Assembly Of Neuronal Circuits Research Articles

Antagonistic action of Siah2 and Pard3/JamC to promote germinal zone exit of differentiated cerebellar granule neurons by modulating Ntn1 signaling via Dcc.

Exiting a germinal zone (GZ) initiates a cascade of events that promote neuronal maturation and circuit assembly. Developing neurons and their progenitors must interpret various niche signals-such as morphogens, guidance molecules, extracellular matrix components, and adhesive cues-to navigate this region. How differentiating neurons integrate and adapt to multiple cell-extrinsic niche cues with their cell-intrinsic machinery in exiting a GZ is unknown. We establish cooperation between cell polarity-regulated adhesion and Netrin-1 (Ntn-1) signaling comprises a coincidence detection circuit repelling maturing neurons from their GZ. In this circuit, the Partitioning defective 3 (Pard3) polarity protein and Junctional adhesion molecule-C (JamC) adhesion protein promote, while the Seven in absentia 2 (Siah2) ubiquitin ligase inhibits, Deleted in colorectal cancer (Dcc) receptor surface recruitment to gate differentiation linked repulsion to GZ Ntn-1. These results demonstrate cell polarity as a central integrator of adhesive- and guidance cues cooperating to spur GZ exit.

Neuronal Wiring Receptors Dprs and DIPs Are GPI Anchored and This Modification Contributes to Their Cell Surface Organization.

The Drosophila Dpr and DIP proteins belong to the immunoglobulin superfamily of cell surface proteins (CSPs). Their hetero- and homophilic interactions have been implicated in a variety of neuronal functions, including synaptic connectivity, cell survival, and axon fasciculation. However, the signaling pathways underlying these diverse functions are unknown. To gain insight into Dpr-DIP signaling, we sought to examine how these CSPs are associated with the membrane. Specifically, we asked whether Dprs and DIPs are integral membrane proteins or membrane anchored through the addition of glycosylphosphatidylinositol (GPI) linkage. We demonstrate that most Dprs and DIPs are GPI anchored to the membrane of insect cells and validate these findings for some family members in vivo using Drosophila larvae, where GPI anchor cleavage results in loss of surface labeling. Additionally, we show that GPI cleavage abrogates aggregation of insect cells expressing cognate Dpr-DIP partners. To test if the GPI anchor affects Dpr and DIP localization, we replaced it with a transmembrane domain and observed perturbation of subcellular localization on motor neurons and muscles. These data suggest that membrane anchoring of Dprs and DIPs through GPI linkage is required for localization and that Dpr-DIP intracellular signaling likely requires transmembrane coreceptors.

From signalling to form: the coordination of neural tube patterning.

The development of the vertebrate spinal cord involves the formation of the neural tube and the generation of multiple distinct cell types. The process starts during gastrulation, combining axial elongation with specification of neural cells and the formation of the neuroepithelium. Tissue movements produce the neural tube which is then exposed to signals that provide patterning information to neural progenitors. The intracellular response to these signals, via a gene regulatory network, governs the spatial and temporal differentiation of progenitors into specific cell types, facilitating the assembly of functional neuronal circuits. The interplay between the gene regulatory network, cell movement, and tissue mechanics generates the conserved neural tube pattern observed across species. In this review we offer an overview of the molecular and cellular processes governing the formation and patterning of the neural tube, highlighting how the remarkable complexity and precision of vertebrate nervous system arises. We argue that a multidisciplinary and multiscale understanding of the neural tube development, paired with the study of species-specific strategies, will be crucial to tackle the open questions.

Selective activation and down-regulation of Trk receptors by neurotrophins in human neurons co-expressing TrkB and TrkC.

In the central nervous system, most neurons co‐express TrkB and TrkC, the tyrosine kinase receptors for brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down‐regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.

Input-specific control of interneuron numbers in nascent striatal networks

The assembly of functional neuronal circuits requires appropriate numbers of distinct classes of neurons, but the mechanisms through which their relative proportions are established remain poorly defined. Investigating the mouse striatum, we found that the two most prominent subtypes of striatal interneurons, parvalbumin-expressing (PV+) GABAergic and cholinergic (ChAT+) interneurons, undergo extensive programmed cell death between the first and second postnatal weeks. Remarkably, the survival of PV+ and ChAT+ interneurons is regulated by distinct mechanisms mediated by their specific afferent connectivity. While long-range cortical inputs control PV+ interneuron survival, ChAT+ interneuron survival is regulated by local input from the medium spiny neurons. Our results identify input-specific circuit mechanisms that operate during the period of programmed cell death to establish the final number of interneurons in nascent striatal networks.

The enteric nervous system of the C. elegans pharynx is specified by the Sine oculis-like homeobox gene ceh-34.

