Migration Of Cortical Interneurons Research Articles

The role of Slit-Robo signaling in the generation, migration and morphological differentiation of cortical interneurons

Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1 −/−, but not Robo2 −/−, animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1 −/− mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1 −/−/Slit2 −/− mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1 −/−, Robo2 −/− and Slit1 −/−/Slit2 −/−, but not in Slit1 −/− mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons.

Symposium: ‘A celebration of neuroanatomy in the centennial of Cajal's Nobel Prize’

By 1889, at the age of 37 years, Cajal had published two papers on neuroanatomy in the Revista trimestral de Histologia normal y patologica, which he had founded (and subsidised) the previous year. However, his ideas received hardly any attention from his colleagues and he became a ‘little alarmed by their silence’ (Ramon y Cajal, 1981). He admitted that most histologists had not read his papers since ‘it is true that Spanish is an unknown language to the educated’ and he thought that the ‘direct objective demonstration’ of facts would be more convincing. Therefore, he first translated his principal papers into French and then decided to show his histological preparations to scientific colleagues, in order to demonstrate his findings and ideas. To this end, Cajal applied for membership of the Anatomische Gesellschaft, asked his Rector for permission to travel, collected all of his savings and set out for Berlin, where the Anatomische Gesellschaft met in October 1889.

Slit–Robo interactions during cortical development

Interneurons are an integral part of cortical neuronal circuits. During the past decade, numerous studies have shown that these cells, unlike their pyramidal counterparts that are derived from the neuroepithelium along the lumen of the lateral ventricles, are generated in the ganglionic eminences in the subpallium. They use tangential migratory paths to reach the cortex, guided by intrinsic and extrinsic cues. Evidence is now emerging which suggests that the family of Slit proteins, acting through Robo receptors, play a role not only in axon guidance in the developing forebrain, but also as guiding signals in the migration of cortical interneurons. Here we describe the patterns of expression of Slit and Robo at different stages of forebrain development and review the evidence in support of their role in cortical interneuron migration. Slit-Robo signal transduction mechanisms are also important during normal development in a number of systems in the body and in disease states, making them potential therapeutic targets for the treatment of neurological disorders and certain types of cancer.

Cortical Interneurons Refuse to Follow the PAK

The tangential migration of GABAergic interneurons to the cortex requires the activity of several transcription factors, but the mechanisms involved have remained elusive. In this issue of Neuron, Rubenstein and colleagues demonstrate that the homeobox proteins Dlx1 and Dlx2 promote interneuron migration in part by preventing the premature expression of PAK3, a serine/threonine kinase that is later involved in neurite outgrowth and other aspects of interneuron maturation.

Neuregulin-1 Enhances Depolarization-Induced GABA Release

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.

Both Doublecortin and Doublecortin-Like Kinase Play a Role in Cortical Interneuron Migration

Type I lissencephaly, a genetic disease characterized by disorganized cortical layers and gyral abnormalities, is associated with severe cognitive impairment and epilepsy. Two genes, LIS1 and doublecortin (DCX), have been shown to be responsible for a large proportion of cases of type I lissencephaly. Both genes encode microtubule-associated proteins that have been shown to be important for radial migration of cortical pyramidal neurons. To investigate whether DCX also plays a role in cortical interneuron migration, we inactivated DCX in the ganglionic eminence of rat embryonic day 17 brain slices using short hairpin RNA. We found that, when DCX expression was blocked, the migration of interneurons from the ganglionic eminence to the cerebral cortex was slowed but not absent, similar to what had previously been reported for radial neuronal migration. In addition, the processes of DCX-deficient migrating interneurons were more branched than their counterparts in control experiments. These effects were rescued by DCX overexpression, confirming the specificity to DCX inactivation. A similar delay in interneuron migration was observed when Doublecortin-like kinase (DCLK), a microtubule-associated protein related to DCX, was inactivated, although the morphology of the cells was not affected. The importance of these genes in interneuron migration was confirmed by our finding that the cortices of Dcx, Dclk, and Dcx/Dclk mutant mice contained a reduced number of such cells in the cortex and their distribution was different compared with wild-type controls. However, the defect was different for each group of mutant animals, suggesting that DCX and DCLK have distinct roles in cortical interneuron migration.

