Viral-mediated Labeling and Transplantation of Medial Ganglionic Eminence (MGE) Cells for In Vivo Studies

GABAergic cortical interneurons, derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE), are functionally and morphologically diverse. Inroads have been made in understanding the roles of distinct cortical interneuron subgroups, however, there are still many mechanisms to be worked out that may contribute to the development and maturation of different types of GABAergic cells. Moreover, altered GABAergic signaling may contribute to phenotypes of autism, schizophrenia and epilepsy. Specific Cre-driver lines have begun to parcel out the functions of unique interneuron subgroups. Despite the advances in mouse models, it is often difficult to efficiently study GABAergic cortical interneuron progenitors with molecular approaches in vivo. One important technique used to study the cell autonomous programming of these cells is transplantation of MGE cells into host cortices. These transplanted cells migrate extensively, differentiate, and functionally integrate. In addition, MGE cells can be efficiently transduced with lentivirus immediately prior to transplantation, allowing for a multitude of molecular approaches. Here we detail a protocol to efficiently transduce MGE cells before transplantation for in vivo analysis, using available Cre-driver lines and Cre-dependent expression vectors. This approach is advantageous because it combines precise genetic manipulation with the ability of these cells to disperse after transplantation, permitting greater cell-type specific resolution in vivo.     

Ephrins guide migrating cortical interneurons in the basal telencephalon

Cortical interneurons are born in the proliferative zones of the ganglionic eminences in the subpallium and migrate to the developing cortex along well-defined tangential routes. The mechanisms regulating interneuron migration are not completely understood. Here we examine the role of class-A members of the Eph/ephrin system in directing the migration of interneurons. In situ hybridizations demonstrated that ephrin A3 is expressed in the developing striatum, an area that is strictly avoided by migrating cortical interneurons in vivo, which express the EphA4 receptor. We then examined interneuron migration in grafting experiments, where explants of the medial ganglionic eminence (MGE) from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. After blocking ephrin-A ligands, many interneurons invaded the striatal anlage. Moreover, stripe assay experiments revealed that ephrin-A3 acts as a repellent cue for neurons from the medial ganglionic eminence. Downregulation of the EphA4 receptor via siRNA transfection reduced the repulsive effect of ephrin-A3, indicating that EphA4 mediates at least in part the repulsive effect of ephrin A3 on these cells. Together, these results suggest that ephrin-A3 acts as a repulsive cue that restricts cortical interneurons from entering inappropriate regions and thus contributes to define the migratory route of cortical interneurons.     

Ephrins guide migrating cortical interneurons in the basal telencephalon

Cortical interneurons are born in the proliferative zones of the ganglionic eminences in the subpallium and migrate to the developing cortex along well-defined tangential routes. The mechanisms regulating interneuron migration are not completely understood. Here we examine the role of class-A members of the Eph/ephrin system in directing the migration of interneurons. In situ hybridizations demonstrated that ephrin-A3 is expressed in the developing striatum, an area that is strictly avoided by migrating cortical interneurons in vivo, which express the EphA4 receptor. We then examined interneuron migration in grafting experiments, where explants of the medial ganglionic eminence (MGE) from enhanced green fluorescent protein-expressing transgenic mice were homotopically grafted into host slices from wild-type littermate embryos. After blocking ephrin-A ligands, many interneurons invaded the striatal anlage. Moreover, stripe assay experiments revealed that ephrin-A3 acts as a repellent cue for neurons from the medial ganglionic eminence. Downregulation of the EphA4 receptor via siRNA transfection reduced the repulsive effect of ephrin-A3, indicating that EphA4 mediates at least in part the repulsive effect of ephrin-A3 on these cells. Together, these results suggest that ephrin-A3 acts as a repulsive cue that restricts cortical interneurons from entering inappropriate regions and thus contributes to define the migratory route of cortical interneurons.Key words: interneuron migration, cortical development, neuronal guidance cues, ephrin, Eph receptors, organotypic slice cultures     