Overarching themes in the terminal differentiation of the enteric nervous system, an autonomously acting unit of animal nervous systems, have so far eluded discovery. We describe here the overall regulatory logic of enteric nervous system differentiation of the nematode Caenorhabditis elegans that resides within the foregut (pharynx) of the worm. A C. elegans homolog of the Drosophila Sine oculis homeobox gene, ceh-34, is expressed in all 14 classes of interconnected pharyngeal neurons from their birth throughout their life time, but in no other neuron type of the entire animal. Constitutive and temporally controlled ceh-34 removal shows that ceh-34 is required to initiate and maintain the neuron type-specific terminal differentiation program of all pharyngeal neuron classes, including their circuit assembly. Through additional genetic loss of function analysis, we show that within each pharyngeal neuron class, ceh-34 cooperates with different homeodomain transcription factors to individuate distinct pharyngeal neuron classes. Our analysis underscores the critical role of homeobox genes in neuronal identity specification and links them to the control of neuronal circuit assembly of the enteric nervous system. Together with the pharyngeal nervous system simplicity as well as its specification by a Sine oculis homolog, our findings invite speculations about the early evolution of nervous systems.

Dysregulation of GABAergic Signaling in Neurodevelomental Disorders: Targeting Cation-Chloride Co-transporters to Re-establish a Proper E/I Balance.

The construction of the brain relies on a series of well-defined genetically and experience- or activity -dependent mechanisms which allow to adapt to the external environment. Disruption of these processes leads to neurological and psychiatric disorders, which in many cases are manifest already early in postnatal life. GABA, the main inhibitory neurotransmitter in the adult brain is one of the major players in the early assembly and formation of neuronal circuits. In the prenatal and immediate postnatal period GABA, acting on GABAA receptors, depolarizes and excites targeted cells via an outwardly directed flux of chloride. In this way it activates NMDA receptors and voltage-dependent calcium channels contributing, through intracellular calcium rise, to shape neuronal activity and to establish, through the formation of new synapses and elimination of others, adult neuronal circuits. The direction of GABAA-mediated neurotransmission (depolarizing or hyperpolarizing) depends on the intracellular levels of chloride [Cl−]i, which in turn are maintained by the activity of the cation-chloride importer and exporter KCC2 and NKCC1, respectively. Thus, the premature hyperpolarizing action of GABA or its persistent depolarizing effect beyond the postnatal period, leads to behavioral deficits associated with morphological alterations and an excitatory (E)/inhibitory (I) imbalance in selective brain areas. The aim of this review is to summarize recent data concerning the functional role of GABAergic transmission in building up and refining neuronal circuits early in development and its dysfunction in neurodevelopmental disorders such as Autism Spectrum Disorders (ASDs), schizophrenia and epilepsy. In particular, we focus on novel information concerning the mechanisms by which alterations in cation-chloride co-transporters (CCC) generate behavioral and cognitive impairment in these diseases. We discuss also the possibility to re-establish a proper GABAA-mediated neurotransmission and excitatory (E)/inhibitory (I) balance within selective brain areas acting on CCC.

A Role for Frizzled and Their Post-Translational Modifications in the Mammalian Central Nervous System.

The Wnt pathway is a key signalling cascade that regulates the formation and function of neuronal circuits. The main receptors for Wnts are Frizzled (Fzd) that mediate diverse functions such as neurogenesis, axon guidance, dendritogenesis, synapse formation, and synaptic plasticity. These processes are crucial for the assembly of functional neuronal circuits required for diverse functions ranging from sensory and motor tasks to cognitive performance. Indeed, aberrant Wnt–Fzd signalling has been associated with synaptic defects during development and in neurodegenerative conditions such as Alzheimer’s disease. New studies suggest that the localisation and stability of Fzd receptors play a crucial role in determining Wnt function. Post-translational modifications (PTMs) of Fzd are emerging as an important mechanism that regulates these Wnt receptors. However, only phosphorylation and glycosylation have been described to modulate Fzd function in the central nervous system (CNS). In this review, we discuss the function of Fzd in neuronal circuit connectivity and how PTMs contribute to their function. We also discuss other PTMs, not yet described in the CNS, and how they might modulate the function of Fzd in neuronal connectivity. PTMs could modulate Fzd function by affecting Fzd localisation and stability at the plasma membrane resulting in local effects of Wnt signalling, a feature particularly important in polarised cells such as neurons. Our review highlights the importance of further studies into the role of PTMs on Fzd receptors in the context of neuronal connectivity.

The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning.

The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for defined aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most, if not all, tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific, roles in different genetic pathways and may be involved in the regulation or localization of transmembrane and secreted proteins to specific subcellular compartments.

