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Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development. [ABSTRACT FROM AUTHOR]

Neurons are post‐mitotic cells, with microtubules playing crucial roles in axonal transport and growth. Kinesin family member 2c (KIF2C), a member of the Kinesin‐13 family, possesses the ability to depolymerize microtubules and is involved in remodelling the microtubule lattice. Myocyte enhancer factor 2c (MEF2C) was initially identified as a regulator of muscle differentiation but has recently been associated with neurological abnormalities such as severe cognitive impairment, stereotyping, epilepsy and brain malformations when mutated or deleted. However, further investigation is required to determine which target genes MEF2C acts upon to influence neuronal function as a transcription regulator. Our data demonstrate that knockdown of both Mef2c and Kif2c significantly impacts spinal motor neuron development and behaviour in zebrafish. Luciferase reporter assays and chromosome immunoprecipitation assays, along with down/upregulated expression analysis, revealed that MFE2C functions as a novel transcription regulator for the Kif2c gene. Additionally, the knockdown of either Mef2c or Kif2c expression in E18 cortical neurons substantially reduces the number of primary neurites and axonal branches during neuronal development in vitro without affecting neurite length. Finally, depletion of Kif2c eliminated the effects of overexpression of Mef2c on the neurite branching. Based on these findings, we provided novel evidence demonstrating that MEF2C regulates the transcription of the Kif2c gene thereby influencing the axonal branching. [ABSTRACT FROM AUTHOR]

Fidgetin, a microtubule-severing enzyme, regulates neurite outgrowth, axonal regeneration, and cell migration by trimming off the labile domain of microtubule polymers. Because maintenance of the microtubule labile domain is essential for axon initiation, elongation, and navigation, it is of interest to determine whether augmenting the microtubule labile domain via depletion of fidgetin serves as a therapeutic approach to promote axonal regrowth in spinal cord injury. In this study, we constructed rat models of spinal cord injury and sciatic nerve injury. Compared with spinal cord injury, we found that expression level of tyrosinated microtubules in the labile portion of microtubules continuously increased, whereas fidgetin decreased after peripheral nerve injury. Depletion of fidgetin enhanced axon regeneration after spinal cord injury, whereas expression level of end binding protein 3 (EB3) markedly increased. Next, we performed RNA interference to knockdown EB3 or fidgetin. We found that deletion of EB3 did not change fidgetin expression. Conversely, deletion of fidgetin markedly increased expression of tyrosinated microtubules and EB3. Deletion of fidgetin increased the amount of EB3 at the end of neurites and thereby increased the level of tyrosinated microtubules. Finally, we deleted EB3 and overexpressed fidgetin. We found that fidgetin trimmed tyrosinated tubulins by interacting with EB3. When fidgetin was deleted, the labile portion of microtubules was elongated, and as a result the length of axons and number of axon branches were increased. These findings suggest that fidgetin can be used as a novel therapeutic target to promote axonal regeneration after spinal cord injury. Furthermore, they reveal an innovative mechanism by which fidgetin preferentially severs labile microtubules., Competing Interests: None

Npr2 (natriuretic peptide receptor 2) affects bifurcation of neural crest or placode-derived afferents upon entering the brain stem/spinal cord, leading to a lack of either rostral or caudal branches. Previous work has shown that early embryonic growth of cochlear and vestibular afferents is equally affected in this mutant but later work on postnatal Npr2 point mutations suggested some additional effects on the topology of afferent projections and mild functional defects. Using multicolor lipophilic dye tracing, we show that absence of Npr2 has little to no effect on the initial patterning of inner ear afferents with respect to their dorsoventral cochleotopic-specific projections. However, in contrast to control animals, we found a variable degree of embryonic extension of auditory afferents beyond the boundaries of the anterior cochlear nucleus into the cerebellum that emanates only from apical spiral ganglion neurons. Such expansion has previously only been reported for Hox gene mutants and implies an unclear interaction of Hox codes with Npr2-mediated afferent projection patterning to define boundaries. Some vestibular ganglion neurons expand their projections to reach the cochlear apex and the cochlear nuclei, comparable to previous findings in Neurod1 mutant mice. Before birth, such expansions are reduced or lost leading to truncated projections to the anteroventral cochlear nucleus and expansion of low-frequency fibers of the apex to the posteroventral cochlear nucleus. [ABSTRACT FROM AUTHOR]

