsequently, reactivity for Ca ), and many convertedastroglial cellsmaintain Tbr1expression thereafter. However, neither Tbr2 O 4 PHYSI 1 Neurog2 . µ g ml -transduced astroglia acquire MAP2immunoreactivity asasign of dendritic maturation Neurog2 µ

− M) orthe AMPA (

1 amphotericin Bfor perforation. et al. -transduced cells, indicating thattheir expression iseither transient orrestricted O -transduced astroglia exhibit Ca L 2

Dlx2 Neurog2 + 4

O calmodulin-dependent kinase II 0

− . Forfurther details onwhole-cell recording using the amphotericin Bperforated patch-

1 GICAL REC withextracellular solution containing 150mMNaCl, 3mMKCl, 3mMCaCl or µ M) todetermine the neurotransmitter released atthe autapse. 4 Mash1 -transduced cellsstarttoexpress Tbr1reaching up to48.2%at7dafter 2 , step-depolarize the cellinvoltage-clamp for 1ms from α Ω -amino-3-hydroxyl-5-methyl-4-isoxazole-propionate)/kainate receptor antagonist . are considerably smaller insizeand often bipolar( O RDINGS 2 in vitro

+

-calmodulin-dependent kinase II α , neurons derived from (see below), and therefore some of the vGluT1- Fig. 4c – d ). By2–3weeksaftertransduction, Neurog2 Neurog2 Neurog2

−

70 mVto 4 . α -transduced immunoreactivity. Fig. 5h A -transduced receptor antago Fig. 5 -induced 2 , 2mMMgCl

+ 38,39

). 30mVata Fig. 5e,g, f

). It is following

2

,

h 2

- ,

). -

© 2011 Nature America, Inc. All rights reserved. 2. 1. com/reprintsandpermissions/. http://npg.nature. at online available is information permissions and Reprints Published online at http://www.natureprotocols.com/. interests. CO astroglia-derived neurons and preparation of the manuscript. B.B. contributed to protocol design, electrophysiological characterization of the conversion and contributed to the current protocol design and the manuscript; lapse video microscopy. M.G. pioneered this protocol for astroglia-to-neuron culture and immunocytochemistry; and T.S. to single-cell tracking by time- astroglia-derived neurons; A.L. and R.S. to viral vector design; T.S.-E. to astroglia tracking by time-lapse video microscopy; G.M. to the characterization of the manuscript; S.G. to the design of the transfection procedure and single-cell and characterization of the astroglia-derived neurons and preparation of the AUT and the Arts (ForNeuroCell) to B.B. and M.G. We thank S. Bauer for T.S.-E.virus production. (SCHR 1142/1-1), and by the Bavarian State Ministry of Sciences, ForschungsgemeinschaftResearch to B.B. and M.G. (BE 4182/1-3 and GO 640/9-1) and A to d.p.i.). 1.4 ± 26.9 recordedat ofthe (6.1% rates induceslower generationthe drastically ofat GABAergic although synapses, nature( glutamatergic ofa exclusively are that connections synaptic or autaptic form silencing-resistant retroviral construct (pCAG), a substantial number of astroglia-derived neurons ± d.p.i.)0.9 at 24.6 (58.3% neurons cortical embryonic co-cultured from input synaptic ofreceiving neurons capable were astroglia-derived connections, ofalthough synaptic Neurog2 (see electrophysiology (see immunocytochemistry by neurons, assessed as astroglia-derived formationby synapse that Weobserved S as wellion channel density ( glia-derived neurons acquire spiking characteristics of mature neurons thatalsocorrespond tothe neuronal subtypeidentity in current clamptypically range from small spikelets tosingle mature action potentials. Withmaturation proceeding, astro ing tetrodotoxin-sensitive Na are largely unexcitable the cellsalsochange overthe courseof astroglia-to-neuron conversion (see Along withthe morphological and immunocytochemical metamorphosis of astroglia into neurons, the electrical properties of E 35.9 ± 13.0% in neurosphere cells versus adherent astroglia, respectively). neurosphere conditions greatly enhances the efficiency of assessed by Neurog2 may reflect the overall lower degree of functionality of neurons derived from vesicular Consistent with their GABAergic identity, 33.7 ± 3.6% of the astroglia-derived neurons following Mash1 lation of Fig. 5g, cknowle ynapse formation lectrophysiological changes

