Cortical interneuron specification and diversification in the era of big data.

Inhibition in the mammalian cerebral cortex is mediated by a small population of highly diverse GABAergic interneurons. These largely local neurons are interspersed among excitatory projection neurons and exert pivotal regulation on the formation and function of cortical circuits. We are beginning to understand the extent of GABAergic neuron diversity and how this is generated and shaped during brain development in mice and humans. In this review, we summarise recent findings and discuss how new technologies are being used to further advance our knowledge. Understanding how inhibitory neurons are generated in the embryo is an essential pre-requisite of stem cell therapy, an evolving area of research, aimed at correcting human disorders that result in inhibitory dysfunction.

Rac1 and Rac3 GTPases and TPC2 are required for axonal outgrowth and migration of cortical interneurons.

Rho GTPases, among them Rac1 and Rac3, are major transducers of extracellular signals and are involved in multiple cellular processes. In cortical interneurons, the neurons that control the balance between excitation and inhibition of cortical circuits, Rac1 and Rac3 are essential for their development. Ablation of both leads to a severe reduction in the numbers of mature interneurons found in the murine cortex, which is partially due to abnormal cell cycle progression of interneuron precursors and defective formation of growth cones in young neurons. Here, we present new evidence that upon Rac1 and Rac3 ablation, centrosome, Golgi complex and lysosome positioning is significantly perturbed, thus affecting both interneuron migration and axon growth. Moreover, for the first time, we provide evidence of altered expression and localization of the two-pore channel 2 (TPC2) voltage-gated ion channel that mediates Ca2+ release. Pharmacological inhibition of TPC2 negatively affected axonal growth and migration of interneurons. Our data, taken together, suggest that TPC2 contributes to the severe phenotype in axon growth initiation, extension and interneuron migration in the absence of Rac1 and Rac3.

The role of selective SATB1 deletion in somatostatin expressing interneurons on endogenous network activity and the transition to epilepsy.

Somatostatin (SST) expressing interneurons are the second most abundant group of inhibitory neurons in the neocortex. They mainly target the apical dendrites of excitatory pyramidal cells and are implicated in feedforward and feedback inhibition. In the present study, we employ a conditional knockout mouse, in which the transcription factor Satb1 is selectively deleted in SST-expressing interneurons resulting to the reduction of their number across the somatosensory barrel field. Our goal was to investigate the effect of the reduced number of Satb1 mutant SST-interneurons on (i) the endogenous cortical network activity (spontaneously recurring Up/Down states), and (ii) the transition to epileptiform activity. By conducting LFP recordings in acute brain slices from young male and female mice, we demonstrate that mutant animals exhibit significant changes in network excitability, reflected in increased Up state occurrence, decreased Up state duration and higher levels of extracellular spiking activity. Epileptiform activity was induced through two distinct and widely used in vitro protocols: the low magnesium and the 4-Aminopyridine (4-AP) model. In the former, slices from mutant animals manifested shorter latency for the expression of stable seizure-like events. In contrast, when epilepsy was induced by 4-AP, no significant differences were reported. We conclude that normal SST-interneuron function has a significant role both in the regulation of the endogenous network activity, and in the transition to seizure-like discharges in a context-dependent manner.

MTG8 interacts with LHX6 to specify cortical interneuron subtype identity.

Cortical interneurons originating in the embryonic medial ganglionic eminence (MGE) diverge into a range of different subtypes found in the adult mouse cerebral cortex. The mechanisms underlying this divergence and the timing when subtype identity is set up remain unclear. We identify the highly conserved transcriptional co-factor MTG8 as being pivotal in the development of a large subset of MGE cortical interneurons that co-expresses Somatostatin (SST) and Neuropeptide Y (NPY). MTG8 interacts with the pan-MGE transcription factor LHX6 and together the two factors are sufficient to promote expression of critical cortical interneuron subtype identity genes. The SST-NPY cortical interneuron fate is initiated early, well before interneurons migrate into the cortex, demonstrating an early onset specification program. Our findings suggest that transcriptional co-factors and modifiers of generic lineage specification programs may hold the key to the emergence of cortical interneuron heterogeneity from the embryonic telencephalic germinal zones.

The developmental changes in intrinsic and synaptic properties of prefrontal neurons enhance local network activity from the second to the third postnatal weeks in mice.