Bifurcation of axons from dorsal root ganglion (DRG) and cranial sensory ganglion (CSG) neurons is mediated by a cGMP-dependent signaling pathway composed of the ligand C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2 and the cGMP-dependent protein kinase I (cGKI). Here, we demonstrate that mesencephalic trigeminal neurons (MTN) which are the only somatosensory neurons whose cell bodies are located within the CNS co-express Npr2 and cGKI. Afferents of MTNs form Y-shaped branches in rhombomere 2 where the ligand CNP is expressed. Analyzing mouse mutants deficient for CNP or Npr2 we found that in the absence of CNP-induced cGMP signaling MTN afferents no longer bifurcate and instead extend either into the trigeminal root or caudally in the hindbrain. Since MTNs provide sensory information from jaw closing muscles and periodontal ligaments we measured the bite force of conditional mouse mutants of Npr2 (Npr2flox/flox;Engr1Cre) that lack bifurcation of MTN whereas the bifurcation of trigeminal afferents is normal. Our study revealed that the maximal biting force of both sexes is reduced in Npr2flox/flox;Engr1Cre mice as compared to their Npr2flox/flox littermate controls. In conclusion sensory feedback mechanisms from jaw closing muscles or periodontal ligaments might be impaired in the absence of MTN axon bifurcation.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr27A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr27A,7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth. [ABSTRACT FROM AUTHOR]

Bifurcation of axons from dorsal root ganglion (DRG) and cranial sensory ganglion (CSG) neurons is mediated by a cGMP-dependent signaling pathway composed of the ligand C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2 and the cGMP-dependent protein kinase I (cGKI). Here, we demonstrate that mesencephalic trigeminal neurons (MTN) which are the only somatosensory neurons whose cell bodies are located within the CNS co-express Npr2 and cGKI. Afferents of MTNs form Y-shaped branches in rhombomere 2 where the ligand CNP is expressed. Analyzing mouse mutants deficient for CNP or Npr2 we found that in the absence of CNP-induced cGMP signaling MTN afferents no longer bifurcate and instead extend either into the trigeminal root or caudally in the hindbrain. Since MTNs provide sensory information from jaw closing muscles and periodontal ligaments we measured the bite force of conditional mouse mutants of Npr2 (Npr2flox/flox;Engr1Cre) that lack bifurcation of MTN whereas the bifurcation of trigeminal afferents is normal. Our study revealed that the maximal biting force of both sexes is reduced in Npr2flox/flox;Engr1Cre mice as compared to their Npr2flox/flox littermate controls. In conclusion sensory feedback mechanisms from jaw closing muscles or periodontal ligaments might be impaired in the absence of MTN axon bifurcation. [ABSTRACT FROM AUTHOR]

Light touch sensation begins with activation of low-threshold mechanoreceptor (LTMR) endings in the skin and propagation of their signals to the spinal cord and brainstem. We found that the clustered protocadherin gamma (Pcdhg) gene locus, which encodes 22 cell-surface homophilic binding proteins, is required in somatosensory neurons for normal behavioral reactivity to a range of tactile stimuli. Developmentally, distinct Pcdhg isoforms mediate LTMR synapse formation through neuron-neuron interactions and peripheral axonal branching through neuron-glia interactions. The Pcdhgc3 isoform mediates homophilic interactions between sensory axons and spinal cord neurons to promote synapse formation in vivo and is sufficient to induce postsynaptic specializations in vitro. Moreover, loss of Pcdhgs and somatosensory synaptic inputs to the dorsal horn leads to fewer corticospinal synapses on dorsal horn neurons. These findings reveal essential roles for Pcdhg isoform diversity in somatosensory neuron synapse formation, peripheral axonal branching, and stepwise assembly of central mechanosensory circuitry. [Display omitted] • Pcdhg isoforms have compartmentalized roles in touch sensory neuron development • Pcdhgc3 promotes synapse formation between sensory neurons and spinal cord neurons • Pcdhgs promote peripheral axonal branching through neuron-glia interactions • Sensory inputs to the spinal cord regulate the assembly of spinal cord circuitry Meltzer et al. show that the clustered protocadherin gamma (Pcdhg) gene locus regulates somatosensory neuron synapse formation and peripheral axonal branching. Pcdhgc3 is the only isoform that promotes synapse formation between sensory neurons and spinal cord neurons. Further, they found that somatosensory inputs regulate the assembly of spinal cord circuitry. [ABSTRACT FROM AUTHOR]

