Embryonic Cortical Slices Research Articles

Metabolic regulation and glucose sensitivity of cortical radial glial cells

The primary stem cells of the cerebral cortex are the radial glial cells (RGCs), and disturbances in their operation lead to myriad brain disorders in all mammals from mice to humans. Here, we found in mice that maternal gestational obesity and hyperglycemia can impair the maturation of RGC fibers and delay cortical neurogenesis. To investigate potential mechanisms, we used optogenetic live-imaging approaches in embryonic cortical slices. We found that Ca2+ signaling regulates mitochondrial transport and is crucial for metabolic support in RGC fibers. Cyclic intracellular Ca2+ discharge from localized RGC fiber segments detains passing mitochondria and ensures their proper distribution and enrichment at specific sites such as endfeet. Impairment of mitochondrial function caused an acute loss of Ca2+ signaling, while hyperglycemia decreased Ca2+ activity and impaired mitochondrial transport, leading to degradation of the RGC scaffold. Our findings uncover a physiological mechanism indicating pathways by which gestational metabolic disturbances can interfere with brain development.

Facilitation of axon outgrowth via a Wnt5a-CaMKK-CaMKIα pathway during neuronal polarization.

BackgroundWnt5a, originally identified as a guidance cue for commissural axons, activates a non-canonical pathway critical for cortical axonal morphogenesis. The molecular signaling cascade underlying this event remains obscure.ResultsThrough Ca2+ imaging in acute embryonic cortical slices, we tested if radially migrating cortical excitatory neurons that already bore primitive axons were sensitive to Wnt5a. While Wnt5a only evoked brief Ca2+ transients in immature neurons present in the intermediate zone (IZ), Wnt5a-induced Ca2+ oscillations were sustained in neurons that migrated out to the cortical plate (CP). We wondered whether this early Wnt5a-Ca2+ signaling during neuronal polarization has a morphogenetic consequence. During transition from round to polarized shape, Wnt5a administration to immature cultured cortical neurons specifically promoted axonal, but not dendritic, outgrowth. Pharmacological and genetic inhibition of the CaMKK-CaMKIα pathway abolished Wnt5a-induced axonal elongation, and rescue of CaMKIα in CaMKIα-knockdown neurons restored Wnt5a-mediated axon outgrowth.ConclusionsThis study suggests that Wnt5a activates Ca2+ signaling during a neuronal morphogenetic time window when axon outgrowth is critically facilitated. Furthermore, the CaMKK-CaMKIα cascade is required for the axonal growth effect of Wnt5a during neuronal polarization.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0189-3) contains supplementary material, which is available to authorized users.

CB2 Cannabinoid Receptors Promote Neural Progenitor Cell Proliferation via mTORC1 Signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.

Generation of Glutamatergic Neurons from Postnatal and Adult Subventricular Zone with Pyramidal-Like Morphology

The mammalian subventricular zone (SVZ) contains progenitors derived from cerebral cortex radial glia cells, which give rise to glutamatergic pyramidal neurons during embryogenesis. However, during postnatal life, SVZ generates neurons that migrate and differentiate into olfactory bulb γ-aminobutyric acid (GABA)ergic interneurons. In this work, we tested if SVZ cells are able to produce glutamatergic neurons if confronted with the embryonic cortical ventricular zone environment. Different from typical SVZ chain migration, cells from P9-P11 SVZ explants migrate into embryonic cortical slices individually, many of which radially oriented. An average of 82.5% of green fluorescent protein-positive cells were immunolabeled for neuronal marker class III β-tubulin. Invading cells differentiate into multiple morphologies, including a pyramidal-like morphotype. A subset of these cells are GABAergic; however, about 28% of SVZ-derived cells are immunoreactive for glutamate. Adult SVZ explants also give rise to glutamatergic neurons in these conditions. Taken together, our results indicate that SVZ can be a source of glutamatergic cortical neurons when submitted to proper environmental cues.

α3β1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

We show that alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.

Fate Sealed by Age

Neurons and glia are derived from a common set of precursor stem cells. Morrow et al. used a cortical slice assay to examine the signals required to specify neuronal versus glial cell fate and differentiation. Dissociated embryonic neural stem cells from a green fluorescent protein (GFP)-expressing strain of mice were plated with cortical slices from mice of different ages (embryonic through postnatal). Cell fate and differentiation were monitored by changes in the morphology and expression of marker proteins for neurons and glia. Incubation of neural stem cells with embryonic cortical slices led to the development of mostly neuronal cells and incubation with postnatal cortical slices produced mostly glial cells, suggesting that different factors are produced at different stages of development and that the stem cells are poised to respond to either set of signals. Analysis of clones of the GFP-expressing cells showed that the signals were predominantly instructive and not trophic signals. The steps of glial cell fate specification and glial differentiation could be separated. The effects of postnatal cortical slices could be recapitulated by culture of the stem cells with fibroblast growth factor 2 (FGF2) and ciliary neurotrophic factor (CNTF), with FGF2 providing the signal for glial fate specification and CNTF providing the glial differentiation signal. T. Morrow, M-R. Song, A. Ghosh, Sequential specification of neurons and glia by developmentally regulated extracellular factors. Development 128 , 3585-3594 (2001). [Online Journal]

Sequential specification of neurons and glia by developmentally regulated extracellular factors.

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.

Migrating neurons cross a reelin-rich territory to form an organized tissue out of embryonic cortical slices.

In this study we show that the radial migration of neuronal precursors out of cerebral cortex of embryonic brain slices cultured for 4-7 days gives rise to an organized tissue that forms de novo off developing slices. In our in vitro preparations, migrating neuronal precursors overshot the marginal zone, as did the elongation of radial glial processes out of the slices. These cells detached from radial glia at a distance from the cortex and differentiated into pyramidal and nonpyramidal profiles that expressed different neuronal markers. Glial precursors were shown to proliferate in the slice and in the neotissue, and to differentiate into astrocytes. We show that cells expressing reelin in the marginal zone of embryonic cortical slices persist after a week in culture, which implies that neuronal migration is not necessarily hindered by the presumed stop signals provided by reelin in the marginal zone. Furthermore, our results provide a new model for in vitro studies of migration and differentiation during cortical development.