RGMa Regulates Cortical Interneuron Migration and Differentiation

The etiology of neuropsychiatric disorders, including schizophrenia and autism, has been linked to a failure to establish the intricate neural network comprising excitatory pyramidal and inhibitory interneurons during neocortex development. A large proportion of cortical inhibitory interneurons originate in the medial ganglionic eminence (MGE) of the ventral telencephalon and then migrate through the ventral subventricular zone, across the corticostriatal junction, into the embryonic cortex. Successful navigation of newborn interneurons through the complex environment of the ventral telencephalon is governed by spatiotemporally restricted deployment of both chemorepulsive and chemoattractive guidance cues which work in concert to create a migratory corridor. Despite the expanding list of interneuron guidance cues, cues responsible for preventing interneurons from re-entering the ventricular zone of the ganglionic eminences have not been well characterized. Here we provide evidence that the chemorepulsive axon guidance cue, RGMa (Repulsive Guidance Molecule a), may fulfill this function. The ventricular zone restricted expression of RGMa in the ganglionic eminences and the presence of its receptor, Neogenin, in the ventricular zone and on newborn and maturing MGE-derived interneurons implicates RGMa-Neogenin interactions in interneuron differentiation and migration. Using an in vitro approach, we show that RGMa promotes interneuron differentiation by potentiating neurite outgrowth. In addition, using in vitro explant and migration assays, we provide evidence that RGMa is a repulsive guidance cue for newborn interneurons migrating out of the ganglionic eminence ventricular zone. Intriguingly, the alternative Neogenin ligand, Netrin-1, had no effect on migration. However, we observed complete abrogation of RGMa-induced chemorepulsion when newborn interneurons were simultaneously exposed to RGMa and Netrin-1 gradients, suggesting a novel mechanism for the tight regulation of RGMa-guided interneuron migration. We propose that during peak neurogenesis, repulsive RGMa-Neogenin interactions drive interneurons into the migratory corridor and prevent re-entry into the ventricular zone of the ganglionic eminences.     

In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain

Recent studies suggest that neurons born in the developing basal forebrain migrate long distances perpendicularly to radial glia and that many of these cells reach the developing neocortex. This form of tangential migration, however, has not been demonstrated in vivo, and the sites of origin, pathways of migration and final destinations of these neurons in the postnatal brain are not fully understood. Using ultrasound-guided transplantation in utero, we have mapped the migratory pathways and fates of cells born in the lateral and medial ganglionic eminences (LGE and MGE) in 13.5-day-old mouse embryos. We demonstrate that LGE and MGE cells migrate along different routes to populate distinct regions in the developing brain. We show that LGE cells migrate ventrally and anteriorly, and give rise to the projecting medium spiny neurons in the striatum, nucleus accumbens and olfactory tubercle, and to granule and periglomerular cells in the olfactory bulb. By contrast, we show that the MGE is a major source of neurons migrating dorsally and invading the developing neocortex. MGE cells migrate into the neocortex via the neocortical subventricular zone and differentiate into the transient subpial granule neurons in the marginal zone and into a stable population of GABA-, parvalbumin- or somatostatin-expressing interneurons throughout the cortical plate.     

Distinct cortical migrations from the medial and lateral ganglionic eminences

Recent evidence suggests that projection neurons and interneurons of the cerebral cortex are generally derived from distinct proliferative zones. Cortical projection neurons originate from the cortical ventricular zone (VZ), and then migrate radially into the cortical mantle, whereas most cortical interneurons originate from the basal telencephalon and migrate tangentially into the developing cortex. Previous studies using methods that label both proliferative and postmitotic cells have found that cortical interneurons migrate from two major subdivisions of the developing basal telencephalon: the medial and lateral ganglionic eminences (MGE and LGE). Since these studies labeled cells by methods that do not distinguish between the proliferating cells and those that may have originated elsewhere, we have studied the contribution of the MGE and LGE to cortical interneurons using fate mapping and genetic methods. Transplantation of BrdU-labeled MGE or LGE neuroepithelium into the basal telencephalon of unlabeled telencephalic slices enabled us to follow the fate of neurons derived from each of these primordia. We have determined that early in neurogenesis GABA-expressing cells from the MGE tangentially migrate into the cerebral cortex, primarily via the intermediate zone, whereas cells from the LGE do not. Later in neurogenesis, LGE-derived cells also migrate into the cortex, although this migration occurs primarily through the subventricular zone. Some of these LGE-derived cells invade the cortical plate and express GABA, while others remain within the cortical proliferative zone and appear to become mitotically active late in gestation. In addition, by comparing the phenotypes of mouse mutants with differential effects on MGE and LGE migration, we provide evidence that the MGE and LGE may give rise to different subtypes of cortical interneurons.     

A spatial bias for the origins of interneuron subgroups within the medial ganglionic eminence

Although it is well established that the ventral telencephalon is the primary source of GABAergic cortical interneurons in rodents, little is known about the specification of specific interneuron subtypes. It is also unclear whether the potential to achieve a given fate is established at their place of origin or by signals received during their migration to or during their maturation within the cerebral cortex. Using both in vivo and in vitro transplantation techniques, we find that two major interneuron subgroups have largely distinct origins within the MGE. Somatostatin (SST)-expressing interneurons are primarily generated within the dorsal MGE, while parvalbumin (PV)-expressing interneurons primarily originate from the ventral MGE. In addition, we show that significant heterogeneity exists between gene expression patterns in the dorsal and ventral MGE. These results suggest that, like the spinal cord, neuronal fate determination in the ventral telencephalon is largely the result of spatially segregated, molecularly distinct microdomains arranged on the dorsal-ventral axis.     

Two specific populations of GABAergic neurons originating from the medial and the caudal ganglionic eminences aid in proper navigation of callosal axons

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE‐ and CGE‐derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT‐PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE‐ and CGE‐derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 647–672, 2013     

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.     

