Prenatal carbon monoxide impairs migration of interneurons into the cerebral cortex.

Prenatal exposure to carbon monoxide (CO) disrupts brain development, however little is known about effects on neocortical maturation. We exposed pregnant mice to CO from embryonic day 7 (E7) until birth. To study the effect of CO on neuronal migration into the neocortex we injected BrdU during corticogenesis and observed misplaced BrdU+ cells. The majority of cells not in their proper layer colocalized with GAD65/67, suggesting impairment of interneuron migration; interneuron subtypes were also affected. We subsequently followed interneuron migration from E15 organotypic cultures of mouse neocortex exposed to CO; the leading process length of migrating neurons diminished. To examine an underlying mechanism, we assessed the effects of CO on the cellular cascade mediating the cytoskeletal protein vasodilator-stimulated phosphoprotein (VASP). CO exposure resulted in decreased cGMP and in a downstream target, phosphorylated VASP. Organotypic cultures grown in the presence of the phosphodiesterase inhibitor IBMX resulted in a recovery of the leading processes. These data support the idea that CO acts as a signaling molecule and impairs function and neuronal migration by acting through the CO/NO-cGMP pathway. In addition, treated mice demonstrated functional impairment in behavioral tests.

Fine-tuning of neurogenesis is essential for the evolutionary expansion of the cerebral cortex.

We used several animal models to study global and regional cortical surface expansion: The lissencephalic mouse, gyrencephalic normal ferrets, in which the parietal cortex expands more than the temporal cortex, and moderately lissencephalic ferrets, showing a similar degree of temporal and parietal expansion. We found that overall cortical surface expansion is achieved when specific events occur prior to surpragranular layer formation. (1) The subventricular zone (SVZ) shows substantial growth, (2) the inner SVZ contains an increased number of outer radial glia and intermediate progenitor cells expressing Pax6, and (3) the outer SVZ contains a progenitor cell composition similar to the combined VZ and inner SVZ. A greater parietal expansion is also achieved by eliminating the latero-dorsal neurogenic gradient, so that neurogenesis displays a similar developmental degree between parietal and temporal regions. In contrast, mice or lissencephalic ferrets show more advanced neurogenesis in the temporal region. In conclusion, we propose that global and regional cortical surface expansion rely on similar strategies consisting in altering the timing of neurogenic events prior to the surpragranular layer formation, so that more progenitor cells, and ultimately more neurons, are produced. This hypothesis is supported by findings from a ferret model of lissencephaly obtained by transiently blocking neurogenesis during the formation of layer IV.

CXCR7 Receptor Controls the Maintenance of Subpial Positioning of Cajal-Retzius Cells.

Cajal-Retzius (CR) cells are essential for cortical development and lamination. These pioneer neurons arise from distinct progenitor sources, including the cortical hem and the ventral pallium at pallium-subpallium boundary (PSB). CXCR4, the canonical receptor for the chemokine CXCL12, controls the superficial location of hem-derived CR cells. However, recent studies showed that CXCR7, a second CXCL12 receptor, is also expressed in CR cells at early developmental stages. We thus investigated the role of CXCR7 during CR cell development using multiple loss-of-function approaches. Cxcr7 gene inactivation led to aberrant localization of Reelin-positive cells within the pallium. In addition, Cxcr7(-/-) mice were characterized by significant accumulation of ectopic CR cells in the lateral part of the dorsal pallium compared with Cxcr4 knockout mice. Loss-of-function approaches, using either gene targeting or pharmacological receptor inhibition, reveal that CXCR7 and CXCR4 act both in CR positioning. Finally, conditional Cxcr7 deletion in cells derived from Dbx1-expressing progenitors indicates an essential role of CXCR7 in controlling the positioning of a subpopulation of PSB-derived CR cells. Our data demonstrate that CXCR7 has a role in the positioning of hem and PSB-derived CR cells, CXCL12 regulating CR cell subpial localization through the combined action of CXCR4 and CXCR7.

Populations of radial glial cells respond differently to reelin and neuregulin1 in a ferret model of cortical dysplasia.

Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not.

Regulation of neural progenitor cell development in the nervous system.

The mammalian telencephalon, which comprises the cerebral cortex, olfactory bulb, hippocampus, basal ganglia, and amygdala, is the most complex and intricate region of the CNS. It is the seat of all higher brain functions including the storage and retrieval of memories, the integration and processing of sensory and motor information, and the regulation of emotion and drive states. In higher mammals such as humans, the telencephalon also governs our creative impulses, ability to make rational decisions, and plan for the future. Despite its massive complexity, exciting work from a number of groups has begun to unravel the developmental mechanisms for the generation of the diverse neural cell types that form the circuitry of the mature telencephalon. Here, we review our current understanding of four aspects of neural development. We first begin by providing a general overview of the broad developmental mechanisms underlying the generation of neuronal and glial cell diversity in the telencephalon during embryonic development. We then focus on development of the cerebral cortex, the most complex and evolved region of the brain. We review the current state of understanding of progenitor cell diversity within the cortical ventricular zone and then describe how lateral signaling via the Notch-Delta pathway generates specific aspects of neural cell diversity in cortical progenitor pools. Finally, we review the signaling mechanisms required for development, and response to injury, of a specialized group of cortical stem cells, the radial glia, which act both as precursors and as migratory scaffolds for newly generated neurons.

Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex.

Numerous functions related to neuronal migration are linked to the glycoprotein reelin. Reelin also elongates radial glia, which are disrupted in mutant reeler mice. Our lab developed a model of cortical dysplasia in ferrets that shares features with the reeler mouse, including impaired migration of neurons into the cerebral cortex and disrupted radial glia. Explants of normal ferret cortex in coculture with dysplastic ferret cortex restore the deficits in this model. To determine if reelin is integral to the repair, we used explants of P0 mouse cortex either of the wild type (WT) or heterozygous (het) for the reelin gene, as well as P0 reeler cortex (not containing reelin), in coculture with organotypic cultures of dysplastic ferret cortex. This arrangement revealed that all types of mouse cortical explants (WT, het, reeler) elongated radial glia in ferret cortical dysplasia, indicating that reelin is not required for proper radial glial morphology. Migration of cells into ferret neocortex, however, did not improve with explants of reeler cortex, but was almost normal after pairing with WT or het explants. We also placed an exogenous source of reelin in ferret cultures at the pial surface to reveal that migrating cells move toward the reelin source in dysplastic cortex; radial glia in these cultures were also improved toward normal. Our results demonstrate that the normotopic position of reelin is important for proper neuronal positioning, and that reelin is capable of elongating radial glial cells but is not the only radialization factor.

Alteration of interneuron migration in a ferret model of cortical dysplasia.

During cerebral cortical development, gamma-aminobutyric acidergic (GABAergic) interneurons arise from a different site than projection neurons. GABAergic cells are generated in the subpallial ganglionic eminence (GE), while excitatory projection neurons arise from the neocortical ventricular zone. Our laboratory studies a model of cortical dysplasia that displays specific disruption of GABAergic mechanisms and an alteration in the overall balance of excitation in the neocortex. To produce this model, the birth of neurons on a specific gestational day in ferrets (embryonic day 33 [E33]) is interrupted by injection of the antimitotic methylazoxymethanol (MAM). We hypothesized that migration of interneurons might be disrupted in this cortical dysplasia paradigm. We observed that although interneurons migrate into the neocortex in both normal and dysplastic cortex, the migrating cells become disoriented over time after E33 MAM treatment. Coculture experiments using normal GE and MAM-treated cortex (and vice versa) demonstrate that cues dictating proper orientation of migrating interneurons arise from the cortex and are not intrinsic to the migrating cells. As a consequence, interneurons in mature brains of MAM-treated animals are abnormally distributed. We report that GABA(A) receptor activation is crucial to the proper positioning of interneurons migrating into the cortex from the GE in normal and MAM-treated animals.

A normal radial glial scaffold is necessary for migration of interneurons during neocortical development.

The relationship between radial glia and neurons migrating tangentially from the ganglionic eminence (GE) has been suggested but not firmly established. To study this relationship we used a ferret model of cortical dysplasia where radial glia are highly disorganized. To produce this, an antimitotic, methylazoxy methanol (MAM) is injected on the 24th day of gestation (E24 MAM). Neurons migrating away from the GE in MAM-treated animals tend to remain in the intermediate zone (IZ) and do not reach the cortical plate (CP) as they do in normal ferret slices. We recently observed that the disrupted radial glia after MAM treatment could be restored toward their normal morphology by exogenous application of neuregulin1 (NRG1). We demonstrate here that when E24 MAM slices are treated with NRG1, the distribution of cells arising from the GE was similar to normal slices. In a second paradigm, we disrupted radial glia by adding ciliary neurotrophic factor (CNTF) to the culture media of normal ferret slices; CNTF induces acute differentiation of radial glia into astrocytes. After CNTF exposure, few tangentially migrating cells reach the CP compared to untreated slices. These results show that interneurons fail to reach the CP by disrupted normal radial glia and restoring the normal radial glial scaffold is sufficient to allow migrating cells to invade the CP. Our results suggest an important role for radial glia by controlling directly or indirectly the migration of interneurons to the CP, their main target.

AMPA-evoked ion influx is strongest in tangential neurons of the rat neocortical intermediate zone close to the front of the migratory stream.

In addition to the classically described radially migrating neurons, embryonic cortical areas receive neurons originating from the basal ganglia. One of the migration routes is in the intermediate zone. The front of this migration moves toward the hippocampus synchronously with the edge of the dorsally extending cortical plate. We investigated whether cells close to the front have specific properties compared with those at less advanced positions. Activation of AMPA receptors in the presence of cobalt showed that a strong influx of divalent cations could be triggered in front cells by low agonist concentration, whereas the less advanced cells needed a higher concentration to incorporate detectable amounts of cobalt. As shown by in situ hybridization, this discrepancy was not due to differential expression of GluR-2 (known to reduce permeability for divalent cations). In vivo, release of an endogenous agonist presumably affects more, or differently, the tangential cells close to the front.

AMPA receptor activation induces GABA release from neurons migrating tangentially in the intermediate zone of embryonic rat neocortex.

In the intermediate zone of the embryonic rodent neocortex, neurons migrating tangentially from the basal ganglia express both functional amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and gamma-aminobutyric acid (GABA). To test the hypothesis of GABA release triggered by AMPA receptor activation, we used whole-hemisphere cultures prepared from rat embryos (day 15). We observed a marked decrease in the number of detectable GABA-positive cells in the intermediate zone after exposure to T-AMPA. This effect was blocked by coapplying GYKI 53655, an AMPA receptor antagonist. The decrease in GABA immunolabelling induced by T-AMPA did not require extracellular calcium. In contrast, it was abolished after sodium substitution by choline, or after coapplication of nipecotic acid, a GABA transporter inhibitor. Exposure to high potassium reduced the number of detectable GABA-positive cells. These results are compatible with carrier-mediated GABA release consecutive to sodium influx. GABA released from neurons migrating tangentially in the intermediate zone after AMPA receptor activation may influence neighbouring elements including radially migrating postmitotic neurons, proliferating progenitors and possibly the tangential cells themselves.