Dlx Homeobox Genes Promote Cortical Interneuron Migration from the Basal Forebrain by Direct Repression of the Semaphorin Receptor Neuropilin-2

Dlx homeobox genes play an important role in vertebrate forebrain development. Dlx1/Dlx2 null mice die at birth with an abnormal cortical phenotype, including impaired differentiation and migration of GABAergic interneurons to the neocortex. However, the molecular basis for these defects downstream of loss of Dlx1/Dlx2 function is unknown. Neuropilin-2 (NRP-2) is a receptor for Class III semaphorins, which inhibit neuronal migration. Herein, we show that Neuropilin-2 is a specific DLX1 and DLX2 transcriptional target by applying chromatin immunoprecipitation to embryonic forebrain tissues. Both homeobox proteins repress Nrp-2 expression in vitro, confirming the functional significance of DLX binding. Furthermore, the homeodomain of DLX1 and DLX2 is necessary for DNA binding and this binding is essential for Dlx repression of Nrp-2 expression. Of importance, there is up-regulated and aberrant expression of NRP-2 in the forebrains of Dlx1/Dlx2 null mice. This is the first report that DLX1 or DLX2 can function as transcriptional repressors. Our data show that DLX proteins specifically mediate the repression of Neuropilin-2 in the developing forebrain. As well, our results support the hypothesis that down-regulation of Neuropilin-2 expression may facilitate tangential interneuron migration from the basal forebrain.

In vivo time-lapse imaging of migration of cortical interneurons during postnatal development

In vivo time-lapse imaging of migration of cortical interneurons during postnatal development

Robo1 regulates the development of major axon tracts and interneuron migration in the forebrain

The Slit genes encode secreted ligands that regulate axon branching, commissural axon pathfinding and neuronal migration. The principal identified receptor for Slit is Robo (Roundabout in Drosophila). To investigate Slit signalling in forebrain development, we generated Robo1 knockout mice by targeted deletion of exon 5 of the Robo1 gene. Homozygote knockout mice died at birth, but prenatally displayed major defects in axon pathfinding and cortical interneuron migration. Axon pathfinding defects included dysgenesis of the corpus callosum and hippocampal commissure, and abnormalities in corticothalamic and thalamocortical targeting. Slit2 and Slit1/2 double mutants display malformations in callosal development, and in corticothalamic and thalamocortical targeting, as well as optic tract defects. In these animals, corticothalamic axons form large fasciculated bundles that aberrantly cross the midline at the level of the hippocampal and anterior commissures, and more caudally at the medial preoptic area. Such phenotypes of corticothalamic targeting were not observed in Robo1 knockout mice but, instead, both corticothalamic and thalamocortical axons aberrantly arrived at their respective targets at least 1 day earlier than controls. By contrast, in Slit mutants, fewer thalamic axons actually arrive in the cortex during development. Finally, significantly more interneurons (up to twice as many at E12.5 and E15.5) migrated into the cortex of Robo1 knockout mice, particularly in both rostral and parietal regions, but not caudal cortex. These results indicate that Robo1 mutants have distinct phenotypes, some of which are different from those described in Slit mutants, suggesting that additional ligands, receptors or receptor partners are likely to be involved in Slit/Robo signalling.

Cell and molecular mechanisms involved in the migration of cortical interneurons

Since the discovery that the vast majority of the GABA-containing interneurons of the cerebral cortex arise in the subpallium, considerable effort has been put into the description of the precise origin of these neurons in subdivisions of the ganglionic eminence and in the migratory routes they follow on their way to the developing cortex. More recently, studies have focused on the molecular and cellular mechanisms that guide their migration. Investigations of the molecular mechanisms involved have demonstrated important roles for numerous transcription factors, motogenic factors and guidance molecules. Here, we review results of very recent analyses of the underlying cellular mechanisms and specifically of the movement of the nucleus, cytoplasmic components and neuritic processes during interneuron migration.

