Abstract
Research in the field of cortical development has benefited from technical advances in recent years, and tools are now available to label, monitor, and modulate cohorts of cerebral cortical neurons using in vivo approaches. Substantial populations of cerebral cortical neurons are generated in a specific sequence by the radial glia progenitors that line the ventricular surface during development. These radial progenitors self-renew and generate intermediate progenitors or neurons in a precisely choreographed fashion. Electroporation or electropermeabilization is a method that uses electric pulses to deliver molecules into cells and tissues. The in utero electroporation method has enabled the field to administer plasmids to these neural progenitors, allowing temporal and cell type-specific control for the manipulation of gene expression. For this reason, in utero electroporation has become a central technique in the study of key aspects of neural development, such as progenitor proliferation, neurogenesis, neuronal migration, and circuit formation. This method has also facilitated the exploitation of cell lineage and optogenetic techniques in various species from chick to gyrencephalic higher mammals. This chapter provides a description of the method and gives some examples for its utility in the study of cerebral cortical development and evolution.
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References
Rakic P (1995) A small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution. Trends Neurosci 18(9):383–388
Kriegstein AR, Noctor SC (2004) Patterns of neuronal migration in the embryonic cortex. Trends Neurosci 27(7):392–399
Marín O, Rubenstein JL (2003) Cell migration in the forebrain. Annu Rev Neurosci 26:441–483
Rakic P (2000) Molecular and cellular mechanisms of neuronal migration: relevance to cortical epilepsies. Adv Neurol 84:1–14
Weigmann A, Corbeil D, Hellwig A, Huttner WB (1997) Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells. Proc Natl Acad Sci U S A 94(23):12425–12430
Dubreuil V, Marzesco AM, Corbeil D, Huttner WB, Wilsch-Bräuninger M (2007) Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1. J Cell Biol 176(4):483–495
Walsh C, Cepko CL (1993) Clonal dispersion in proliferative layers of developing cerebral cortex. Nature 362(6421):632–635
Molnár Z, Blakey D, Bystron I, Carney R (2006) Tract-tracing in developing systems and in post-mortem human material, Chapter 12. In: Zaborszky L, Wouterlood FG, Lanciego JL (eds) Neuroanatomical tract-tracing 3: molecules - neurons – systems. Springer, New York, NY, pp 336–393
Kriegstein AR (2005) Constructing circuits: neurogenesis and migration in the developing neocortex. Epilepsia 46(Suppl 7):15–21
de Carlos JAJ, López-Mascaraque LL, Valverde FF (1996) Dynamics of cell migration from the lateral ganglionic eminence in the rat. J Neurosci 16:6146–6156
Anderson S (1997) Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 278:474–476
Wonders CP, Anderson SA (2006) The origin and specification of cortical interneurons. Nat Rev Neurosci 7(9):687–696
Fukuchi-Shimogori T, Grove EA (2001) Neocortex patterning by the secreted signaling molecule FGF8. Science 294(5544):1071–1074
Tabata H, Nakajima K (2003) Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex. J Neurosci 23(31):9996–10001
Shimogori T (2006) Micro in utero electroporation for efficient gene targeting in mouse embryos. In: Friedmann T, Rossi J (eds) Gene transfer: delivery and expression of DNA and RNA, a laboratory manual. Cold Spring Harbor Laboratory Press, p 427–432
Shimogori T, Ogawa M (2008) Gene application with in utero electroporation in mouse embryonic brain. Dev Growth Differ 50(6):499–506
Matsui A, Yoshida AC, Kubota M, Ogawa M, Shimogori T (2011) Mouse in utero electroporation: controlled spatiotemporal gene transfection. J Vis Exp (54): pii: 3024
García-Moreno F, Vasistha NA, Begbie J, Molnár Z (2014) CLoNe is a new method to target single progenitors and study their progeny in mouse and chick. Development 141(7):1589–1598
Vasistha NA, García-Moreno F, Arora S, Cheung AF, Arnold SJ, Robertson EJ, Molnár Z (2014) Cortical and clonal contribution of Tbr2 expressing progenitors in the developing mouse brain. Cereb Cortex. pii: bhu125.