The prefrontal cortex (PFC) is characterized by protracted maturation. The cellular mechanisms controlling the early development of prefrontal circuits are still largely unknown. Our study delineates the developmental cellular processes in the mouse medial PFC (mPFC) during the second and the third postnatal weeks and characterizes their contribution to the changes in network activity. We show that spontaneous inhibitory postsynaptic currents (sIPSC) are increased, whereas spontaneous excitatory postsynaptic currents (sEPSC) are reduced from the second to the third postnatal week. Drug application suggested that the increased sEPSC frequency in mPFC at postnatal day 10 (P10) is due to depolarizing γ-aminobutyric acid (GABA) type A receptor function. To further validate this, perforated patch-clamp recordings were obtained and the expression levels of K-Cl cotransporter 2 (KCC2) protein were examined. The reversal potential of IPSCs in response to current stimulation was significantly more depolarized at P10 than P20 while KCC2 expression is decreased. Moreover, the number of parvalbumin-expressing GABAergic interneurons increases and their intrinsic electrophysiological properties significantly mature in the mPFC from P10 to P20. Using computational modeling, we show that the developmental changes in synaptic and intrinsic properties of mPFC neurons contribute to the enhanced network activity in the juvenile compared with neonatal mPFC.

The development of MGE-derived cortical interneurons: An Lhx6 tale.

The cerebral cortex contains two main neuronal cell populations: the excitatory pyramidal neurons and the inhibitory interneurons, which constitute 20-30% of all cortical neurons. Cortical interneurons are characterized by a remarkable morphological, molecular and functional diversity. A swathe of research activity over the last 20 years has sought to determine how cortical interneurons acquire their mature cellular and functional features, and has identified a number of transcription factors that function at different stages of interneuron development. Here, we review all current knowledge concerning the multiple functions of the "master regulator" - LIM-Homeodomain transcription factor Lhx6 - a gene expressed in the medial ganglionic eminence of the basal telencephalon that controls the development of somatostatin and parvalbumin expressing interneurons.

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of γ-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences - MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Homeostatic Regulation of Interneuron Apoptosis During Cortical Development.

The mammalian cortex consists of two main neuronal types: the principal excitatory pyramidal neurons (PNs) and the inhibitory interneurons (INs). The interplay between these two neuronal populations - which drive excitation and inhibition (E/I balance), respectively - is crucial for controlling the overall activity in the brain. A number of neurological and psychiatric disorders have been associated with changes in E/I balance. It is not surprising, therefore, that neural networks employ several different mechanisms to maintain their firing rates at a stable level, collectively referred as homeostatic forms of plasticity. Here, we share our views on how the size of IN populations may provide an early homeostatic checkpoint for controlling brain activity. In a recent paper published in Cell Reports, we demonstrate that the extent of IN apoptosis during a critical early postnatal period is plastic, cell type specific, and can be reduced in a cell-autonomous manner by acute increases in neuronal activity. We propose that a critical interplay between the physiological state of the network and its cellular units fine-tunes the size of IN populations with the aim of stabilizing network activity.

Modulation of Apoptosis Controls Inhibitory Interneuron Number in the Cortex.

Cortical networks are composed of excitatory projection neurons and inhibitory interneurons. Finding the right balance between the two is important for controlling overall cortical excitation and network dynamics. However, it is unclear how the correct number of cortical interneurons (CIs) is established in the mammalian forebrain. CIs are generated in excess from basal forebrain progenitors, and their final numbers are adjusted via an intrinsically determined program of apoptosis that takes place during an early postnatal window. Here, we provide evidence that the extent of CI apoptosis during this critical period is plastic and cell-type specific and can be reduced in a cell-autonomous manner by acute increases in neuronal activity. We propose that the physiological state of the emerging neural network controls the activity levels of local CIs to modulate their numbers in a homeostatic manner.

Corrigendum: Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

This corrects the article DOI: 10.1038/nature23663.

Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep-wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.

Rac-GTPases Regulate Microtubule Stability and Axon Growth of Cortical GABAergic Interneurons.