Neurons in the ventral tegmental area (VTA) that innervate the nucleus accumbens (Acb) constitute the so-called mesoaccumbens system. Increased activity by these neurons is correlated with the expectation and achievement of reward. The mesoaccumbens projection neurons are regarded as a central node in the brain networks that regulate drive and hedonic experience, and their dysregulation is a common pathophysiological step in addictive behaviors as well as major depression. Despite previous anatomical studies that have analyzed the origin of the mesoaccumbens axons within the VTA, regarded as a unit, the exact contributions of the various cytoarchitectural subdivisions of the VTA to this innervation is still unexplored; understanding these contributions would help further our understanding of their precise anatomical organization. With the aim of deciphering the contribution of the various VTA subdivisions to accumbal innervation, the present study has used retrograde tracer microinjections in the Acb to map the location within the various VTA subdivisions of neurons targeting either the shell or core compartments of the Acb in mice. Furthermore, the dopaminergic nature of these projections has also been analyzed using tyrosine-hydroxylase immunohistochemistry. We demonstrate here that small territories of the Acb core and shell are innervated simultaneously by many VTA subdivisions, contributing dopaminergic as well as nondopaminergic axons to the accumbal innervation. In fact, single VTA subdivisions harbor both dopaminergic and non-dopaminergic neurons that project to the same accumbal territory. The most medial VTA subnuclei, like the caudal linear nucleus, project abundantly to medial aspects of the Acb core, whereas more lateral territories of the Acb are preferentially targeted by neurons located in the parabrachial pigmented and paranigral nuclei. Overall, about half of the mesoaccumbens neurons are putatively dopaminergic in mice. Anterograde single-cell labeling (Sindbis-pal-eGFP vector) of a limited sample of neurons revealed that mesoaccumbens neurons form profuse terminal arborizations to cover large volumes of either the Acb core or shell, and, unlike other VTA projection neuron populations, they do not branch to other striatal or extrastriatal structures. These anatomical observations are consistent with reports of an intense response in many Acb neurons after stimulation of very few VTA cells. [ABSTRACT FROM AUTHOR]

Axonal branching is one of the key processes within the enormous complexity of the nervous system to enable a single neuron to send information to multiple targets. However, the molecular mechanisms that control branch formation are poorly understood. In particular, previous studies have rarely addressed the mechanisms underlying axonal bifurcation, in which axons form new branches via splitting of the growth cone. We demonstrate that DISCO Interacting Protein 2 (DIP2) is required for precise axonal bifurcation in Drosophila mushroom body (MB) neurons by suppressing ectopic bifurcation and regulating the guidance of sister axons. We also found that DIP2 localize to the plasma membrane. Domain function analysis revealed that the AMP-synthetase domains of DIP2 are essential for its function, which may involve exerting a catalytic activity that modifies fatty acids. Genetic analysis and subsequent biochemical analysis suggested that DIP2 is involved in the fatty acid metabolization of acyl-CoA. Taken together, our results reveal a function of DIP2 in the developing nervous system and provide a potential functional relationship between fatty acid metabolism and axon morphogenesis. [ABSTRACT FROM AUTHOR]

Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10-20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 Hz up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells’ signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for many different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations.

Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iotophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F+BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four subtypes could be distinguished among the FPN cells based on their projection targets: 1) Mesocorticolimbic FPN projecting to both neocortex and basal forebrain; 2) Mesocortical FPN innervating the neocortex almost exclusively; 3) Mesolimbic FPN projecting to the basal forebrain, accumbens and caudateputamen; and 4) Mesostriatal FPN targeting only the caudateputamen. While the F+BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and brainstem structures.

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors.

