Regional Cellular Environment Shapes Phenotypic Variations of Hippocampal and Neocortical Chandelier Cells.

Different cortical regions processing distinct information, such as the hippocampus and the neocortex, share common cellular components and circuit motifs but form unique networks by modifying these cardinal units. Cortical circuits include diverse types of GABAergic interneurons (INs) that shape activity of excitatory principal neurons (PNs). Canonical IN types conserved across distinct cortical regions have been defined by their morphological, electrophysiological, and neurochemical properties. However, it remains largely unknown whether canonical IN types undergo specific modifications in distinct cortical regions and display "regional variants." It is also poorly understood whether such phenotypic variations are shaped by early specification or regional cellular environment. The chandelier cell (ChC) is a highly stereotyped IN type that innervates axon initial segments of PNs and thus serves as a good model with which to address this issue. Here, we show that Cadherin-6 (Cdh6), a homophilic cell adhesion molecule, is a reliable marker of ChCs and Cdh6-CreER mice (both sexes) provide genetic access to hippocampal ChCs (h-ChCs). We demonstrate that, compared with neocortical ChCs (nc-ChCs), h-ChCs cover twice as much area and innervate twice as many PNs. Interestingly, a subclass of h-ChCs exhibits calretinin (CR) expression, which is not found in nc-ChCs. Furthermore, we find that h-ChCs appear to be born earlier than nc-ChCs. Surprisingly, despite the difference in temporal origins, ChCs display host-region-dependent axonal/synaptic organization and CR expression when transplanted heterotopically. These results suggest that local cellular environment plays a critical role in shaping terminal phenotypes of regional IN variants in the hippocampus and the neocortex.SIGNIFICANCE STATEMENT Canonical interneuron (IN) types conserved across distinct cortical regions such as the hippocampus and the neocortex are defined by morphology, physiology, and gene expression. However, it remains unknown whether they display phenotypic variations in different cortical regions. In addition, it is unclear whether terminal phenotypes of regional IN variants belonging to a canonical IN type are determined intrinsically or extrinsically. Our results provide evidence of striking differences in axonal/synaptic organization and calretinin expression between hippocampal chandelier cells (ChCs) and neocortical ChCs. They also reveal that local cellular environment in distinct cortical regions regulates these terminal phenotypes. Therefore, our study suggests that local cortical environment shapes the phenotypes of regional IN variants, which may be required for unique circuit operations in distinct cortical regions.

EphA/ephrin A reverse signaling promotes the migration of cortical interneurons from the medial ganglionic eminence.

Inhibitory interneurons control the flow of information and synchronization in the cerebral cortex at the circuit level. During embryonic development, multiple subtypes of cortical interneurons are generated in different regions of the ventral telencephalon, such as the medial and caudal ganglionic eminence (MGE and CGE), as well as the preoptic area (POA). These neurons then migrate over long distances towards their cortical target areas. Diverse families of diffusible and cell-bound signaling molecules, including the Eph/ephrin system, regulate and orchestrate interneuron migration. Ephrin A3 and A5, for instance, are expressed at the borders of the pathway of MGE-derived interneurons and prevent these cells from entering inappropriate regions via EphA4 forward signaling. We found that MGE-derived interneurons, in addition to EphA4, also express ephrin A and B ligands, suggesting Eph/ephrin forward and reverse signaling in the same cell. In vitro and in vivo approaches showed that EphA4-induced reverse signaling in MGE-derived interneurons promotes their migration and that this effect is mediated by ephrin A2 ligands. In EphA4 mutant mice, as well as after ephrin A2 knockdown using in utero electroporation, we found delayed interneuron migration at embryonic stages. Thus, besides functions in guiding MGE-derived interneurons to the cortex through forward signaling, here we describe a novel role of the ephrins in driving these neurons to their target via reverse signaling.

Disrupted-in-Schizophrenia 1 (DISC1) is necessary for the correct migration of cortical interneurons.

Disrupted-in-Schizophrenia 1 (DISC1) is a prominent susceptibility gene for major psychiatric disorders. Previous work indicated that DISC1 plays an important role during neuronal proliferation and differentiation in the cerebral cortex and that it affects the positioning of radial migrating pyramidal neurons. Here we show that in mice, DISC1 is necessary for the migration of the cortical interneurons generated in the medial ganglionic eminence (MGE). RT-PCR, in situ hybridizations, and immunocytochemical data revealed expression of DISC1 transcripts and protein in MGE-derived cells. To study the possible functional role of DISC1 during tangential migration, we performed in utero and ex utero electroporation to suppress DISC1 in the MGE in vivo and in vitro. Results indicate that after DISC1 knockdown, the proportion of tangentially migrating MGE neurons that reached their cortical target was strongly reduced. In addition, there were profound alterations in the morphology of DISC1-deficient neurons, which exhibited longer and less branched leading processes than control cells. These findings provide a possible link between clinical studies reporting alterations of cortical interneurons in schizophrenic patients and the current notion of schizophrenia as a neurodevelopmental disorder.