Immediate effects of maternal separation on the development of interneurons derived from medial ganglionic eminence in the neonatal mouse hippocampus

Aversive experiences, including maternal separation (MS), have been known as a risk for abnormal hippocampus development. Given that impairment of GABA inhibitory system is known as one of the common features of the abnormal hippocampal development induced by MS, we examined whether the MS on 4‐day‐old (P4) mice for 24 hr abolishes the interneuron development. We observed that the MS reduced the volume of dorsal hippocampus on P14 as long‐term effects. In addition, the MS decreased the number of parvalbumin (PV)‐positive interneuron on P14 and P28 in the dorsal hippocampus. We further examined the immediate effects of MS by measuring the percentage of glutamic acid decarboxylase (GAD) 67‐positive interneurons among the immature interneurons derived from medial ganglionic eminence (MGE) progenitors marked in nkx2.1cre;β‐geo EGFP mice. During normal development from P4 to P5, the percentage of GAD67‐positive interneurons among the MGE‐derived interneurons in the dorsal hippocampus was significantly increased from 42.29% to 70.73% in the stratum pyramidale of the CA1 and increased from 46.4% to 56.99% in the stratum pyramidale of the CA2/3 region. However, the increase was not observed on P5 among the mice treated with the MS. These results suggest that the maturation of interneurons was suppressed by the MS. The suppressed maturation of interneurons may be one of the causes of the reduced volume of the hippocampus and PV+ interneurons observed in the hippocampus on P14 and P28 as a consequence of the MS during neonatal stage.     

Interneurons in the developing human neocortex

Cortical interneurons play a crucial role in the functioning of cortical microcircuitry as they provide inhibitory input to projection (pyramidal) neurons. Despite their involvement in various neurological and psychiatric disorders, our knowledge about their development in human cerebral cortex is still incomplete. Here we demonstrate that at the beginning of corticogenesis, at embryonic 5 gestation weeks (gw, Carnegie stage 16) in human, early neurons could be labeled with calretinin, calbindin, and GABA antibodies. These immunolabeled cells show a gradient from the ganglionic eminences (GE) toward the neocortex, suggesting that GE is a well conserved source of early born cortical interneurons from rodents to human. At mid-term (20 gw), however, a subset of calretinin(+) cells proliferates in the cortical subventricular zone (SVZ), suggesting a second set of interneuron progenitors that have neocortical origin. Neuropeptide Y, somatostatin, or parvalbumin cells are sparse in mid-term cerebral cortex. In addition to the early source of cortical interneurons in the GE and later in the neocortical SVZ, other regions, such as the subpial granular layer, may also contribute to the population of human cortical interneurons. In conclusion, our findings from cryosections and previous in vitro results suggest that cortical interneuron progenitor population is more complex in humans relative to rodents. The increased complexity of progenitors is probably evolutionary adaptation necessary for development of the higher brain functions characteristic to humans.     

Characterization of a new Gsx2‐cre line in the developing mouse telencephalon

In this study, we generated a transgenic mouse line driving Cre and EGFP expression with two putative cis‐regulatory modules (CRMs) (i.e., hs687 and hs678) upstream of the homeobox gene Gsx2 (formerly Gsh2), a critical gene for establishing lateral ganglionic eminence (LGE) identity. The combination of these two CRMs drives transgene expression within the endogenous Gsx2 expression domains along the anterior–posterior neuraxis. By crossing this transgenic line with the Rosa^tdTomato (Ai14) reporter mouse line, we observed a unique recombination pattern in the lateral ventral telencephalon, namely the LGE and the dorsal half of the medial GE (MGE), but not in the septum. We found robust recombination in many cell types derived from these embryonic regions, including olfactory bulb and amygdala interneurons and striatal projection neurons from the LGE, as well as cortical interneurons from the MGE and caudal GE (CGE). In summary, this transgenic mouse line represents a new tool for genetic manipulation in the LGE/CGE and the dorsal half of MGE.     

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.     

Arx acts as a regional key selector gene in the ventral telencephalon mainly through its transcriptional repression activity

The homeobox-containing gene Arx is expressed during ventral telencephalon development and required for correct GABAergic interneuron tangential migration from the ganglionic eminences to the olfactory bulbs, cerebral cortex and striatum. Its human ortholog is associated with a variety of neurological clinical manifestations whose symptoms are compatible with the loss of cortical interneurons and altered basal ganglia-related activities. Herein, we report the identification of a number of genes whose expression is consistently altered in Arx mutant ganglionic eminences. Our analyses revealed a striking ectopic expression in the ganglionic eminences of several of these genes normally at most marginally expressed in the ventral telencephalon. Among them, Ebf3 was functionally analyzed. Thus, its ectopic expression in ventral telencephalon was found to prevent neuronal tangential migration. Further, we showed that Arx is sufficient to repress Ebf3 endogenous expression and that its silencing in Arx mutant tissues partially rescues tangential cell movement. Together, these data provide new insights into the molecular pathways regulated by Arx during telencephalon development.     

Striatal interneurons in dissociated cell culture

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague–Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks.