Local circuit amplification of spatial selectivity in the hippocampus.

Local circuit architecture facilitates the emergence of feature selectivity in the cerebral cortex1. In the hippocampus, it remains unknown whether local computations supported by specific connectivity motifs2 regulate the spatial receptive fields of pyramidal cells3. Here we developed an in vivo electroporation method for monosynaptic retrograde tracing4 and optogenetics manipulation at single-cell resolution to interrogate the dynamic interaction of place cells with their microcircuitry during navigation. We found a local circuit mechanism in CA1 whereby the spatial tuning of an individual place cell can propagate to a functionally recurrent subnetwork5 to which it belongs. The emergence of place fields in individual neurons led to the development of inverse selectivity in a subset of their presynaptic interneurons, and recruited functionally coupled place cells at that location. Thus, the spatial selectivity of single CA1 neurons is amplified through local circuit plasticity to enable effective multi-neuronal representations that can flexibly scale environmental features locally without degrading the feedforward input structure.

Commissural neurons transgress the CNS/PNS boundary in absence of ventricular zone-derived netrin 1.

During the development of the central nervous system (CNS), only motor axons project into peripheral nerves. Little is known about the cellular and molecular mechanisms that control the development of a boundary at the CNS surface and prevent CNS neuron emigration from the neural tube. It has previously been shown that a subset of spinal cord commissural axons abnormally invades sensory nerves in Ntn1 hypomorphic embryos and Dcc knockouts. However, whether netrin 1 also plays a similar role in the brain is unknown. In the hindbrain, precerebellar neurons migrate tangentially under the pial surface, and their ventral migration is guided by netrin 1. Here, we show that pontine neurons and inferior olivary neurons, two types of precerebellar neurons, are not confined to the CNS in Ntn1 and Dcc mutant mice, but that they invade the trigeminal, auditory and vagus nerves. Using a Ntn1 conditional knockout, we show that netrin 1, which is released at the pial surface by ventricular zone progenitors is responsible for the CNS confinement of precerebellar neurons. We propose, that netrin 1 distribution sculpts the CNS boundary by keeping CNS neurons in netrin 1-rich domains.

Slit-Robo signaling.

Slits are secreted proteins that bind to Roundabout (Robo) receptors. Slit-Robo signaling is best known for mediating axon repulsion in the developing nervous system. However, in recent years the functional repertoire of Slits and Robo has expanded tremendously and Slit-Robo signaling has been linked to roles in neurogenesis, angiogenesis and cancer progression among other processes. Likewise, our mechanistic understanding of Slit-Robo signaling has progressed enormously. Here, we summarize new insights into Slit-Robo evolutionary and system-dependent diversity, receptor-ligand interactions, signaling crosstalk and receptor activation.

Conformational dynamics of the focal adhesion targeting domain control specific functions of focal adhesion kinase in cells.

Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr(925) facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr(925) phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr(861), located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser(910) by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule.

Inhibition of the NOTCH pathway using γ-secretase inhibitor RO4929097 has limited antitumor activity in established glial tumors.

Notch signaling is altered in many cancers. Our previous findings in primary pediatric ependymoma support a role for NOTCH in glial oncogenesis. The present study evaluates the γ-secretase inhibitor RO4929097 in glial tumor models. The expression of Notch pathway genes was evaluated using real-time RT-PCR in 21 ependymoma and glioma models. NOTCH1 mutations were analyzed by DNA sequencing. RO4929097 activity was evaluated in vitro and in vivo, as a single agent and in combination, in glioma and ependymoma models. Notch pathway genes are overexpressed in ependymomas and gliomas along with FBXW7 downregulation. NOTCH1 mutations in the TAD domain were observed in 20% (2/10) of ependymoma primary cultures. Blocking the Notch pathway with the γ-secretase inhibitor RO4929097 reduced cell density and viability in ependymoma short-term cultures. When combined with chemotherapeutic agents, RO4929097 enhanced temozolomide effects in ependymoma short-term cultures and potentiated the cytotoxicity of etoposide, cisplatinum, and temozolomide in glioma cells. RO4929097, in combined treatment with mTOR inhibition, potentiated cytotoxicity in vitro, but did not enhance antitumor effects in vivo. In contrast, RO4929097 enhanced irradiation effects in glioma and ependymoma xenografts and showed tumor growth inhibition in advanced-stage IGRG121 glioblastoma xenografts. RO4929097-mediated effects were independent of NOTCH1 mutation status or expression levels, but associated with low IL-6 levels. In established glial tumor models, NOTCH inhibition had limited effects as a single agent, but enhanced efficacy when combined with DNA-interfering agents. These preclinical data need to be considered for further clinical development of NOTCH inhibitors in glial tumors.

Preconfigured dynamics in the hippocampus are guided by embryonic birthdate and rate of neurogenesis.

The incorporation of new information into the hippocampal network is likely to be constrained by its innate architecture and internally generated activity patterns. However, the origin, organization and consequences of such patterns remain poorly understood. In the present study we show that hippocampal network dynamics are affected by sequential neurogenesis. We birthdated CA1 pyramidal neurons with in utero electroporation over 4 embryonic days, encompassing the peak of hippocampal neurogenesis, and compared their functional features in freely moving adult mice. Neurons of the same birthdate displayed distinct connectivity, coactivity across brain states and assembly dynamics. Same-birthdate neurons exhibited overlapping spatial representations, which were maintained across different environments. Overall, the wiring and functional features of CA1 pyramidal neurons reflected a combination of birthdate and the rate of neurogenesis. These observations demonstrate that sequential neurogenesis during embryonic development shapes the preconfigured forms of adult network dynamics.

Reorganization of CA1 dendritic dynamics by hippocampal sharp-wave ripples during learning.

The hippocampus plays a critical role in memory consolidation, mediated by coordinated network activity during sharp-wave ripple (SWR) events. Despite the link between SWRs and hippocampal plasticity, little is known about how network state affects information processing in dendrites, the primary sites of synaptic input integration and plasticity. Here, we monitored somatic and basal dendritic activity in CA1 pyramidal cells in behaving mice using longitudinal two-photon calcium imaging integrated with simultaneous local field potential recordings. We found immobility was associated with an increase in dendritic activity concentrated during SWRs. Coincident dendritic and somatic activity during SWRs predicted increased coupling during subsequent exploration of a novel environment. In contrast, somatic-dendritic coupling and SWR recruitment varied with cells' tuning distance to reward location during a goal-learning task. Our results connect SWRs with the stabilization of information processing within CA1 neurons and suggest that these mechanisms may be dynamically biased by behavioral demands.

Compartment-specific tuning of dendritic feature selectivity by intracellular Ca2+ release.

Dendritic calcium signaling is central to neural plasticity mechanisms that allow animals to adapt to the environment. Intracellular calcium release (ICR) from the endoplasmic reticulum has long been thought to shape these mechanisms. However, ICR has not been investigated in mammalian neurons in vivo. We combined electroporation of single CA1 pyramidal neurons, simultaneous imaging of dendritic and somatic activity during spatial navigation, optogenetic place field induction, and acute genetic augmentation of ICR cytosolic impact to reveal that ICR supports the establishment of dendritic feature selectivity and shapes integrative properties determining output-level receptive fields. This role for ICR was more prominent in apical than in basal dendrites. Thus, ICR cooperates with circuit-level architecture in vivo to promote the emergence of behaviorally relevant plasticity in a compartment-specific manner.

Local feedback inhibition tightly controls rapid formation of hippocampal place fields.

Hippocampal place cells underlie spatial navigation and memory. Remarkably, CA1 pyramidal neurons can form new place fields within a single trial by undergoing rapid plasticity. However, local feedback circuits likely restrict the rapid recruitment of individual neurons into ensemble representations. This interaction between circuit dynamics and rapid feature coding remains unexplored. Here, we developed "all-optical" approaches combining novel optogenetic induction of rapidly forming place fields with 2-photon activity imaging during spatial navigation in mice. We find that induction efficacy depends strongly on the density of co-activated neurons. Place fields can be reliably induced in single cells, but induction fails during co-activation of larger subpopulations due to local circuit constraints imposed by recurrent inhibition. Temporary relief of local inhibition permits the simultaneous induction of place fields in larger ensembles. We demonstrate the behavioral implications of these dynamics, showing that our ensemble place field induction protocol can enhance subsequent spatial association learning.

A homogeneous fluorescence resonance energy transfer system for monitoring the activation of a protein switch in real time.

A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.

Developmental mechanisms underlying circuit wiring: Novel insights and challenges ahead.

Synaptic connectivity within neural circuits is characterized by high degrees of cellular and subcellular specificity. This precision arises from the combined action of several classes of molecular cues, transmembrane receptors, secreted cues and extracellular matrix components, coordinating transitions between axon guidance, dendrite patterning, axon branching and synapse specificity. We focus this review on recent insights into some of the molecular and cellular mechanisms controlling these transitions and present the results of large-scale efforts and technological developments aimed at mapping neural connectivity at single cell resolution in the mouse cortex as a mammalian model organism. Finally, we outline some of the technical and conceptual challenges lying ahead as the field is starting to explore one of the most challenging problems in neuroscience: the molecular and cellular logic underlying the emergence of the connectome characterizing specific circuits within the central nervous system of mammals.

Synaptogenic activity of the axon guidance molecule Robo2 underlies hippocampal circuit function.

Synaptic connectivity within adult circuits exhibits a remarkable degree of cellular and subcellular specificity. We report that the axon guidance receptor Robo2 plays a role in establishing synaptic specificity in hippocampal CA1. In vivo, Robo2 is present and required postsynaptically in CA1 pyramidal neurons (PNs) for the formation of excitatory (E) but not inhibitory (I) synapses, specifically in proximal but not distal dendritic compartments. In vitro approaches show that the synaptogenic activity of Robo2 involves a trans-synaptic interaction with presynaptic Neurexins, as well as binding to its canonical extracellular ligand Slit. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in awake behaving mice shows that preventing Robo2-dependent excitatory synapse formation cell autonomously during development alters place cell properties of adult CA1 PNs. Our results identify a trans-synaptic complex linking the establishment of synaptic specificity to circuit function.

Whole-Neuron Synaptic Mapping Reveals Spatially Precise Excitatory/Inhibitory Balance Limiting Dendritic and Somatic Spiking.

The balance between excitatory and inhibitory (E and I) synapses is thought to be critical for information processing in neural circuits. However, little is known about the spatial principles of E and I synaptic organization across the entire dendritic tree of mammalian neurons. We developed a new open-source reconstruction platform for mapping the size and spatial distribution of E and I synapses received by individual genetically-labeled layer 2/3 (L2/3) cortical pyramidal neurons (PNs) in vivo. We mapped over 90,000 E and I synapses across twelve L2/3 PNs and uncovered structured organization of E and I synapses across dendritic domains as well as within individual dendritic segments. Despite significant domain-specific variation in the absolute density of E and I synapses, their ratio is strikingly balanced locally across dendritic segments. Computational modeling indicates that this spatially precise E/I balance dampens dendritic voltage fluctuations and strongly impacts neuronal firing output.

Activity-Dependent Nucleation of Dynamic Microtubules at Presynaptic Boutons Controls Neurotransmission.