CHN1 and duane retraction syndrome: Expanding the phenotype to cranial nerves development disease

Duane retraction syndrome is a congenital eye movement disorder characterized by a failure of abducens nerve to develop normally, resulting in restriction or absence of abduction, adduction, or both, and narrowing of the palpebral fissure and retraction of the globe on attempted adduction. There is a genetic heterogeneity in Duane retraction syndrome (DURS). DURS maps to chromosome 8q13 in some patients, and pathogenic variants in CHN1 and MAFB genes are known to lead to DURS. We report here a child and his father with Duane retraction syndrome, associated to swallowing difficulties and unilateral trapeze aplasia. A whole exome sequencing revealed a heterozygous missense variant in CHN1 gene. This gene encodes GTPase-activating protein and is involved in the assembly of neuronal locomotor circuits. A patient with a 8q deletion has previously been described with a Duane retraction syndrome associated to trapeze aplasia. We provide an additional description to support the role in cranial nerves development of the CHN1 gene.

Axonal branch patterning and neuronal shape diversity: roles in developmental circuit assembly: Axonal branch patterning and neuronal shape diversity in developmental circuit assembly.

Recent progress in human genetics and single cell sequencing rapidly expands the list of molecular factors that offer important new contributions to our understanding of brain wiring. Yet many new molecular factors are being discovered that have never been studied in the context of neuronal circuit development. This is clearly asking for increased efforts to better understand the developmental mechanisms of circuit assembly [1]. Moreover, recent studies characterizing the developmental causes of some psychiatric diseases show impressive progress in reaching cellular resolution in their analysis. They provide concrete support emphasizing the importance of axonal branching and synapse formation as a hotspot for potential defects. Inspired by these new studies we will discuss progress but also challenges in understanding how neurite branching and neuronal shape diversity itself impacts on specificity of neuronal circuit assembly. We discuss the idea that neuronal shape acquisition itself is a key specificity factor in neuronal circuit assembly.

Familiar growth factors have diverse roles in neural network assembly.

The assembly of neuronal circuits during development depends on guidance of axonal growth cones by molecular cues deposited in their environment. While a number of families of axon guidance molecules have been identified and reviewed, important and diverse activities of traditional growth factors are emerging. Besides clear and well recognized roles in the regulation of cell division, differentiation and survival, new research shows later phase roles for a number of growth factors in promoting neuronal migration, axon guidance and synapse formation throughout the nervous system.

Subcellular sorting of neuregulins controls the assembly of excitatory-inhibitory cortical circuits.

The assembly of specific neuronal circuits relies on the expression of complementary molecular programs in presynaptic and postsynaptic neurons. In the cerebral cortex, the tyrosine kinase receptor ErbB4 is critical for the wiring of specific populations of GABAergic interneurons, in which it paradoxically regulates both the formation of inhibitory synapses as well as the development of excitatory synapses received by these cells. Here, we found that Nrg1 and Nrg3, two members of the neuregulin family of trophic factors, regulate the inhibitory outputs and excitatory inputs of interneurons in the mouse cerebral cortex, respectively. The differential role of Nrg1 and Nrg3 in this process is not due to their receptor-binding EGF-like domain, but rather to their distinctive subcellular localization within pyramidal cells. Our study reveals a novel strategy for the assembly of cortical circuits that involves the differential subcellular sorting of family-related synaptic proteins.

Local externalization of phosphatidylserine mediates developmental synaptic pruning by microglia

Neuronal circuit assembly requires the fine balance between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized as key players, the neuronal molecular components that specify synapses to be eliminated are still undefined. Here, we show that exposed phosphatidylserine (PS) represents a neuronal “eat‐me” signal involved in microglial‐mediated pruning. In hippocampal neuron and microglia co‐cultures, synapse elimination can be partially prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, PS exposure at both hippocampal and retinogeniculate synapses and engulfment of PS‐labeled material by microglia occurs during established developmental periods of microglial‐mediated synapse elimination. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, have elevated presynaptic PS exposure and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial‐mediated synapse pruning and identify a novel role of developmentally regulated neuronal PS exposure that is common among developing brain structures.

Functional mosaic organization of neuroligins in neuronal circuits.

Complex brain circuitry with feedforward and feedback systems regulates neuronal activity, enabling neural networks to process and drive the entire spectrum of cognitive, behavioral, sensory, and motor functions. Simultaneous orchestration of distinct cells and interconnected neural circuits is underpinned by hundreds of synaptic adhesion molecules that span synaptic junctions. Dysfunction of a single molecule or molecular interaction at synapses can lead to disrupted circuit activity and brain disorders. Neuroligins, a family of cell adhesion molecules, were first identified as postsynaptic-binding partners of presynaptic neurexins and are essential for synapse specification and maturation. Here, we review recent advances in our understanding of how this family of adhesion molecules controls neuronal circuit assembly by acting in a synapse-specific manner.