Cortical interneurons are characterized by extraordinary functional and morphological diversity. Although tremendous progress has been made in uncovering molecular and cellular mechanisms implicated in interneuron generation and function, several questions still remain open. Rho-GTPases have been implicated as intracellular mediators of numerous developmental processes such as cytoskeleton organization, vesicle trafficking, transcription, cell cycle progression, and apoptosis. Specifically in cortical interneurons, we have recently shown a cell-autonomous and stage-specific requirement for Rac1 activity within proliferating interneuron precursors. Conditional ablation of Rac1 in the medial ganglionic eminence leads to a 50% reduction of GABAergic interneurons in the postnatal cortex. Here we examine the additional role of Rac3 by analyzing Rac1/Rac3 double-mutant mice. We show that in the absence of both Rac proteins, the embryonic migration of medial ganglionic eminence-derived interneurons is further impaired. Postnatally, double-mutant mice display a dramatic loss of cortical interneurons. In addition, Rac1/Rac3-deficient interneurons show gross cytoskeletal defects in vitro, with the length of their leading processes significantly reduced and a clear multipolar morphology. We propose that in the absence of Rac1/Rac3, cortical interneurons fail to migrate tangentially towards the pallium due to defects in actin and microtubule cytoskeletal dynamics.

The LIM homeodomain protein Lhx6 regulates maturation of interneurons and network excitability in the mammalian cortex.

Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst(+) interneurons, but relatively minor and localized changes in Pva(+) interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst(+) interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

Maturation-promoting activity of SATB1 in MGE-derived cortical interneurons.

The generation of cortical interneuron subtypes is controlled by genetic programs that are activated in the ventral forebrain and unfold during the prolonged period of inhibitory neuron development. The LIM-homeodomain protein LHX6 is critical for the development of all cortical interneurons originating in the medial ganglionic eminence, but the molecular mechanisms that operate downstream of LHX6 to control the terminal differentiation of somatostatin- and parvalbumin-expressing interneurons within the cortex remain unknown. Here, we provide evidence that the nuclear matrix and genome organizer protein SATB1 is induced by neuronal activity and functions downstream of Lhx6 to control the transition of tangentially migrating immature interneurons into the terminally differentiated Somatostatin (SST)-expressing subtype. Our experiments provide a molecular framework for understanding the genetic and epigenetic mechanisms by which specified but immature cortical interneurons acquire the subtype-defining molecular and morphophysiological characteristics that allow them to integrate and function within cortical circuits.

The cell-intrinsic requirement of Sox6 for cortical interneuron development.

We describe the role of Sox6 in cortical interneuron development, from a cellular to a behavioral level. We identify Sox6 as a protein expressed continuously within MGE-derived cortical interneurons from postmitotic progenitor stages into adulthood. Both its expression pattern and null phenotype suggests that Sox6 gene function is closely linked to that of Lhx6. In both Lhx6 and Sox6 null animals, the expression of PV and SST and the position of both basket and Martinotti neurons are abnormal. We find that Sox6 functions downstream of Lhx6. Electrophysiological analysis of Sox6 mutant cortical interneurons revealed that basket cells, even when mispositioned, retain characteristic but immature fast-spiking physiological features. Our data suggest that Sox6 is not required for the specification of MGE-derived cortical interneurons. It is, however, necessary for their normal positioning and maturation. As a consequence, the specific removal of Sox6 from this population results in a severe epileptic encephalopathy.

The LIM homeodomain transcription factors Lhx6 and Lhx7 are key regulators of mammalian dentition.

Genes encoding LIM homeodomain transcription factors are implicated in cell type specification and differentiation during embryogenesis. Two closely related members of this family, Lhx6 and Lhx7, are expressed in the ectomesenchyme of the maxillary and mandibular processes and have been suggested to control patterning of the first branchial arch (BA1) and odontogenesis. However, mice homozygous for single mutations either have no cranial defects (Lhx6) or show only cleft palate (Lhx7). To reveal the potential redundant activities of Lhx6 and Lhx7 in cranial morphogenesis, we generated mice with all combinations of wild-type and mutant alleles. Double homozygous mice have characteristic defects of the cranial skeleton and die shortly after birth, most likely because of cleft palate. In addition, Lhx6/7 deficient embryos lack molar teeth. The absence of molars in double mutants is not due to patterning defects of BA1 but results from failure of specification of the molar mesenchyme. Despite molar agenesis, Lhx6/7-deficient animals have normal incisors which, in the maxilla, are flanked by a supernumerary pair of incisor-like teeth. Our experiments demonstrate that the redundant activities of the LIM homeodomain proteins Lhx6 and Lhx7 are critical for craniofacial development and patterning of mammalian dentition.

Lhx6 activity is required for the normal migration and specification of cortical interneuron subtypes.