M As in H PET 600–613 (2009). 600–613 transition. fate astrogenicpromote to cells precursor neural cells. stem neural Hirabayashi, Y.Hirabayashi, adult and embryonic of nature glial The A. Alvarez-Buylla, & A. Kriegstein, OR I - or

N CONTR Neurog2 G G FI . Of note, combining was expressed from a silencing-prone retroviral vector construct (pCLIG), we failed to observe the establishment establishment the observe to failed we (pCLIG), construct vector retroviral silencing-prone a from expressed was dgm h β γ ). At the same time, inputresistance valuesdecrease, presumably reflecting anincrease in neuronal cellbody size -aminobutyric acid (GABA) transporter vGaT at 22.0 ± 0.6 d.p.i. This rather low number of vGaT-positive neurons Dlx2 III tubulin or MAP2. However, although using the same vector backbones to drive expression, only ~40% of the NANC ents IB β III tubulin immunoreactivity at 10.7 ± 2.0 d.p.i. ( UT -transduced astroglia generate neurons ( I et al. et

-induced astroglia-to-neuron conversion, I This work was supported by grants from the Deutsche AL ONS Annu. Rev. Neurosci. Rev. Annu. I NTERESTS Polycomb limits the neurogenic competence of competenceneurogenic the limits Polycomb C.H. C.H. contributed to protocol design, generation 5 . Subsequently (6–10d),cells undergoing astroglia-to-neuron conversion show progressively increas Box 3 Box 5 The authors declare no competing financial declare no The financial authors competing . In contrast, when a stronger and more persistent stronger moreanda when persistent contrast, In . Mash1

), highly depends on the overall expression level ofWhenneurogenicthe determinant. fate level expression overall the on depends highly ), + 4,5

-currents inresponse tostepdepolarization .

32 and , 149–184 (2009). 149–184 , Dlx2

increases astroglia-to-neuron conversion efficiency up to 93.0 ± 3.1%, as

Neuron Mash1

63 Mash1 Dlx2 , : 33.5 ± 17.8%, -mediated astroglia-to-neuron conversion (94.7 ± 0.3% versus or Fig. 4 8. 7. 6. 5. 4. 3. 14. 13. 12. 11. 10. 9.

Dlx2

and bHLH genes in specification of a retinal cell type. cell retinal a of specification in genes bHLH and CNS. the in progeny development. brain (2007). vitro in neurons. functional specific subtype Pax6. factor Neurosci. cells. stem neural adult expanded from derived neurones of cells. stem-like of populations glia. and neurons another. factors. defined by hippocampus. mouse adult the in maturationneuron granule dentate of stages morphological (2001). 1313–1322 Hatakeyama, J., Tomita, K., Inoue, T. & Kageyama, R. Roles of homeobox of Roles R. Kageyama, & T.Inoue, K., Tomita, J., Hatakeyama, diverse of heterogeneity—thesource cell glial Radial M. Götz, & L. Pinto, vertebrate for cells multi-purpose glia: Radial M. Götz, & K. Campbell, Berninger,B. C. Heinrich, N. Heins, Berninger, B., Guillemot, F. & Götz, M. Directing neurotransmitter identityneurotransmitter Directing M. Götz, & F.Guillemot, Berninger,B., B.V.Varga, T.Glaser, into cell one converting makeover: Extreme D.A. Melton, & Q. Zhou, T.Vierbuchen, DistinctF.H. Gage, & G.L. Ming,Jr., R.G., Summers, E.M., Teng, C., Zhao, d ). In addition, prior expansion of astroglial cells under induce a rapid loss of GFAP expression, and upregu reprogrammed postnatal astroglia. postnatalreprogrammed 5 . Accordingly, responses tostep-current injection

Cell Stem Cell Stem Cell 25 et al. et et al. et Dlx2 Box 3 Dlx2 et al. et , 2581–2590 (2007). 2581–2590 , et al. et Nat. Neurosci. Nat. et al. et et al. et Adult germ line stem cells as a source of functional of source a as cells stem line germ Adult Neurog2 Glial cells generate neurons: the role of the transcription the of role the neurons:generate cells Glial -transduced astroglia compared with Generation of diverse neuronal subtypes in cloned in subtypes neuronal diverse of Generation : 35.9 ± 13.0% at 10.7 ± 2.0 d.p.i.; Directing astroglia from the cerebral cortex into cortex cerebral the from astroglia Directing Stem Cells Stem Functional properties of neurons derived from derived neurons of propertiesFunctional ). At 4dafter Nature Trends Neurosci. Trends Direct conversion of fibroblasts to functional neurons functional to fibroblasts of conversion Direct Prog. Neurobiol. Prog. J. Neurosci. J.

