Embryo Brain Research Articles

Accumulation and effects of the UV-filter octocrylene in adult and embryonic zebrafish (Danio rerio)

Wide application of the UV-filter octocrylene (OC) in cosmetics leads to contamination of the aquatic environment, but effects of OC remain unclear. Here we determine bioaccumulation and molecular effects of OC. Adult male zebrafish were exposed to 22, 209 and 383μg/L and embryos to 69, 293 and 925μg/L OC. OC accumulated in fish up to 17μg/g. Calculated BCF varied between 41 and 136. Microarray analysis in brain and liver following exposure to 383μg/L OC revealed alteration of 628 and 136 transcripts, respectively. Most prominent GO processes included developmental processes, organ development, hematopoiesis, formation of blood vessels, blood circulation, fat cell differentiation and metabolism. Validation by RT-qPCR in brain and liver of adult fish and embryos included a series of genes. Blood levels of 11-ketotestosterone were not altered. The transcriptomics data suggest that OC mainly affects transcription of genes related to developmental processes in the brain and liver as well as metabolic processes in the liver.

Molecular cloning and knockdown of galactocerebrosidase in zebrafish: New insights into the pathogenesis of Krabbe's disease

The lysosomal hydrolase galactocerebrosidase (GALC) catalyzes the removal of galactose from galactosylceramide and from other sphingolipids. GALC deficiency is responsible for globoid cell leukodystrophy (GLD), or Krabbe's disease, an early lethal inherited neurodegenerative disorder characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system (CNS). The poor outcome of current clinical treatments calls for novel model systems to investigate the biological impact of GALC down-regulation and for the search of novel therapeutic strategies in GLD. Zebrafish (Danio rerio) represents an attractive vertebrate model for human diseases. Here, lysosomal GALC activity was demonstrated in the brain of zebrafish adults and embryos. Accordingly, we identified two GALC co-orthologs (named galca and galcb) dynamically co-expressed in CNS during zebrafish development. Both genes encode for lysosomal enzymes endowed with GALC activity. Single down-regulation of galca or galcb by specific antisense morpholino oligonucleotides results in a partial decrease of GALC activity in zebrafish embryos that was abrogated in double galca/galcb morphants. However, no psychosine accumulation was observed in galca/galcb double morphants. Nevertheless, double galca/galcb knockdown caused reduction and partial disorganization of the expression of the early neuronal marker neuroD and an increase of apoptotic events during CNS development. These observations provide new insights into the pathogenesis of GLD, indicating that GALC loss-of-function may have pathological consequences in developing CNS independent of psychosine accumulation. Also, they underscore the potentiality of the zebrafish system in studying the pathogenesis of lysosomal neurodegenerative diseases, including GLD.

Influence of Maternal Alcoholism on the Brain Benzodiazepine Receptor Development in Human Embryo and Fetus during Ontogeny

Graphical Abstract: Influence of maternal alcoholism on the brain benzodiazepine receptor development in human’s embryo and fetus showed decreases their affinity and increases density as compensatory adaptation of the fetal nervous system to the effects of alcohol. Abstract: Signaling mechanisms that are required for proper neuronal development and how these processes are impaired by ethanol resulting in harmful consequences to brain development are very important for our understanding. The aim of the present work was to study the development of synaptic Benzodiazepine Receptors (BzDR) (functionally associated with the brain GABAergic system) in the brains of embryos and fetuses aged 8-15 weeks obtained from alcoholic female patients. Material and method: Abortive material (samples of brain tissue) was obtained from 33 women with grade II alcoholism (ICD-10 F10.201 and F10.202) and 30 control women were studied. The properties of BzDR were studied by radioreceptor binding with the selective ligand [3H]-flunitrazepam using a crude embryo brain synaptosomal fraction. Results: In contrast to controls, brain cells developing in conditions of prenatal alcoholization showed formation of synaptic benzodiazepine receptors and alterations in their properties: decreases in their affinity and increases in their density. A decrease in the ability of receptors to bind agonist ligands impairs the ligand: receptor protein ratio, leading to decreased binding of the major neurotransmitter GABA and impairment to synaptic transmission. Conclusion: These are interpreted as compensatory reactions promoting adaptation of the fetal nervous system to the effects of alcohol and functional deficiency of the GABAergic system.