Control of microtubule (MT) nucleation and dynamics is critical for neuronal function. Whether MT nucleation is regulated at presynaptic boutons and influences overall presynaptic activity remains unknown. By visualizing MT plus-end dynamics at individual excitatory en passant boutons in axons of cultured hippocampal neurons and in hippocampal slices expressing EB3-EGFP and vGlut1-mCherry, we found that dynamic MTs preferentially grow from presynaptic boutons, show biased directionality in that they are almost always oriented toward the distal tip of the axon, and can be induced by neuronal activity. Silencing of γ-tubulin expression reduced presynaptic MT nucleation, and depletion of either HAUS1 or HAUS7-augmin subunits increased the percentage of retrograde comets initiated at boutons, indicating that γ-tubulin and augmin are required for activity-dependent de novo nucleation of uniformly distally oriented dynamic MTs. We analyzed the dynamics of a wide range of axonal organelles as well as synaptic vesicles (SVs) relative to vGlut1+ stable presynaptic boutons in a time window during which MT nucleation at boutons is promoted upon induction of neuronal activity, and we found that γ-tubulin-dependent presynaptic MT nucleation controls bidirectional (SV) interbouton transport and regulates evoked SV exocytosis. Hence, en passant boutons act as hotspots for activity-dependent de novo MT nucleation, which controls neurotransmission by providing dynamic tracks for bidirectional delivery of SVs between sites of neurotransmitter release.

Circuit Wiring: Neurite Speed Dating versus Stable Synaptic Matchmaking.

Understanding the mechanisms establishing the complex but precise pattern of connectivity characterizing neural circuits remains an immense challenge. In a recent issue of Neuron, Mao and colleagues (2018) provide new insights by showing that the activation kinetics of EphB2, a transmembrane receptor tyrosine kinase, control whether dendritic filopodia makes a synapse with candidate axons.

Fly Dscams Can Also Help You Find the Right Partners.

The modular reiterative pattern of the fly visual system makes it an ideal model to study axon guidance and synaptogenesis. In this issue of Neuron, Tadros et al. (2016) show that Dscam2/4 promote the anchoring of dendrites to their targets.

The multifaceted roles of Slits and Robos in cortical circuits: from proliferation to axon guidance and neurological diseases.

Slit repulsion, mediated by Robo receptors, is known to play a major role in axon guidance in the nervous system. However, recent studies have revealed that in the mammalian cortex these molecules are highly versatile and that their function extends far beyond axon guidance. They act at all phases of development to control neurogenesis, neuronal migration, axon patterning, dendritic outgrowth and spinogenesis. The expression of Robo receptors in cortical and thalamocortical axons (TCAs) is tightly regulated by a combination of transcription factors (TFs), proteases and activity. These findings also suggest that Slit and Robos have influenced the evolution of cortical circuits. Last, novel genetic evidence associates various neurological disorders, such as autism, to abnormal Slit/Robo signaling.

Signaling switch of the axon guidance receptor Robo3 during vertebrate evolution.

Development of neuronal circuits is controlled by evolutionarily conserved axon guidance molecules, including Slits, the repulsive ligands for roundabout (Robo) receptors, and Netrin-1, which mediates attraction through the DCC receptor. We discovered that the Robo3 receptor fundamentally changed its mechanism of action during mammalian evolution. Unlike other Robo receptors, mammalian Robo3 is not a high-affinity receptor for Slits because of specific substitutions in the first immunoglobulin domain. Instead, Netrin-1 selectively triggers phosphorylation of mammalian Robo3 via Src kinases. Robo3 does not bind Netrin-1 directly but interacts with DCC. Netrin-1 fails to attract pontine neurons lacking Robo3, and attraction can be restored in Robo3(-/-) mice by expression of mammalian, but not nonmammalian, Robo3. We propose that Robo3 evolution was key to sculpting the mammalian brain by converting a receptor for Slit repulsion into one that both silences Slit repulsion and potentiates Netrin attraction.

Dystroglycan adds more sugars to the midline cocktail.

In this issue of Neuron, Wright et al. (2012) identify two novel mediators of α-dystroglycan glycosylation in mouse and unravel a novel function of glycosylated dystroglycan in axon guidance by providing evidence for direct binding of α-DG to the midline chemorepellent Slit2.