A cell-autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration

Precise cell cycle regulation is critical for nervous system development. To assess the role of the cell cycle regulator, retinoblastoma (Rb) protein, in forebrain development, we studied mice with telencephalon-specific Rb deletions. We examined the role of Rb in neuronal specification and migration of diverse neuronal populations. Although layer specification occurred at the appropriate time in Rb mutants, migration of early-born cortical neurons was perturbed. Consistent with defects in radial migration, neuronal cell death in Rb mutants specifically affected Cajal-Retzius neurons. In the ventral telencephalon, although calbindin- and Lhx6-expressing cortical neurons were generated at embryonic day 12.5, their tangential migration into the neocortex was dramatically and specifically reduced in the mutant marginal zone. Cell transplantation assays revealed that defects in tangential migration arose owing to a cell-autonomous loss of Rb in migrating interneurons and not because of a defective cortical environment. These results revealed a cell-autonomous role for Rb in regulating the tangential migration of cortical interneurons. Taken together, we reveal a novel requirement for the cell cycle protein, Rb, in the regulation of neuronal migration.

Sonic hedgehog maintains the identity of cortical interneuron progenitors in the ventral telencephalon

Fate determination in the mammalian forebrain, where mature phenotypes are often not achieved until postnatal stages of development, has been an elusive topic of study despite its relevance to neuropsychiatric disease. In the ventral telencephalon, major subgroups of cerebral cortical interneurons originate in the medial ganglionic eminence (MGE), where the signaling molecule sonic hedgehog (Shh) continues to be expressed during the period of neuronogenesis. To examine whether Shh regulates cortical interneuron specification, we studied mice harboring conditional mutations in Shh within the neural tube. At embryonic day 12.5, NestinCre:Shh(Fl/Fl) mutants have a relatively normal index of S-phase cells in the MGE, but many of these cells do not co-express the interneuron fate-determining gene Nkx2.1. This effect is reproduced by inhibiting Shh signaling in slice cultures, and the effect can be rescued in NestinCre:Shh(Fl/Fl) slices by the addition of exogenous Shh. By culturing MGE progenitors on a cortical feeder layer, cell fate analyses suggest that Shh signaling maintains Nkx2.1 expression and cortical interneuron fate determination by MGE progenitors. These results are corroborated by the examination of NestinCre:Shh(Fl/Fl) cortex at postnatal day 12, in which there is a dramatic reduction in cell profiles that express somatostatin or parvalbumin. By contrast, analyses of Dlx5/6Cre:Smoothened(Fl/Fl) mutant mice suggest that cell-autonomous hedgehog signaling is not crucial to the migration or differentiation of most cortical interneurons. These results combine in vitro and ex vivo analyses to link embryonic abnormalities in Shh signaling to postnatal alterations in cortical interneuron composition.

The adhesion molecule TAG-1 is required for proper migration of the superficial migratory stream in the medulla but not of cortical interneurons

The neural cell adhesion molecule TAG-1 has been implicated in the tangential migration of neurons of the caudal medulla and of cortical interneurons. In the former case, protein is expressed by the neurons as they migrate, and blocking its function results in altered and reduced migration in vitro. In the latter case, protein is expressed, in part, by the pathway the interneurons use to reach the cortex, and in vitro experiments propose a role for TAG-1 in this system, as well. However, the in vivo requirement of TAG-1 in these migrations has not been investigated. In this report, we analyze the developmental phenotype of TAG-1-deficient animals in these two migratory systems. We show that mutant mice have smaller lateral reticular nuclei as a result of increased cell death in the superficial migratory stream of the caudal medulla. On the other hand, the absence of TAG-1 does not affect the number, morphology, timing and routes of GABAergic interneurons that migrate from the ganglionic eminences to the cortex. Therefore, TAG-1 function is required for the survival of the neurons of some precerebellar nuclei, while it is not required for cortical interneuron migration in vivo.

Short- and long-range attraction of cortical GABAergic interneurons by neuregulin-1.