In contrast to the central nervous system (CNS) nerve fibers do regenerate in the peripheral nervous system (PNS) although in a clinically unsatisfying manner. A major problem is excessive sprouting of regenerating axons which results in aberrant reinnervation of target tissue and impaired functional recovery. In the CNS, the reticulon protein Nogo-A has been identified as a prominent oligodendrocyte expressed inhibitor of long-distance growth of regenerating axons. We show here that the related isoform Nogo-B is abundantly expressed in Schwann cells in the PNS. Other than Nogo-A in oligodendrocytes, Nogo-B does not localize to the myelin sheath but is detected in the ER and the plasma membrane of Schwann cells. Adult sensory neurons that are cultured on nogo-a/b deficient Schwann cells form significantly fewer axonal branches vs. those on wildtype Schwann cells, while their maximal axonal extension is unaffected. We demonstrate that this effect of Nogo-B on neuronal morphology is restricted to undifferentiated Schwann cells and is mediated by direct physical contact between these two cell types. Moreover, we show that blocking the Nogo-B specific receptor NgBR, which we find expressed on sensory neurons and to interact with Schwann cell expressed Nogo-B, produces the same branching phenotype as observed after deletion of Nogo-B. These data provide evidence for a novel function of the nogo gene that is implemented by the Nogo-B isoform. The remarkably specific effects of Nogo-B/NgBR on axonal branching, while leaving axonal extension unaffected, are of potential clinical relevance in the context of excessive axonal sprouting after peripheral nerve injury. Main Points • Nogo-B is prominently expressed in Schwann cells and localizes to the ER and plasma membrane. It distributes to the external cytoplasmic compartment of Schwann cells in vivo, but is absent from the myelin sheath. • Genetic deletion of Nogo-B in Schwann cells reduces axonal branching, but not long-distance growth, of co-cultured adult sensory neurons. • Schwann cell expressed Nogo-B interacts with neuronal NgBR. Blockade of NgBR mimics the loss-of-nogo branching phenotype. [ABSTRACT FROM AUTHOR]

The connectivity patterns of many neural circuits are highly ordered and often impressively complex. The intricate order and complexity of neuronal wiring remain not only a challenge for questions related to circuit functions but also for our understanding of how they develop with such an apparent precision. The chemotropic guidance of the growing axon by target-derived cues represents a central paradigm for how neurons get connected with the correct target cells. However, many studies reveal a remarkable variety of important target-independent wiring mechanisms. These mechanisms include axonal sorting, axonal tiling, growth cone polarization, as well as cell-intrinsic mechanisms underlying growth cone sprouting, and neurite branching. Our review focuses on target independent wiring mechanisms and in particular on recent progress emerging from studies on three different sensory systems: olfactory, visual, and somatosensory. We discuss molecular mechanisms that operate during axon-axon interactions or constitute axon-intrinsic functions and outline how they complement the well-known target-dependent wiring mechanisms. [ABSTRACT FROM AUTHOR]

Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iontophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F + BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four "subtypes" could be distinguished among the FPN cells based on their projection targets: (1) "Mesocorticolimbic" FPN projecting to both neocortex and basal forebrain; (2) "Mesocortical" FPN innervating the neocortex almost exclusively; (3) "Mesolimbic" FPN projecting to the basal forebrain, accumbens and caudateputamen; and (4) "Mesostriatal" FPN targeting only the caudateputamen. While the F + BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and brainstem structures. [ABSTRACT FROM AUTHOR]

Axonal branching is a prerequisite for the establishment of complex neuronal circuits and their capacity for parallel information processing. Previously, we have identified a cGMP signaling pathway composed of the ligand C-type natriuretic peptide (CNP), its receptor, the guanylyl cyclase natriuretic peptide receptor 2 (Npr2), and the cGMP-dependent kinase Iα (cGKIα) that regulates axon bifurcation of dorsal root ganglion (DRG) neurons in the spinal cord. Now we asked whether this cascade also controls axon bifurcation elsewhere in the nervous system. An Npr2-lacZ reporter mouse line was generated to clarify the pattern of the CNP receptor expression. It was found that during the period of axonal outgrowth, Npr2 and cGKIα were strongly labeled in neurons of all cranial sensory ganglia (gV, gVII, gVIII, gIX, and gX). In addition, strong complementary expression of CNP was detected in the hindbrain at the entry zones of sensory afferents. To analyze axon branching in individual Npr2-positive neurons, we generated a mouse mutant expressing a tamoxifen-inducible variant of Cre recombinase expressed under control of the Npr2-promoter (Npr2-CreERT2). After crossing this strain with conditional reporter mouse lines, we revealed that the complete absence of Npr2 activity indeed prohibited the bifurcation of cranial sensory axons in their entrance region. Consequently, axons only turned in either an ascending or descending direction, while collateral formation and growth of the peripheral arm was not affected. These findings indicate that in neurons of the cranial sensory ganglia, as in DRG neurons, cGMP signals are necessary for the execution of an axonal bifurcation program. [ABSTRACT FROM AUTHOR]