Marques-Smith A (2014) Using optical stimulation to study the developing thalamocortical circuit in mouse somatosensory cortex. D Phil thesis, University of Oxford
Borrell V, Yoshimura Y, Callaway EM (2005) Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J Neurosci Methods 143(2):151–158
de Marco Garcia NV, Fishell G (2014) Subtype-selective electroporation of cortical interneurons. J Vis Exp. (90): e51518
Nakahira E, Yuasa S (2005) Neuronal generation, migration, and differentiation in the mouse hippocampal primordium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol 483(3):329–340
dal Maschio M, Ghezzi D, Bony G, Alabastri A, Deidda G, Brondi M, Sato SS, Zaccaria RP, Di Fabrizio E, Ratto GM, Cancedda L (2012) High-performance and site-directed in utero electroporation by a triple-electrode probe. Nat Commun 3:960
Okada T, Keino-Masu K, Masu M (2007) Migration and nucleogenesis of mouse precerebellar neurons visualized by in utero electroporation of a green fluorescent protein gene. Neurosci Res 57(1):40–49
Navarro-Quiroga I, Chittajallu R, Gallo V, Haydar TF (2007) Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation. J Neurosci 27(19):5007–5011
Ito H, Morishita R, Iwamoto I, Nagata K (2014) Establishment of an in vivo electroporation. Hippocampus 24(12):1449–1457
García-Frigola C, Carreres MI, Vegar C, Herrera E (2007) Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev Biol 7:103
LoTurco J, Manent JB, Sidiqi F (2009) New and improved tools for in utero electroporation studies of developing cerebral cortex. Cereb Cortex 19(Suppl 1):i120–i125
García-Moreno F, Pedraza M, Di Giovannantonio LG, Di Salvio M, López-Mascaraque L, Simeone A, De Carlos JA (2010) A neuronal migratory pathway crossing from diencephalon to telencephalon populates amygdala nuclei. Nat Neurosci 13:680–689
Langevin LM, Mattar P, Scardigli R, Roussigné M, Logan C, Blader P, Schuurmans C (2007) Validating in utero electroporation for the rapid analysis of gene regulatory elements in the murine telencephalon. Dev Dyn 236(5):1273–1286
Saito T (2006) In vivo electroporation in the embryonic mouse central nervous system. Nat Protoc 1(3):1552–1558
Walantus W, Castaneda D, Elias L, Kriegstein A (2007) In utero intraventricular injection and electroporation of E15 mouse embryos. J Vis Exp. (6):239
Dixit R, Lu F, Cantrup R, Gruenig N, Langevin LM, Kurrasch DM, Schuurmans C (2011) Efficient gene delivery into multiple CNS territories using in utero electroporation. J Vis Exp (52): pii: 2957
Rana ZA, Ekmark M, Gundersen K (2004) Coexpression after electroporation of plasmid mixtures into muscle in vivo. Acta Physiol Scand 181:233–238
Paracchini S, Thomas A, Castro S, Lai C, Paramasivam M, Wang Y, Keating BJ, Taylor JM, Hacking DF, Scerri T, Francks C, Richardson AJ, Wade-Martins R, Stein JF, Knight JC, Copp AJ, Loturco J, Monaco AP (2006) The chromosome 6p22 haplotype associated with dyslexia reduces the expression of KIAA0319, a novel gene involved in neuronal migration. Hum Mol Genet 15(10):1659–1666
García-Marqués J, López-Mascaraque L (2013) Clonal identity determines astrocyte cortical heterogeneity. Cereb Cortex 23(6):1463–1472
Siddiqi F, Chen F, Aron AW, Fiondella CG, Patel K, LoTurco JJ (2014) Fate mapping by piggyBac transposase reveals that neocortical GLAST+ progenitors generate more astrocytes than Nestin+ progenitors in rat neocortex. Cereb Cortex 24:508
García-Marqués J, Nunez-Llaves R, López-Mascaraque L (2014) NG2-glia from pallial progenitors produce the largest clonal clusters of the brain: time frame of clonal generation in cortex and olfactory bulb. J Neurosci 34:2305–2313
Chen F, Maher BJ, LoTurco JJ (2014) piggyBac transposon-mediated cellular transgenesis in mammalian forebrain by in utero electroporation. Cold Spring Harb Protoc 2014(7):741–749
Johns DC, Marx R, Mains RE, O’Rourke B, Marbán E (1999) Inducible genetic suppression of neuronal excitability. J Neurosci 19(5):1691–1697