3 Fig. 5 Fig. , 382–388 (2008). 382–388 , natureprotocols expression is induced by using a using induced by is expression

3 5 , 1035–1041 (2010). 1035–1041 ,

, 308–315 (2002). 308–315 , 26 BMC Dev. Biol. Dev. BMC , 2434–2443 (2008). 2434–2443 , e

26 , inset). In contrast, contrast, In inset). ,

, 3–11 (2006). 3–11 , Dlx2 25 PLoS Biol. PLoS Dlx2 83 Neurog2 , 235–238 (2002). 235–238 , , 2–23 (2007). 2–23 , transduction express the -expressing neurons -expressing J. Neurosci. J.

8

, 89 (2008). 89 , | 8 VOL.6 NO.2VOL.6 transduction, cells , e1000373 (2010). e1000373 ,

protocol 27 Box 2 Box Development , 8654–8664 , Eur. J. Eur. | Dlx2 2011 ) or or ) Fig. 4

128 | -

227 - d

- , ).

© 2011 Nature America, Inc. All rights reserved. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 228 protocol

3049–3055 (2006). 3049–3055 neurons newborn adult in technique knockout retroviruses. high-titer,recombinanthelper-free, of production rapid system: cells. glial cortical rat postnatal (2000). brain. mouse adult and immature the in cell stem astrocytic multipotent a Identification of (2008). 3581–3586 brain. injured the in cells multipotent Discov. Drug Rev. Nat. Cell Stem Cell Pax6. lacking cells stem embryonic from generated neurons misspecified cells. progenitorindividual of lineages cells. stem embryonic cells. stem embryonic from cells. stem embryonic mouse cells. stem embryonic mouse from neurons and progenitors CNS of population uniform and lineage. neuronal defined cells. stem 28 GDNF: and BDNF with treatment and 1 neurogenin of expressiontransient after cells stem embryonic Tashiro, A., Zhao, C. & Gage, F.H. Retrovirus-mediated single-cell gene single-cell Retrovirus-mediatedF.H.Gage, & C. Zhao, A., Tashiro, vector pCL The I.M. Verma, & M. Haas, E., Costanzi, R.K., Naviaux, R. Blum, Steindler,D.A. & P.,Kukekov,E.C. V.G.,Holland,Rakic, E.D., Laywell, A. Buffo, development. drug preclinical in brain human adult The Dragunow,M. V.Nikoletopoulou, Q. Shen, N. Gaspard, Watanabe,K. C. Chatzi, defined a of GenerationY.A.Barde, & E. Lacroix, Richter,J., M., Bibel, M. Bibel, N. Gaspard, J.H. Reyes, | VOL.6 NO.2VOL.6 , 12622–12631 (2008). 12622–12631 , et al. et et al. et et al. et et al. et et al. et Nat. Protoc. Nat. et al. et et al. et al. et J. Virol. J.

et al. et 1 The timing of cortical neurogenesis is encoded within encoded is neurogenesiscortical of timing The Neuronal network formation from reprogrammed early reprogrammedfrom formation network Neuronal | Differentiation of mouse embryonic stem cells into a into cells stem embryonic mouse ofDifferentiation Origin and progeny of reactive gliosis: a source of source a gliosis: reactive of progeny and Origin , 529–540 (2007). 529–540 , 2011 Derivation of homogeneous GABAergic neurons from GABAergicneuronshomogeneous of Derivation Glutamatergic neuronal differentiation of mouse ofdifferentiation neuronalGlutamatergic An intrinsic mechanism of corticogenesis from corticogenesis of mechanism intrinsic An embryonic mouse from neurons cortical of Generation et al. et Directed differentiation of telencephalic precursors telencephalic ofdifferentiation Directed

70 7 | Nature Protoc. Nat.