Effect of prenatal chronic noise exposure on the growth and development of body and brain of chick embryo

Context:Noise acts as an environmental stressor as has been demonstrated by an increased brain acetyl cholinesterase activity as well as elevated plasma corticosterone and adrenocorticotropic hormone levels. Noise can lead to neurodegenerative changes in the brain and in the ear.Aim:This study was undertaken to investigate the effect of chronic noise on growth and development during the sensitive period of embryonic life.Materials and Methods:In this study, we analyzed the body weight, brain weight and brain size following prenatal chronic noise exposure. Fertilized eggs of domestic chicks were exposed to chronic excessive acoustic stimulation with frequency of the sound ranging from 30 to 3000 Hz with a peak at 2700 Hz was given at 110 dB sound pressure level from embryonic day (E) 10 until hatching.Results:An appreciable decrease in body weight, brain weight and brain size was evident in the experimental group exposed to noise. A generalized decrease in the neuronal nuclear size and increase in the density of neurons was also observed.Conclusion:These observations could be an indicator of growth and developmental retardation following exposure to noise.

Peg3 Mutational Effects on Reproduction and Placenta-Specific Gene Families

Peg3 (paternally expressed gene 3) is an imprinted gene encoding a DNA-binding protein. This gene plays important roles in controlling fetal growth rates and nurturing behaviors. In the current study, a new mutant mouse model has been generated to further characterize the functions of this DNA-binding protein. Besides known phenotypes, this new mutant model also revealed potential roles of Peg3 in mammalian reproduction. Female heterozygotes produce a much smaller number of mature oocytes than the wild-type littermates, resulting in reduced litter sizes. According to genome-wide expression analyses, several placenta-specific gene families are de-repressed in the brain of Peg3 heterozygous embryos, including prolactin, cathepsin and carcinoembryonic antigen cell adhesion molecule (Ceacam) families. The observed de-repression is more pronounced in females than in males. The de-repression of several members of these gene families is observed even in the adult brain, suggesting potential defects in epigenetic setting of the placenta-specific gene families in the Peg3 mutants. Overall, these results indicate that Peg3 likely controls the transcription of several placenta-specific gene families, and further suggest that this predicted transcriptional control by Peg3 might be mediated through unknown epigenetic mechanisms.

Strong Bones for a Strong Mind

A hormone secreted by osteoblasts protects mice from depression and anxiety and promotes learning.

Notch1-Induced Brain Tumor Models the Sonic Hedgehog Subgroup of Human Medulloblastoma

While activation of the Notch pathway is observed in many human cancers, it is unknown whether elevated Notch1 expression is sufficient to initiate tumorigenesis in most tissues. To test the oncogenic potential of Notch1 in solid tumors, we expressed an activated form of Notch1 (N1ICD) in the developing mouse brain. N1ICD;hGFAP-cre mice were viable but developed severe ataxia and seizures, and died by weaning age. Analysis of transgenic embryo brains revealed that N1ICD expression induced p53-dependent apoptosis. When apoptosis was blocked by genetic deletion of p53, 30% to 40% of N1ICD;GFAP-cre;p53(+/-) and N1ICD;GFAP-cre;p53(-/-) mice developed spontaneous medulloblastomas. Interestingly, N1ICD-induced medulloblastomas most closely resembled the sonic hedgehog subgroup of human medulloblastoma at the molecular level. Surprisingly, N1ICD-induced tumors do not maintain high levels of the Notch pathway gene expression, except for Notch2, showing that initiating oncogenic events may not be decipherable by analyzing growing tumors in some cases. In summary, this study shows that Notch1 has an oncogenic potential in the brain when combined with other oncogenic hits, such as p53 loss, and provides a novel mouse model of medulloblastoma. Cancer Res; 73(17); 5381-90. ©2013 AACR.