Mire E, Mezzera C, Leyva-Díaz E, Paternain AV, Squarzoni P, Bluy L, Castillo-Paterna M, López MJ, Peregrín S, Tessier-Lavigne M, Garel S, Galcerán J, Lerma J, López-Bendito G (2012) Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth. Nat Neurosci 15(8):1134–1143
Suárez R, Fenlon LR, Marek R, Avitan L, Sah P, Goodhill GJ, Richards LJ (2014) Balanced interhemispheric cortical activity is required for correct targeting of the corpus callosum. Neuron 82(6):1289–1298
Crick FH (1979) Thinking about the brain. Sci Am 241:219–232
Fenno L, Yizhar O, Deisseroth K (2011) The development and application of optogenetics. Annu Rev Neurosci 34(1):389–412
Miesenböck G (2011) Optogenetic control of cells and circuits. Annu Rev Cell Dev Biol 27:731–758
Bernstein JG, Garrity PA, Boyden ES (2012) Optogenetics and thermogenetics: technologies for controlling the activity of targeted cells within intact neural circuits. Curr Opin Neurobiol 22:61–71
Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8:1263–1268
Zemelman BV, Lee GA, Ng M, Miesenböck G (2002) Selective photostimulation of genetically chARGed neurons. Neuron 33:15–22
Zemelman BV, Nesnas N, Lee GA, Miesenböck G (2003) Photochemical gating of heterologous ion channels: remote control over genetically designated populations of neurons. Proc Natl Acad Sci U S A 100:1352–1357
Lima SQ, Miesenböck G (2005) Remote control of behavior through genetically targeted photostimulation of neurons. Cell 121:141–152
Zhang F, Wang L-P, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A et al (2007) Multimodal fast optical interrogation of neural circuitry. Nature 446:633–639
Rickgauer JP, Tank DW (2009) Two-photon excitation of channelrhodopsin-2 at saturation. Proc Natl Acad Sci U S A 106:15025–15030
Vaziri A, Emiliani V (2012) Reshaping the optical dimension in optogenetics. Curr Opin Neurobiol 22:128–137
Packer AM, Peterka DS, Hirtz JJ, Prakash R, Deisseroth K, Yuste R (2012) Two-photon optogenetics of dendritic spines and neural circuits. Nat Methods 9:1202–1205
Packer AM, Roska B, Häusser M (2013) Targeting neurons and photons for optogenetics. Nat Neurosci 16:805–815
Petreanu L, Huber D, Sobczyk A, Svoboda K (2007) Channelrhodopsin-2–assisted circuit mapping of long-range callosal projections. Nat Neurosci 10:663–668
Petreanu L, Mao T, Sternson SM, Svoboda K (2009) The subcellular organization of neocortical excitatory connections. Nature 457:1142–1145
Kätzel D, Buetfering C, Wölfel M, Miesenböck G (2010) The columnar and laminar organization of inhibitory connections to neocortical excitatory cells. Nat Neurosci 14:100–107
Pfeffer CK, Xue M, He M, Huang ZJ, Scanziani M (2013) Inhibition of inhibition in visual cortex: the logic of connections between molecularly distinct interneurons. Nat Neurosci 16:1068–1076
Atallah BV, Bruns W, Carandini M, Scanziani M (2012) Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli. Neuron 73:159–170
Wilson NR, Runyan CA, Wang FL, Sur M (2012) Division and subtraction by distinct cortical inhibitory networks in vivo. Nature 488:343
Lee S-H, Kwan AC, Zhang S, Phoumthipphavong V, Flannery JG, Masmanidis SC, Taniguchi H, Huang ZJ, Zhang F, Boyden ES et al (2012) Activation of specific interneurons improves V1 feature selectivity and visual perception. Nature 488:379
Dalva MB, Katz LC (1994) Rearrangements of synaptic connections in visual cortex revealed by laser photostimulation. Science 265:255–258
Bureau I, Shepherd GMG, Svoboda K (2004) Precise development of functional and anatomical columns in the neocortex. Neuron 42:789–801
Viswanathan S, Bandyopadhyay S, Kao JPY, Kanold PO (2012) Changing microcircuits in the subplate of the developing cortex. J Neurosci 32:1589–1601
Anastasiades PG, Butt SJB (2012) A role for silent synapses in the development of the pathway from layer 2/3 to 5 pyramidal cells in the neocortex. J Neurosci 32:13085–13099
Grant E, Hoerder-Suabedissen A, Molnár Z (2012) Development of the corticothalamic projections. Front Neurosci 6:53