An adaptive behavioral control motif mediated by cortical axo-axonic inhibition.

Genetically defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We found that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, with tuning refined through suppression of irrelevant neuronal activity. ChCs contribute to learning-dependent refinements by providing selective inhibitory control over individual pyramidal neurons rather than global suppression. Quantitative analysis of structural plasticity across axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a targeted cell-specific manner.

An adaptive behavioral control motif mediated by cortical axo-axonic inhibition.

Neural circuits are reorganized with specificity during learning. Genetically-defined subgroups of inhibitory interneurons are thought to play distinct roles in learning, but heterogeneity within these subgroups has limited our understanding of the scope and nature of their specific contributions to learning. Here we reveal that the chandelier cell (ChC), an interneuron type that specializes in inhibiting the axon-initial segment (AIS) of pyramidal neurons, establishes cortical microcircuits for organizing neural coding through selective axo-axonic synaptic plasticity. We find that organized motor control is mediated by enhanced population coding of direction-tuned premotor neurons, whose tuning is refined through suppression of irrelevant neuronal activity. ChCs are required for learning-dependent refinements via providing selective inhibitory control over pyramidal neurons rather than global suppression. Quantitative analysis on structural plasticity of axo-axonic synapses revealed that ChCs redistributed inhibitory weights to individual pyramidal neurons during learning. These results demonstrate an adaptive logic of the inhibitory circuit motif responsible for organizing distributed neural representations. Thus, ChCs permit efficient cortical computation in a target cell specific manner, which highlights the significance of interneuron diversity.

Bidirectional ephrinB3/EphA4 signaling mediates the segregation of medial ganglionic eminence- and preoptic area-derived interneurons in the deep and superficial migratory stream.

The integration of interneuron subtypes into specific microcircuits is essential for proper cortical function. Understanding to what extent interneuron diversity is regulated and maintained during development might help to reveal the principles that govern their role as synchronizing elements as well as causes for dysfunction. Particular interneuron subtypes are generated in a temporally regulated manner in the medial ganglionic eminence (MGE), the caudal ganglionic eminence, and the preoptic area (POA) of the basal telencephalon. Long-range tangential migration from their site of origin to cortical targets is orchestrated by a variety of attractive, repulsive, membrane-bound, and secreted signaling molecules, to establish the critical balance of inhibition and excitation. It remains unknown whether interneurons deriving from distinct domains are predetermined to migrate in particular routes and whether this process underlies cell type-specific regulation. We found that POA- and MGE-derived cortical interneurons migrate within spatially segregated corridors. EphrinB3, expressed in POA-derived interneurons traversing the superficial route, acts as a repellent signal for deeply migrating interneurons born in the MGE, which is mediated by EphA4 forward signaling. In contrast, EphA4 induces repulsive ephrinB3 reverse signaling in interneurons generated in the POA, restricting this population to the superficial path. Perturbation of this bidirectional ephrinB3/EphA4 signaling in vitro and in vivo leads to a partial intermingling of cells in these segregated migratory pathways. Thus, we conclude that cell contact-mediated bidirectional ephrinB3/EphA4 signaling mediates the sorting of MGE- and POA-derived interneurons in the deep and superficial migratory stream.

GABA through the ages: regulation of cortical function and plasticity by inhibitory interneurons.

Inhibitory interneurons comprise only about 20% of cortical neurons and thus constitute a clear minority compared to the vast number of excitatory projection neurons. They are, however, an influential minority with important roles in cortical maturation, function, and plasticity. In this paper, we will highlight the functional importance of cortical inhibition throughout brain development, starting with the embryonal formation of the cortex, proceeding by the regulation of sensory cortical plasticity in adulthood, and finishing with the GABA involvement in sensory information processing in old age.

Neuromodulatory control of inhibitory network arborization in the developing postnatal neocortex.