Prenatal activation of maternal TLR3 receptors by viral-mimetic poly(I:C) modifies GluN2B expression in embryos and sonic hedgehog in offspring in the absence of kynurenine pathway activation

Activation of the immune system during pregnancy is believed to lead to psychiatric and neurological disorders in the offspring, but the molecular changes responsible are unknown. Polyinosinic:polycytidylic acid (poly(I:C)) is a viral-mimetic double-stranded RNA complex which activates Toll-Like-Receptor-3 and can activate the metabolism of tryptophan through the oxidative kynurenine pathway to compounds that modulate activity of glutamate receptors. The aim was to determine whether prenatal administration of poly(I:C) affects the expression of neurodevelopmental proteins in the offspring and whether such effects were mediated via the kynurenine pathway. Pregnant rats were treated with poly(I:C) during late gestation and the offspring were allowed to develop to postnatal day 21 (P21). Immunoblotting of the brains at P21 showed decreased expression of sonic hedgehog, a key protein in dopaminergic neuronal maturation. Expression of α-synuclein was decreased, while tyrosine hydroxylase was increased. Disrupted in Schizophrenia-1 (DISC-1) and 5-HT2C receptor levels were unaffected, as were the dependence receptors Unc5H1, Unc5H3 and Deleted in Colorectal Cancer (DCC), the inflammation-related transcription factor NFkB and the inducible oxidative enzyme cyclo-oxygenase-2 (COX-2). An examination of embryo brains 5 h after maternal poly(I:C) showed increased expression of GluN2B, with reduced doublecortin and DCC but no change in NFkB. Despite altered protein expression, there were no changes in the kynurenine pathway. The results show that maternal exposure to poly(I:C) alters the expression of proteins in the embryos and offspring which may affect the development of dopaminergic function. The oxidation of tryptophan along the kynurenine pathway is not involved in these effects.

The influence of sustained dual-factor presentation on the expansion and differentiation of neural progenitors in affinity-binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.

Expression, function and regulation of Evi-1 during embryonic avian development

Ecotropical viral integration site 1 (Evi-1) is a transcription factor essential for vascularisation and cell proliferation during embryonic development. The chimeric transcription factor AML1-EVI-1 is activated in leukaemia where it plays a role as a differentiation block and stimulator of proliferation. Here, we cloned chicken Evi-1 and analysed its expression during embryonic development. There was early expression in the pharyngeal arches, in the brain and intermediate mesoderm of chicken embryos at stage 15. Later at stage 20, Evi-1 mesenchymal expression was concentrated in the second pharyngeal arch, and weaker expression was found in the mandibular and maxillary prominences. Facial expression decreased in intensity during development. Evi-1 expression in the limb was also limited to the mesenchyme with the most prominent expression in the anterior margin. Evi-1 was not detectable in the posterior limb bud. At later stages, Evi-1 was expressed in the peripheral mesenchyme of the limb but not in the developing precartilage blastema. At stage 29, the expression became restricted to the perichondrium and interdigital areas; however, the cartilage condensations themselves were negative. To study the function of Evi-1 in chondrogenesis, we knocked down expression in limb micromass cultures using siRNA. Chondrogenesis was significantly reduced in both anterior and posterior cultures. Since Evi-1 was expressed adjacent to the apical ectodermal ridge and this area is a source of FGFs, we tested whether endogenous FGF receptor signalling was necessary to maintain its expression. Inhibitors of FGFRs (PD161570 and SU5402) were applied to wing mesenchyme, and downregulation of Evi-1 expression was observed after treatment with both inhibitors. Therefore, Evi-1 may be a transcription factor mediating the effects of FGF and may also be defining the size of cartilage elements in the limb.