Catalano SM, Shatz CJ (1998) Activity-dependent cortical target selection by thalamic axons. Science 281:559–562
Zhang J, Ackman JB, Xu H-P, Crair MC (2012) Visual map development depends on the temporal pattern of binocular activity in mice. Nat Neurosci 15:298–307
Zhang F, Prigge M, Beyrière F, Tsunoda SP, Mattis J, Yizhar O, Hegemann P, Deisseroth K (2008) Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri. Nat Neurosci 11:631–633
Lin JY, Knutsen PM, Muller A, Kleinfeld D, Tsien RY (2013) ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci 16: 1499–1508
Luhmann HJ, Hanganu I, Kilb W (2003) Cellular physiology of the neonatal rat cerebral cortex. Curr Opin Neurobiol 60:345–353
Minlebaev M, Colonnese M, Tsintsadze T, Sirota A, Khazipov R (2011) Early gamma oscillations synchronize developing thalamus and cortex. Science 334:226–229
Tolner EA, Sheikh A, Yukin AY, Kaila K, Kanold PO (2012) Subplate neurons promote spindle bursts and thalamocortical patterning in the neonatal rat somatosensory cortex. J Neurosci 32:692–702
Gil-Sanz C, Franco SJ, Martinez-Garay I, Espinosa A, Harkins-Perry S, Müller U (2013) Cajal-Retzius cells instruct neuronal migration by coincidence signaling between secreted and contact-dependent guidance cues. Neuron 79(3):461–477
Borrell V (2010) In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation. J Neurosci Methods 186(2):186–195
Kawasaki H, Iwai L, Tanno K (2012) Rapid and efficient genetic manipulation of gyrencephalic carnivores using in utero electroporation Mol. Brain 5:24
Kawasaki H, Toda T, Tanno K (2013) In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation. Biol Open 2(1):95–100
Kawasaki H (2014) Molecular investigations of the brain of higher mammals using gyrencephalic carnivore ferrets. Neurosci Res 86:59, pii: S0168-0102(14)00117-5
Itasaki N, Bel-Vialar S, Krumlauf R (1999) “Shocking” developments in chick embryology: electroporation and in ovo gene expression. Nat Cell Biol 1:E203–E207
Krull CE (2004) A primer on using in ovo electroporation to analyze gene function. Dev Dyn 229:433–439
Croteau LP, Kania A (2011) Optimisation of in ovo electroporation. J Neurosci Methods 201(2):381–384
Nomura T, Takahashi M, Hara Y, Osumi N (2008) Patterns of neurogenesis and amplitude of reelin expression are essential for making a mammalian-type cortex. PLoS One 3:e1454
Suzuki IK, Kawasaki T, Gojobori T, Hirata T (2012) The temporal sequence of the mammalian neocortical neurogenetic program drives mediolateral pattern in the chick pallium. Dev Cell 22:863–870
García-Moreno F, Molnár Z (unpublished) A subset of early radial glial cells with delayed neurogenic program selectively contribute to the development and evolution of callosal connecting neurons
Nomura T, Gotoh H, Ono K (2013) Changes in the regulation of cortical neurogenesis contribute to encephalization during amniote brain evolution. Nat Commun 4:2006
Nomura T, Kawaguchi M, Ono K, Murakami Y (2013) Reptiles: a new model for brain evo-devo research. J Exp Zool B Mol Dev Evol 320:57–73
Wang X, Chang L, Guo Z, Li W, Liu W, Cai B, Wang J (2013) Neonatal SVZ EGFP-labeled cells produce neurons in the olfactory bulb and astrocytes in the cerebral cortex by in-vivo electroporation. Neuroreport 24(7):381–387
Feliciano DM, Lafourcade CA, Bordey A (2013) Neonatal subventricular zone electroporation. J Vis Exp (72): pii:50197
Louise C, Etienne D, Marie-Pierre R (2014) AFM sensing cortical actin cytoskeleton destabilization during plasma membrane electropermeabilization. Cytoskeleton (Hoboken) 71(10):587–594
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Martínez-Garay, I., García-Moreno, F., Vasistha, N., Marques-Smith, A., Molnár, Z. (2016). In Utero Electroporation Methods in the Study of Cerebral Cortical Development. In: Walker, D. (eds) Prenatal and Postnatal Determinants of Development. Neuromethods, vol 109. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3014-2_2
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