GABAergic Restriction of Network Dynamics Regulates Interneuron Survival in the Developing Cortex

During neonatal development, sensory cortices generate spontaneous activity patterns shaped by both sensory experience and intrinsic influences. How these patterns contribute to the assembly of neuronal circuits is not clearly understood. Using longitudinal invivo calcium imaging in un-anesthetized mouse pups, we show that spatially segregated functional assemblies composed of interneurons and pyramidal cells are prominent in the somatosensory cortex by postnatal day (P) 7. Both reduction of GABA release and synaptic inputs onto pyramidal cells erode the emergence of functional topography, leading to increased network synchrony. This aberrant pattern effectively blocks interneuron apoptosis, causing increased survival of parvalbumin and somatostatin interneurons. Furthermore, the effect of GABA on apoptosis is mediated by inputs from medial ganglionic eminence (MGE)-derived but not caudal ganglionic eminence (CGE)-derived interneurons. These findings indicate that immature MGE interneurons are fundamental for shaping GABA-driven activity patterns that balance the number of interneurons integrating into maturing cortical networks.

How prolonged expression of Hunchback, a temporal transcription factor, re-wires locomotor circuits.

How circuits assemble starting from stem cells is a fundamental question in developmental neurobiology. We test the hypothesis that, in neuronal stem cells, temporal transcription factors predictably control neuronal terminal features and circuit assembly. Using the Drosophila motor system, we manipulate expression of the classic temporal transcription factor Hunchback (Hb) specifically in the NB7-1 stem cell, which produces U motor neurons (MNs), and then we monitor dendrite morphology and neuromuscular synaptic partnerships. We find that prolonged expression of Hb leads to transient specification of U MN identity, and that embryonic molecular markers do not accurately predict U MN terminal features. Nonetheless, our data show Hb acts as a potent regulator of neuromuscular wiring decisions. These data introduce important refinements to current models, show that molecular information acts early in neurogenesis as a switch to control motor circuit wiring, and provide novel insight into the relationship between stem cell and circuit.

Subtype Diversification and Synaptic Specificity of Stem Cell-Derived Spinal Interneurons

Neuronal diversification is a fundamental step in the construction of functional neural circuits, but how neurons generated from single progenitor domains acquire diverse subtype identities remains poorly understood.Here we developed an embryonic stem cell (ESC)-based system to model subtype diversification of V1 interneurons, a class of spinal neurons comprising four clades collectively containing dozens of molecularly distinct neuronal subtypes. We demonstrate that V1 subtype diversity can be modified by extrinsic signals. Inhibition of Notch and activation of retinoid signaling results in a switch to MafA clade identity and enriches differentiation of Renshaw cells, a specialized MafA subtype that mediates recurrent inhibition of spinal motor neurons. We show that Renshaw cells are intrinsically programmed to migrate to species-specific laminae upon transplantation and to form subtype-specific synapses with motor neurons. Our results demonstrate that stem cell-derived neuronal subtypes can be used to investigatemechanisms underlying neuronal subtype specification and circuit assembly.

γ-Neurexin and Frizzled Mediate Parallel Synapse Assembly Pathways Antagonized by Receptor Endocytosis

Synapse formation defines neuronal connectivity and is thus essential for neuronal circuit assembly. Trans-synaptic interactions of cell adhesion molecules are thought to induce synapse assembly. Here we demonstrate that a recently discovered and conserved short form of neurexin, γ-neurexin, which lacks canonical extracellular domains, is nonetheless sufficient to promote presynaptic assembly in the nematode C.elegans. γ- but not α-neurexin is required for assembling active zone components, recruiting synaptic vesicles, and clustering calcium channels at release sites to promote evoked synaptic transmission. Furthermore, we find that neurexin functions in parallel with the transmembrane receptor Frizzled, as the absence of both proteins leads to an enhanced phenotype-the loss of most synapses. Frizzled's pro-synaptogenic function is independent of its ligand, Wnt. Wnt binding instead eliminates synapses by inducing Frizzled's endocytosis and the downregulation of neurexin. These results reveal how pro- and anti-synaptogenic factors converge to precisely sculpt circuit formation invivo.

Role of mechanical cues in shaping neuronal morphology and connectivity

Neuronal circuits, the functional building blocks of the nervous system, assemble during development through a series of dynamic processes including the migration of neurons to their final position, the growth and navigation of axons and their synaptic connection with target cells. While the role of chemical cues in guiding neuronal migration and axonal development has been extensively analysed, the contribution of mechanical inputs, such as forces and stiffness, has received far less attention. In this article, we review the in vitro and more recent in vivo studies supporting the notion that mechanical signals are critical for multiple aspects of neuronal circuit assembly, from the emergence of axons to the formation of functional synapses. By combining live imaging approaches with tools designed to measure and manipulate the mechanical environment of neurons, the emerging field of neuromechanics will add a new paradigm in our understanding of neuronal development and potentially inspire novel regenerative therapies.