The dynamics of polycomb group proteins in early embryonic nervous system in mouse and human

Polycomb group (PcG) proteins are transcription regulatory proteins that control the expression of a variety of genes and the antero-posterior neural patterning from early embryogenesis. Although expression of PcG genes in the nervous system has been noticed, but the expression pattern of PcG proteins in early embryonic nervous system is still unclear. In this study, we analyzed the expression pattern of PRC1 complex members (BMI-1 and RING1B) and PRC2 complex members (EED, SUZ12 and EZH2) in early embryonic nervous system in mouse and human by Western blot and Immunohistochemistry. The results of Western blot showed that EED protein was significantly up-regulated with the increase of the day of pregnancy during the early embryogenesis in mouse. BMI-1 protein level was significantly increased from the day 10 of pregnancy, when compared with the day 9 of pregnancy. But the SUZ12, EZH2 and RING1B protein level did not change significantly. From the results of Immunohistochemistry, we found that the four PcG proteins were all expressed in the fetal brain and fetal spinal cord in mouse. In human, the expression of EED, SUZ12, and EZH2 was not significantly different in cerebral cortex and sacral spinal cord, but BMI-1 and RING1B expression was enhanced with the development of embryos in early pregnancy. Collectively, our findings showed that PRC1 and PRC2 were spatiotemporally expressed in brain and spinal cord of early embryos.

Ontogeny of mRNA expression of somatostatin and its receptors in chicken embryos in association with methylation status of their promoters

The present study was designed to investigate the ontogeny and tissue distribution of somatostatin and its five receptor subtypes (SSTR1-5) mRNA expression in embryonic chicken (Gallus gallus). Brain, gonads (male), intestine, kidney, liver, muscle, stomach and yolk sac membrane (YSM) of chicken embryos on the embryonic (E) ages of 10, 16 and 21days (right before hatch) were investigated. Bisulfite sequencing PCR (BSP) was performed to determine the methylation status of the promoter region of all the six genes in the liver. Somatostatin (SST) was predominately expressed in intestine, brain and gonads (male) with different ontogenic patterns. The highest expression in intestine was detected at E10. There was ontogenic shift from intestine to brain as development progressed. Expression pattern of SSTRs in brain, intestine and kidney was similar to human embryonic expression. In liver, the ontogenic expression pattern of SST and its receptors was associated to methylation status of the respective promoters. Methylation of site Sp1 determines expression level of SST, SSTR1, SSTR2 and SSTR3 while site a is important in governing the expression of SSTR4 and SSTR5. The results show that ontogenic expression profile of chicken SST and SSTRs is time and tissue specific.

Influence of nanoparticles of platinum on chicken embryo development and brain morphology

Platinum nanoparticles (NP-Pt) are noble metal nanoparticles with unique physiochemical properties that have recently elicited much interest in medical research. However, we still know little about their toxicity and influence on general health. We investigated effects of NP-Pt on the growth and development of the chicken embryo model with emphasis on brain tissue micro- and ultrastructure. The embryos were administered solutions of NP-Pt injected in ovo at concentrations from 1 to 20 μg/ml. The results demonstrate that NP-Pt did not affect the growth and development of the embryos; however, they induced apoptosis and decreased the number of proliferating cells in the brain tissue. These preliminary results indicate that properties of NP-Pt might be utilized in brain cancer therapy, but potential toxic side effects must be elucidated in extensive follow-up research.

Synaptogenesis and the Formation of Benzodiazepine Receptors in the Human Brain in Conditions of Prenatal Alcoholization

The aim of the present work was to study the relationship between the development of synaptic contacts and benzodiazepine receptors (functionally associated with the brain GABAergic system) in the brains of embryos and fetuses aged 8–15 weeks obtained from alcoholic female patients. Materials from 33 women with grade II alcoholism (ICD-10 F10.201 and F10.202) and 30 control women were studied. In contrast to controls, brain cells developing in conditions of prenatal alcoholization showed slowed formation of synaptic benzodiazepine receptors and increases in their density. These are interpreted as compensatory reactions promoting adaptation of the fetal nervous system to the effects of alcohol and functional deficiency of the GABAergic system.