Interregional neuronal communication is pivotal to instructing and adjusting cortical circuit assembly. Subcortical neuromodulatory systems project long-range axons to the cortex and affect cortical processing. However, their roles and signaling mechanisms in cortical wiring remain poorly understood. Here, we explored whether and how the cholinergic system regulates inhibitory axonal ramification of neocortical chandelier cells (ChCs), which control spike generation by innervating axon initial segments of pyramidal neurons. We found that acetylcholine (ACh) signaling through nicotinic ACh receptors (nAChRs) and downstream T-type voltage-dependent calcium (Ca2+) channels cell-autonomously controls axonal arborization in developing ChCs through regulating filopodia initiation. This signaling axis shapes the basal Ca2+ level range in varicosities where filopodia originate. Furthermore, the normal development of ChC axonal arbors requires proper levels of activity in subcortical cholinergic neurons. Thus, the cholinergic system regulates inhibitory network arborization in the developing neocortex and may tune cortical circuit properties depending on early-life experiences.

Chondroitin sulfate acts in concert with semaphorin 3A to guide tangential migration of cortical interneurons in the ventral telencephalon.

Chondroitin sulfate (CS) carrying proteoglycans (PGs) are widely expressed in the nervous system, and there is increasing evidence that they regulate developmental mechanisms like neurite outgrowth, axonal guidance and neuronal migration. Moreover, they can also act indirectly by organizing and/or modulating growth factors and guidance molecules. We found that chondroitin-4-sulfate is coexpressed with semaphorin 3A (Sema 3A) in the striatal mantle zone (SMZ), a nontarget region of neuropilin (Nrp)-1-expressing cortical interneurons flanking their migratory route in the subpallium. Using in vitro assays, we showed that CS PGs exert a repulsive effect on cortical interneurons, independently of Sema 3A, due to the CS side chains. We further showed that extracellular Sema 3A binds to CS. Disrupting Sema 3A-Nrp-1 signaling led migrating medial ganglionic eminence neurons to inappropriately invade the SMZ and even more so after removal of the CS side chains. Moreover, we found that soluble Sema 3A enhances the CS-induced repulsion in vitro. We concluded that CS acts as a repellent for cortical interneurons and that, in addition, CS restricts secreted Sema 3A within SMZ. Thus, both molecules act in concert to repel cortical interneurons from the SMZ during tangential migration toward the cerebral cortex.

IgSF11 homophilic adhesion proteins promote layer-specific synaptic assembly of the cortical interneuron subtype.

The most prominent structural hallmark of the mammalian neocortical circuitry is the layer-based organization of specific cell types and synaptic inputs. Accordingly, cortical inhibitory interneurons (INs), which shape local network activity, exhibit subtype-specific laminar specificity of synaptic outputs. However, the underlying molecular mechanisms remain unknown. Here, we demonstrate that Immunoglobulin Superfamily member 11 (IgSF11) homophilic adhesion proteins are preferentially expressed in one of the most distinctive IN subtypes, namely, chandelier cells (ChCs) that specifically innervate axon initial segments of pyramidal neurons (PNs), and their synaptic laminar target. Loss-of-function experiments in either ChCs or postsynaptic cells revealed that IgSF11 is required for ChC synaptic development in the target layer. While overexpression of IgSF11 in ChCs enlarges ChC presynaptic boutons, expressing IgSF11 in nontarget layers induces ectopic ChC synapses. These findings provide evidence that synapse-promoting adhesion proteins, highly localized to synaptic partners, determine the layer-specific synaptic connectivity of the cortical IN subtype.

In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected with CRISPR/Cas9.

CRISPR/Cas-based technologies have revolutionized genetic approaches to addressing a wide range of neurobiological questions. The ability of CRISPR/Cas to introduce mutations into target genes allows us to perform in vivo loss-of-function experiments without generating genetically engineered mice. However, the lack of a reliable method to determine genotypes of individual CRISPR/Cas-transfected cells has made it impossible to unambiguously identify the genetic cause of their phenotypes in vivo. Here, we report a strategy for single-cell genotyping in CRISPR/Cas-transfected neurons that were phenotypically characterized in vivo. We show that re-sectioning of cortical slices and subsequent laser microdissection allow us to isolate individual CRISPR/Cas-transfected neurons. Sequencing of PCR products containing a CRISPR/Cas-targeted genomic region in single reference neurons provided genotypes that completely correspond with those deduced from their target protein expression and phenotypes. Thus, our study establishes a powerful strategy to determine the causality between genotypes and phenotypes in CRISPR/Cas-transfected neurons.

