&lt;em&gt;Ex Utero&lt;/em&gt; Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Venkataramanappa, Sathish; Simon, Ralph; Britsch, Stefan

doi:10.3791/52550

Cited by 5 publications

(6 citation statements)

References 17 publications

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“…Organotypic cultures of mouse coronal forebrain slices were prepared following published methods 45 with some modifications. Whole brains from E14–E18 mouse embryos were embedded in 4% low-melting point agarose and slices were cut at 250–300 μm using a Leica VT1200 vibrotome in complete HBSS (100 ml of 10× HBSS without Ca or Mg, 2.5 ml of 1M HEPES buffer at pH 7.4, 30 ml of 1M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1 M NaHCO3).…”

Section: Methodsmentioning

confidence: 99%

Assembly of functionally integrated human forebrain spheroids

Birey

Andersen

Makinson

et al. 2017

Nature

1,052

1,115

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SUMMARY The development of the nervous system involves a coordinated succession of events including the migration of GABAergic neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here, we generate from human pluripotent cells three-dimensional spheroids resembling either the dorsal or ventral forebrain and containing cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled to recapitulate the saltatory migration of interneurons similar to migration in fetal forebrain. Using this system, we find that in Timothy syndrome– a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel, interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

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Section: Methodsmentioning

confidence: 99%

Assembly of functionally integrated human forebrain spheroids

Birey

Andersen

Makinson

et al. 2017

Nature

1,052

1,115

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“…All mouse experiments were carried out in compliance with German law and approved by the respective government offices in Tü bingen. Ex utero electroporation and organotypic slice cultures of Bcl11b flox/flox ; Emx-1Cre as well as control embryonic brains were carried out as described previously (Simon et al, 2012(Simon et al, , 2016Venkataramanappa et al, 2015). Briefly, up to 9 mg DNA was electroporated into the prospective dentate gyrus area of embryonic brains at embryonic Day 15.5 using five pulses at 50 V, brains were cut into 250 mm slices and kept in culture up to in vitro Day 11.…”

Section: Ex Utero Electroporation and Hippocampal Slice Culturementioning

confidence: 99%

“…To gain further insight into the molecular effects of the four premature termination codon mutations likely escaping NMD [i.e. p.(Tyr455*), p.(Glu499*), p.(Thr502Hisfs*15), p.(Arg518Alafs*45), p.(Asp534Thrfs*29), p.(Gly64 9Alafs*67) and p.(Gly820Alafs*27)] we turned to a previously-established mouse model (Simon et al, 2012;Venkataramanappa et al, 2015) and analysed the influence of the most C-terminally located frameshift mutation p.(Gly820Alafs*27) on hippocampal neurogenesis. As expected, hippocampal slice cultures derived from Bcl11b homozygous mutant mice displayed severely reduced progenitor cell proliferation at 11 days in vitro (Simon et al, 2012).…”

Section: Pathogenic Nature Of the C-terminally Located Frameshift Mutmentioning

confidence: 99%

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells

Lessel

Gehbauer

Bramswig

et al. 2018

Brain

Self Cite

101

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The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.

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“…This method allows for the rapid and low-cost early functional investigation of novel candidate genes identified through single cell RNA sequencing of migratory INs 66,67 or through Next Generation Sequencing of patients with neurodevelopmental disorders 68,69,70 . This technique has been extensively used to study migration in other cell populations, including pyramidal cells in the cortex and hippocampus 70,71,72,73 . Once mastered, it could potentially be used to image the recycling and trafficking of membrane proteins, such as receptors and channels using GFP-tagged proteins; the activation of specific cellular signaling cascades using biosensors 74 ; or even the monitoring and manipulation of cellular activity using calcium imaging coupled to optogenetics in cortical INs, but also in other brain areas, such as the hippocampus, the amygdala or even the striatum.…”

mentioning

confidence: 99%

<em>Ex Utero</em> Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Eid

Lachance

Hickson

et al. 2018

JoVE

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GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

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<em>Ex Utero</em> Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Cited by 5 publications

References 17 publications

Assembly of functionally integrated human forebrain spheroids

Assembly of functionally integrated human forebrain spheroids

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells

<em>Ex Utero</em> Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

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