, 659–666 (2008). 659–666 , natureprotocols 4 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. , 5701–5705 (1996). 5701–5705 , Neurotrophin receptor-mediated death of death receptor-mediatedNeurotrophin , 1454–1463 (2009). 1454–1463 , Nat. Neurosci. Nat. Nat. Neurosci. Nat.

Exp. Neurol. Exp. 455 in vitro in Cereb. Cortex Cereb. , 351–357 (2008). 351–357 ,

2 , 1034–1043 (2007). 1034–1043 , Proc. Natl Acad. Sci. USA Sci. Acad. Natl Proc. Nat. Neurosci. Nat.

and 7

, 1003–1009 (2004). 1003–1009 , 217 8 , 288–296 (2005). 288–296 , in vivo in

, 407–416 (2009). 407–416 , 21 in vivo in , 413–424 (2011). 413–424 ,

97 studies.

, 13883–13888 , . 9 Nat. Protoc. Nat. , 743–751 (2006). 743–751 , J. Neurosci. J.

105

1 ,

,

35. 34. 33. 32. 31. 30. 39. 38. 37. 36. 42. 41. 40.

human cord blood cells with only two factors: Oct4 and Sox2. and Oct4 factors: two only with cells blood cord human (1996). 11400–11406 pseudotypes. G virus stomatitis retrovirus/vesicular titer high ofproduction for line cell packaging Neurosci. J. bulb. olfactory adult the in specification neuron periglomerular oxygen. low or technique culture sandwich a of advantages density: low at medium defined in excision.Cre-mediated upon protein fluorescent green enhanced expresses that line mouse reporter (2006). analysis. lineage and functional for tool valuable 439–448 (1997). 439–448 networks. neural simple in depressionsynaptic dependent (1999). adulthumanbrain.thein Neuralcells stem (2009). 217–218 choice.lineage instruct can cytokinesHematopoietic 5 Sci. USA Sci. culture. cell in maintainedneurons hippocampal isolated technique. Protoc. Nat. neurons. DRG mammalian from recordingscurrent-clamp and Voltage-clamp (2000). neurotrophin-inducedpotentiation.presynapticof Giorgetti, A. Giorgetti, human-derived stable A R.C. Mulligan, & B.A.Neugeboren, D.S., Ory, M.S. Brill, neurons hippocampal of growth and C.W.Survival Cotman, & Brewer,G.J. double a Z/EG, C.G. Lobe, & W.,Nagy,A. Yang, C., Guo, A., Novak, T.Mori, Schinder, A.F., Berninger, B. & Poo, M. Postsynaptic target specificity Postsynaptictarget M. Poo, Schinder,& Berninger,A.F.,B. activity- of Propagation M.M. Poo, & H.J. Song, R.M., Fitzsimonds, Johansson,C.B.,Svensson,Wallstedt,Frisen,J. M., Janson, & L.,A.M. Schroeder,T.& Eitelhuber,A.C. B.M., P.S.,Smejkal,Hoppe, Rieger,M.A., Bekkers, J.M. & Stevens, C.F. Excitatory and inhibitory autaptic currents in currents autaptic inhibitory and ExcitatoryC.F. Stevens, & J.M. Bekkers, clamp patch perforated the using recording Whole-cell J.D. Lippiat, S.G. Waxman, & S.D. Dib-Hajj, M., Estacion, A.M., Rush, T.R.,Cummins, , 811–820 (2010). 811–820 ,

et al. et 88 et al. et Methods Mol. Biol. Mol. Methods

, 7834–7838 (1991). 7834–7838 , 28 4 et al. et Inducible gene deletion in astroglia and radial glia—a radial and astroglia in deletion geneInducible , 1103–1112 (2009). 1103–1112 , , 6439–6452 (2008). 6439–6452 , A dlx2- and pax6-dependent transcriptional code for codetranscriptional pax6-dependent and dlx2- A Generation of induced pluripotent stem cells from cells stem pluripotent induced of Generation Brain Res. Brain Genesis

491 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. , 141–149 (2008). 141–149 ,

28 494 , 147–155 (2000). 147–155 , , 65–74 (1989). 65–74 , Exp. Cell Res.CellExp. Glia Neuron

Science 54

253

, 21–34 ,

Proc. Natl. Acad. Natl. Proc. 25 93 Nature

, 151–163 , , 733–736, , 325

Nat. Protoc. Nat.

,

388

,