Neocortical Chandelier Cells Developmentally Shape Axonal Arbors through Reorganization but Establish Subcellular Synapse Specificity without Refinement.

Diverse types of cortical interneurons (INs) mediate various kinds of inhibitory control mechanisms to balance and shape network activity. Distinct IN subtypes develop uniquely organized axonal arbors that innervate different subcellular compartments of excitatory principal neurons (PNs), which critically contribute to determining their output properties. However, it remains poorly understood how they establish this peculiar axonal organization and synaptic connectivity during development. Here, taking advantage of genetic labeling of IN progenitors, we examined developmental processes of axonal arbors and synaptic connections formed by murine chandelier cells (ChCs), which innervate axon initial segments (AISs) of PNs and thus powerfully regulate their spike generation. Our quantitative analysis by light microscopy revealed that ChCs overgrow and subsequently refine axonal branches as well as varicosities. Interestingly, we found that although a significant number of axonal varicosities are formed off AISs in addition to on AISs, presynaptic markers are predominantly colocalized with those on AISs throughout development. Immunoelectron microscopic (IEM) analysis also demonstrated that only varicosities apposed to AISs contain presynaptic profiles. These results suggest that subcellular synapse specificity of ChCs is genetically predetermined while axonal geometry is shaped through remodeling. Molecular cues localized at AISs may regulate target recognition and synapse formation by ChCs.

DISC1 knockdown impairs the tangential migration of cortical interneurons by affecting the actin cytoskeleton.

Disrupted-in-Schizophrenia 1 (DISC1) is a risk gene for a spectrum of major mental disorders. It has been shown to regulate radial migration as well as dendritic arborization during neurodevelopment and corticogenesis. In a previous study we demonstrated through in vitro experiments that DISC1 also controls the tangential migration of cortical interneurons originating from the medial ganglionic eminence (MGE). Here we first show that DISC1 is necessary for the proper tangential migration of cortical interneurons in the intact brain. Expression of EGFP under the Lhx6 promotor allowed us to analyze exclusively interneurons transfected in the MGE after in utero electroporation. After 3 days in utero, DISC1 deficient interneurons displayed prolonged leading processes and, compared to control, fewer neurons reached the cortex. Time-lapse video microscopy of cortical feeder-layers revealed a decreased migration velocity due to a reduction of soma translocations. Immunostainings indicated that DISC1 is co-localized with F-actin in the growth cone-like structure of the leading process. DISC1 knockdown reduced F-actin levels whereas the overall actin level was not altered. Moreover, DISC1 knockdown also decreased levels of phosphorylated Girdin, which cross-links F-actin, as well as the Girdin-activator pAkt. In contrast, using time-lapse video microscopy of fluorescence-tagged tubulin and EB3 in fibroblasts, we found no effects on microtubule polymerization when DISC1 was reduced. However, DISC1 affected the acetylation of microtubules in the leading processes of MGE-derived cortical interneurons. Together, our results provide a mechanism how DISC1 might contribute to interneuron migration thereby explaining the reduced number of specific classes of cortical interneurons in some DISC1 mouse models.

A dual role of EphB1/ephrin-B3 reverse signaling on migrating striatal and cortical neurons originating in the preoptic area: should I stay or go away?

During embryonic development the preoptic area (POA) gives rise to two populations of neurons which are generated at the same time, cortical interneurons and striatal cells. POA-derived cortical interneurons take a superficial path and avoid the developing striatum (Str) when they migrate to their target region. We found that EphB1, which is expressed in the striatal anlage, prevents cortical interneurons from entering the Str via ephrin-B3 reverse signaling. In contrast, for striatal neurons which also express ephrin-B3, EphB1 acts as a stop signal. This dual role of EphB1 is due to differences in ephrin-B3 reverse signaling cascades. For striatal neurons, binding of EphB1 to ephrin-B3 reduces endogenously high levels of pSrc and pFAK, which then causes the cells to stop migration. In contrast, in cortical interneurons EphB1-ephrin-B3 reverse signaling leads to phosphorylation of Src and focal adhesion kinase (FAK) which then mediates repulsion. Consistent with these in vitro findings, in an ephrin-B3 knockout mouse line, we discovered misrouted cortical interneurons in the Str and an over-migration of striatal neurons in their target region. Thus, EphB1/ephrin-B3 reverse signaling has a different impact on two sets of neurons which are generated at the same time and place: it can act as a repulsive cue for migrating neurons or it can terminate neuronal migration, a novel role of the Eph/ephrin system.

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 wildtype 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.