Most cortical interneurons arise from the subcortical telencephalon, but the molecules that control their migration remain largely unidentified. Here, we show that different isoforms of Neuregulin-1 are expressed in the developing cortex and in the route that migrating interneurons follow toward the cortex, whereas a population of the migrating interneurons express ErbB4, a receptor for Neuregulin-1. The different isoforms of Neuregulin-1 act as short- and long-range attractants for migrating interneurons, and perturbing ErbB4 function in vitro decreases the number of interneurons that tangentially migrate to the cortex. In vivo, loss of Neuregulin-1/ErbB4 signaling causes an alteration in the tangential migration of cortical interneurons and a reduction in the number of GABAergic interneurons in the postnatal cortex. These observations provide evidence that Neuregulin-1 and its ErbB4 receptor directly control neuronal migration in the nervous system.

Lhx6 regulates the migration of cortical interneurons from the ventral telencephalon but does not specify their GABA phenotype.

The LIM homeodomain family of transcription factors is involved in many processes in the developing CNS, ranging from cell fate specification to connectivity. A member of this family of transcription factors, lhx6, is expressed in the medial ganglionic eminence (MGE) of the ventral telencephalon, where the vast majority of cortical interneurons are generated. Its expression in the GABA-containing MGE cells that migrate to the cortex suggests that this gene uniquely or in combination with other transcription factors may play a role in the neurochemical identity and migration of these neurons. We performed loss of function studies for lhx6 in mouse embryonic day 13.5 brain slices and dissociated MGE neuronal cultures using Lhx6-targeted small interfering RNA produced by a U6 promoter-driven vector. We found that silencing lhx6 impeded the tangential migration of interneurons into the cortex, although it did not obstruct their dispersion within the ganglionic eminence. Blocking lhx6 expression in dissociated MGE cultured neurons did not interfere with the production of GABA or its synthesizing enzyme. These results indicate that lhx6, unlike the closely related member lhx7, does not regulate neurotransmitter choice in interneurons but plays an important role in their migration from the ventral telencephalon to the neocortex.

Multimodal tangential migration of neocortical GABAergic neurons independent of GPI-anchored proteins.

Neuronal migration is crucial for the construction of neuronal architecture such as layers and nuclei. Most inhibitory interneurons in the neocortex derive from the basal forebrain and migrate tangentially; however, little is known about the mode of migration of these neurons in the cortex. We used glutamate decarboxylase (Gad)67-green fluorescent protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)-ergic neurons and performed time-lapse analysis. In coronal slices, many GFP-positive neurons in the lower intermediate zone (IZ) and subventricular zone (SVZ) showed robust tangential migration from lateral to medial cortex, while others showed radial and non-radial migration mostly towards the pial surface. In flat-mount preparations, GFP-positive neurons of the marginal zone (MZ) showed multidirectional tangential migration. Some of these neurons descended toward the cortical plate (CP). Intracortical migration of these neurons was largely unaffected by a treatment that cleaves glycosylphosphatidylinositol (GPI) anchors. These findings suggest that tangential migration of cortical interneurons from lateral to medial cortex predominantly occurs in the IZ/SVZ and raise the possibility that a part of the pial surface-directed neurons in the IZ/SVZ reach the MZ, whereby they spread into the whole area of the cortex. At least a part of these neurons may descend toward the CP. Our results also suggest that intracortical migration of GABAergic neurons occurs independent of GPI-anchored proteins.

Subpallial origin of a population of projecting pioneer neurons during corticogenesis.

Pyramidal neurons of the mammalian cerebral cortex are generated in the ventricular zone of the pallium whereas the subpallium provides the cortex with inhibitory interneurons. The marginal zone contains a subpial stream of migratory interneurons and two different classes of transient neurons, the pioneer neurons provided with corticofugal axons, and the reelin-expressing Cajal-Retzius cells. We found in cultured slices that the medial ganglionic eminence provides the reelin-negative pioneer neurons of the marginal zone. Pioneer neurons sent long projection axons that went through the cortical plate and reached the subplate and the lateral border of the lateral ganglionic eminence. In the cultured slices, pioneer neurons were functionally mature: they displayed a voltage-gated sodium current, expressed functional alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and showed gamma-aminobutyric acid type A (GABAA) postsynaptic events that were modulated by presynaptic AMPA receptors. Pioneer neurons expressed the adhesion molecules L1 and TAG-1; the latter has been reported to control tangential migrations to the neocortex [Denaxa, M., Chan, C.-H., Schachner, M., Parnavelas, J. & Karagogeos, D. (2001) Development (Cambridge, U.K.) 128, 4635-4644], and we show here that the pioneer neurons of the marginal zone are the cellular substrate of such a function. Finally, we show that, in early corticogenesis, reelin controls both the tangential migration of cortical interneurons toward the cortical plate and the tangential migration of pioneer neurons toward the marginal zone.