The cerebral cortex contains two main neuronal cell populations, the excitatory glutamatergic (pyramidal) neurons and the inhibitory interneurons, which synthesize GABA and constitute 20-30% of all cortical neurons. In contrast to the mostly homogeneous population of projection neurons, cortical interneurons are characterized by remarkable morphological, molecular, and functional diversity. Among the markers that have been used to classify cortical interneurons are the calcium-binding proteins parvalbumin and calretinin and the neuropeptide somatostatin, which in rodents identify mostly nonoverlapping interneuron subpopulations. Pyramidal neurons are born during embryogenesis in the ventricular zone of the dorsal telencephalon, whereas cortical interneurons are generated in the subpallium and reach the cortex by tangential migration. On completion of tangential migration, cortical interneurons switch to a radial mode of migration and enter the cortical plate. Although the mechanisms that control the generation of interneuron diversity are currently unknown, it has been proposed that their site of origin in the ventral forebrain determines their specification into defined neurochemical subgroups. Here, we show that Lhx6, a gene induced in the medial ganglionic eminence and maintained in parvalbumin- and somatostatin-positive interneurons, is required for the specification of these neuronal subtypes in the neocortex and the hippocampus. We also show that Lhx6 activity is required for the normal tangential and radial migration of GABAergic interneurons in the cortex.

Genomic organization and functional characterization of the alcohol dehydrogenase locus of Ceratitis capitata (Medfly).

Approximately 30 kb of genomic DNA enclosing the Adh locus from the medfly, Ceratitis capitata have been cloned and about 15 kb has been structurally and functionally characterized. The locus consists of two genes, Adh-1 and Adh-2, separated by an intergenic region, which is polymorphic in size ranging from approximately 6.4 kb to 8.1 kb. Both genes consist of three exons and two introns. The introns are below 200 bp in size, except the 1st intron of Adh-1, which is unexpectedly long, variable in size and contains a deleted mariner-like element (postdoc). The two genes are transcribed in different orientations. The Adh-2 gene shows the typical pattern of transcription seen in the homologous genes of Drosophilidae presenting high levels of expression in the fat body, gut and ovaries. The Adh-1 gene is only expressed in the body muscle tissues of embryos, larvae and adult flies, raising the question of what its biological function may be. A DNA fragment containing bases -102 to -1666 relative to the first base of the initiating ATG of Adh-1 is sufficient to drive the expression of a reporter gene in body muscles of Drosophila melanogaster embryos, larvae and adult flies. The study provides further insights into the evolution of the Adh genes of higher diptera.

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.

Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1.

Little is known about the molecular mechanisms and intrinsic factors that are responsible for the emergence of neuronal subtype identity. Several transcription factors that are expressed mainly in precursors of the ventral telencephalon have been shown to control neuronal specification, but it has been unclear whether subtype identity is also specified in these precursors, or if this happens in postmitotic neurons, and whether it involves the same or different factors. SOX1, an HMG box transcription factor, is expressed widely in neural precursors along with the two other SOXB1 subfamily members, SOX2 and SOX3, and all three have been implicated in neurogenesis. SOX1 is also uniquely expressed at a high level in the majority of telencephalic neurons that constitute the ventral striatum (VS). These neurons are missing in Sox1-null mutant mice. In the present study, we have addressed the requirement for SOX1 at a cellular level, revealing both the nature and timing of the defect. By generating a novel Sox1-null allele expressing beta-galactosidase, we found that the VS precursors and their early neuronal differentiation are unaffected in the absence of SOX1, but the prospective neurons fail to migrate to their appropriate position. Furthermore, the migration of non-Sox1-expressing VS neurons (such as those expressing Pax6) was also affected in the absence of SOX1, suggesting that Sox1-expressing neurons play a role in structuring the area of the VS. To test whether SOX1 is required in postmitotic cells for the emergence of VS neuronal identity, we generated mice in which Sox1 expression was directed to all ventral telencephalic precursors, but to only a very few VS neurons. These mice again lacked most of the VS, indicating that SOX1 expression in precursors is not sufficient for VS development. Conversely, the few neurons in which Sox1 expression was maintained were able to migrate to the VS. In conclusion, Sox1 expression in precursors is not sufficient for VS neuronal identity and migration, but this is accomplished in postmitotic cells, which require the continued presence of SOX1. Our data also suggest that other SOXB1 members showing expression in specific neuronal populations are likely to play continuous roles from the establishment of precursors to their final differentiation.