Gene Responses in the Central Nervous System of Zebrafish Embryos Exposed to the Neurotoxicant Methyl Mercury

Methyl mercury (MeHg) is a neurotoxicant with adverse effects on the development of the nervous system from fish to man. Despite a detailed understanding of the molecular mechanisms by which MeHg affects cellular homeostasis, it is still not clear how MeHg causes developmental neurotoxicity. We performed here a genome-wide transcriptional analysis of MeHg-exposed zebrafish embryos and combined this with a whole-mount in situ expression analysis of 88 MeHg-affected genes. The majority of the analyzed genes showed tissue- and region-restricted responses in various organs and tissues. The genes were linked to gene ontology terms like oxidative stress, transport and cell protection. Areas even within the central nervous system (CNS) are affected differently resulting in distinct cellular stress responses. Our study revealed an unexpected heterogeneity in gene responses to MeHg exposure in different tissues and neuronal subregions, even though the known molecular action of MeHg would predict a similar burden of exposed cells. The overall structure of the developing brain of MeHg-exposed embryos appeared normal, suggesting that the mechanism leading to differentiation of the CNS is not overtly affected by exposure to MeHg. We propose that MeHg disturbs the function of the CNS by disturbing the cellular homeostasis. As these cellular stress responses comprise genes that are also involved in normal neuronal activity and learning, MeHg may affect the developing CNS in a subtle manner that manifests itself in behavioral deficits.

Spatial expression and functional flexibility of monocarboxylate transporter isoforms in the zebrafish brain

The present study provides in vivo evidence to prove the functional plasticity of monocarboxylate transporters (MCTs) in brains of vertebrates using zebrafish (Danio rerio) as a model. In the mammalian central nervous system (CNS), energy demands are largely met by oxidation of glucose. In recent studies, in addition to glucose, lactate is also considered an energy substrate for the CNS. Astrocytes were demonstrated to play an important role in transporting lactate as metabolic substrate from capillaries to neurons through monocarboxylate transporters (MCTs). The present study was to use zebrafish as an in vivo model to test the hypothesis of whether the various MCT homologs play differential roles in the development and functioning of the CNS. Using RT-PCR and double in situ hybridization coupling with immunocytochemical staining experiments, zebrafish MCTs1–4 were all found to be expressed in brains of embryos, and were further elucidated to be localized in both neurons and astrocytes. Loss-of-functions by morpholino knockdown further provided in vivo evidences to infer that zMCTs1, -2, and -4 may be involved in metabolite transport and functioning in the developing brain. Subsequent rescue experiments with capped mRNAs of specific isoforms further indicated that zMCT2 is an indispensable monocarboxylate-transporting route for CNS development and function in zebrafish. This information is essential for identifying proper candidates of MCT isoforms that are involved in the development and functioning of the CNS.

Prenatal inhibition of the tryptophan–kynurenine pathway alters synaptic plasticity and protein expression in the rat hippocampus

Glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) are important in early brain development, influencing cell proliferation and migration, neuritogenesis, axon guidance and synapse formation. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Rats were treated in late gestation with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]-benzene-sulphonamide (Ro61-8048), an inhibitor of kynurenine-3-monoxygenase which diverts kynurenine metabolism to kynurenic acid. Within 5h of drug administration, there was a significant decrease in GluN2A expression and increased GluN2B in the embryo brains, with changes in sonic hedgehog at 24h. When injected dams were allowed to litter normally, the brains of offspring were removed at postnatal day 21 (P21). Recordings of hippocampal field excitatory synaptic potentials (fEPSPs) showed that prenatal exposure to Ro61-8048 increased neuronal excitability and paired-pulse facilitation. Long-term potentiation was also increased, with no change in long-term depression. At this time, levels of GluN2A, GluN2B and postsynaptic density protein PSD-95 were all increased. Among several neurodevelopmental proteins, the expression of sonic hedgehog was increased, but DISC1 and dependence receptors were unaffected, while raised levels of doublecortin and Proliferating Cell Nuclear Antigen (PCNA) suggested increased neurogenesis. The results reveal that inhibiting the kynurenine pathway in utero leads to molecular and functional synaptic changes in the embryos and offspring, indicating that the pathway is active during gestation and plays a significant role in the normal early development of the embryonic and neonatal nervous system.

1614 – Prenatal alcoholization of human embryos and fetuses alters structure of synapses and properties of benzodiazepine receptors in their brain

Influence of prenatal alcoholization on development of embryo's human brain and process of synaptogenesis and neurotransmission has been studied inadequately. It has been noticed that acute impact of the alcohol reinforces GABAergic transmission and chronic alcoholization increases selectivity of reverse agonists of benzodiazepine receptor (BDR), that indicates presence of relationship between action of ethanol and functioning of GABA-benzodiazepine receptor complex. Objective of present investigation appeared to be a study of dynamic of formation and development of synaptic contacts and benzodiazepine receptors of synapses of the embryonic brain at weeks 7-15 of development obtained from healthy women and alcoholic ones. Investigations with electronic microscope in cells of embryonic brain obtained from women with alcohol addiction have shown slower formation of synaptic structures as compared with the norm. In addition, we revealed increasing of synaptic contacts number during brain development and alteration of properties of benzodiazepine receptors of synaptosomal membranes during embryo development stage that is expressed in decrease of affinity of receptors (decrease of affinity [ 3 H]flunitrazepam binding) and increase of their density with radioreceptors assay. In cells of the embryo brain obtained from women suffering from alcoholism slowing of formation of synaptic structures as compared with norm and transformation of properties of synaptosomal BDR has been found out, what may be a cause of alteration of neuromediator transmission. Provided data indicate presence of relationship between action of ethanol on formation of synaptic structures and functioning of benzodiazepine receptor in human embryo and fetus brain.

Production of chimeras between the Chinese soft-shelled turtle and Peking duck through transfer of early blastoderm cells

Chimeras are useful models for studies of developmental biology and cell differentiation. Intraspecies and interspecies germline chimeras have been produced in previous studies, but the feasibility of producing chimeras between animals of two different classes remains unclear. To address this issue, we attempted to produce chimeras between the Chinese soft-shelled turtle and the Peking duck by transferring stage X blastoderm cells to recipient embryos. We then examined the survival and development of the PKH26-labeled donor cells in the heterologous embryos. At early embryonic stages, both turtle and duck donor cells that were labeled with PKH26 were readily observed in the brain, neural tube, heart and gonads of the respective recipient embryos. Movement of turtle donor-derived cells was observed in the duck host embryos after 48 h of incubation. Although none of the hatchlings presented a chimeric phenotype, duck donor-derived cells were detected in a variety of organs in the hatchling turtles, particularly in the gonads. Moreover, in the hatched turtles, mRNA expression of tissue-specific duck genes MEF2a and MEF2c was detected in many tissues, including the muscle, heart, small and large intestines, stomach and kidney. Similarly, SPAG6 mRNA was detected in a subset of turtle tissues, including the gonad and the small and large intestines. These results suggest that duck donor-derived cells can survive and differentiate in recipient turtles; however, no turtle-derived cells were detected in the hatched ducks. Our findings indicate that chimeras can be produced between animals of two different classes.

Ex utero Electroporation into Mouse Embryonic Neocortex

This technique allows highly efficient and reproducible transfer of DNA/RNA into the embryonic neocortex of rodents across multiple ages. Ex utero electroporation compliments the more technically difficult in utero electroporation technique by maximizing the number of embryos for available for a given experiment, as well as increasing the variety of constructs used in each experiment, thereby helping to reduce their overall numbers. Ex utero electroporation followed by short term organotypic slice culture of embryonic brain sections allows immediate access to multiple slices for choosing optimal ones for live-cell imaging experiments, and characterization of various NSC manipulations in the intact stem cell niche. (see also “In utero Electroporation of Mouse Embryo Brains” (Ge, 2012); “Organotypic Slice Culture of Embryonic Brain Sections” (Calderon de Anda, 2013). Additionally, ex utero electroporated neocortices can be used for in vitro primary cell cultures with further dissection, dissociation into single cells, and plating on cover slips or in multi-well dishes according to standard techniques: note this procedure can be performed immediately after electroporation, prior to the onset of ectopic gene expression, or after overnight slice culturing to collect just the region of electroporated cells.