Blockade of GABA(B) receptors alters the tangential migration of cortical neurons.

To better understand the role of neurotransmitter receptors in neuronal differentiation and maturation a detailed knowledge of their identity, location and function in the plasma membrane of specific neuronal populations during development is required. Combining pre-embedding immunocytochemistry with cell tracking in embryonic brain slice cultures we show that virtually all neurons (approximately 98%) migrating through the lower intermediate zone (LIZ) on their way from the medial ganglionic eminence to the cerebral cortex, express GABA(B)R1. Blockade of GABA(B)Rs with a specific antagonist, CGP52432, resulted in a concentration-dependent accumulation of these tangentially migrating neurons in the ventricular/sub-ventricular zones (VZ/SVZ) of the cortex and fewer cells were observed in the cortical plate/marginal zone (CP/MZ) and LIZ. Moreover, they had significantly shorter leading processes compared with similar migrating cells in control slices. Electrophysiological recording in LIZ and CP cells revealed no direct effect of either CGP52432 or the GABA(B)R agonist, baclofen, on resting membrane properties suggesting that the effect of CGP52432 on migration might be mediated through a metabotropic action or the regulation of release of factors controlling migration. These results suggest that GABA(B)Rs have an important modulatory role in the migration of cortical interneurons.

Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling.

During telencephalic development, cells from the medial ganglionic eminence (MGE) are thought to migrate to the neocortex to give rise to a majority of cortical GABAergic interneurons. By combining time-lapse video-microscopy, immunofluorescence and pharmacological perturbations in a new in vitro migration assay, we find that MGE-derived cells migrate through the entire extent of the cortex and into the CA fields of the hippocampus, but avoid the dentate gyrus. Migrating neurons initially travel within the marginal zone and intermediate zone, and can enter the cortical plate from either location. Tangential migration is strongly stimulated by BDNF and NT4 and attenuated by the Trk-family inhibitor, K252a, suggesting that migration is regulated by TrkB signaling. Furthermore, TrkB-null mice show a significant decrease in the number of calbindin-positive neurons migrating tangentially in the embryonic cortex. BDNF and NT4 cause rapid activation of PI3-kinase in MGE cells, and inhibition of PI3-kinase (but not of MAP kinase or PLCgamma) dramatically attenuates tangential migration. These observations suggest that TrkB signaling, via PI3-kinase activation, plays an important role in controlling interneuron migration in the developing cerebral cortex.

Chapter 6 The origin and migration of cortical neurons

The chapter discusses the origin and migration of cortical neurons. Sources of neurons destined for the cerebral cortex are discovered in the ganglionic eminence, the primordium of the basal ganglia in the ventral telencephalon lateral ventricle tend to migrate to the striatum, pallidum and neocortex, while lateral ganglionic eminence (LGE) cells migrate prodominantly into the striatum and olfactory bulb. Neurons of the ganglionic eminence destined for the developing cerebral cortex express the L&I homeobox gene Lhx6. The molecular mechanisms that guide the migration of interneurons from the ganglionic eminence, around the corticostriatal notch and into the neocortex are unknown. A number of factors have been shown to stimulate motogenic activity in neural and non-neural tissue. One of these molecules, hepatocyte growth factor/scatter factor (HGFISF) and its receptor MET have recently been shown to be important in the migration of cortical interneurons. Disruption of the normal expression of HGFiSF appears to result in undirected scattering of cells from the ganglionic eminence and in a significant reduction of interneurons in the cortex at the time of birth.