The correct wiring of a neural network requires neuron to integrate an incredible repertoire of cues found in their extracellular environment. The astonishing efficiency of this process plays a pivotal role in the correct wiring of the brain during development and axon regeneration. Biologically inspired micro- and nanostructured substrates have been shown to regulate axonal outgrowth. In parallel, several studies investigated graphene's potential as a conductive neural interface, able to enhance cell adhesion, neurite sprouting and outgrowth. Here, we engineered a 3D single- to few-layer fuzzy graphene morphology (3DFG), 3DFG on a collapsed Si nanowire (SiNW) mesh template (NT-3DFGc), and 3DFG on a noncollapsed SiNW mesh template (NT-3DFGnc) as neural-instructive materials. The micrometric protruding features of the NWs templates dictated neuronal growth cone establishment, as well as influencing axon elongation and branching. Furthermore, neurons-to-graphene coupling was investigated with comprehensive view of integrin-mediated contact adhesion points and plasma membrane curvature processes.

A recombinant rabies virus was used as a retrograde tracer to allow complete fi lling of the axonal and dendritic arbors of identifi ed projection neurons in layer 5 of mouse primary somatosensory cortex (S1) in vivo. Previous studies have distinguished three types of layer 5 pyramids in S1: tall-tufted, tall-simple, and short. Layer 5 pyramidal neurons were retrogradely labeled from several known targets: contralateral S1, superior colliculus, and thalamus. The complete dendritic arbors of labeled cells were reconstructed to allow for unambiguous classifi cation of cell type. We confi rmed that the tall-tufted pyramids project to the superior colliculus and thalamus and that short layer 5 pyramidal neurons project to contralateral cortex, as previously described. We found that tall-simple pyramidal neurons contribute to corticocortical connections. Axonal reconstructions show that corticocortical projection neurons have a large superfi cial axonal arborization locally, while the subcortically projecting neurons limit axonal arbors to the deep layers. Furthermore, reconstructions of local axons suggest that tall-simple cell axons have extensive lateral spread while those of the short pyramids are more columnar. These differences were revealed by the ability to completely label dendritic and axonal arbors in vivo and have not been apparent in previous studies using labeling in brain slices.

RhoG is a member of the Rho family of small GTPases sharing highest sequence similarity with Rac and Cdc42. Mig-2 and Mtl represent the functional equivalents of RhoG in Caenorhabditis elegans and Drosophila, respectively. RhoG has attracted great interest because it plays a central role in the regulation of cytoskeletal reorganization in various physiological and pathophysiological situations. For example, it is fundamental to phagocytotic processes, is able to regulate gene expression, cell survival and proliferation, and is involved in cell migration, and in the invasion of pathogenic bacteria. The activation of Rac1 via an ELMO/Dock180 module has been elaborated to be important for RhoG signaling. Although a stimulatory role for neurite outgrowth in the pheochromocytoma PC12 cell line has been assigned to RhoG, the exact function of this GTPase for the development of the processes of primary neurons remains to be clarified. In this view, we discuss the impact of RhoG on axonal and dendritic differentiation, its role as a conductor of Rac1 and Cdc42 activity, and the functional regulation of RhoG expression by the microRNA miR-124. [ABSTRACT FROM AUTHOR]

The article discusses the signalling mechanisms that regulate axonal branching in vivo in neuronal systems. It focuses on several neuronal systems like the projection of sensory axons into the spinal cord, the corticopontine projection and the retinotectal system. It cites the dorsal root ganglion (DRG) neurons and the retinal ganglion cells (RGC).

Abstract: Receptor tyrosine phosphatases (RPTPs) are required for axon guidance during embryonic development in Drosophila. Here we examine the roles of four RPTPs during development of the larval mushroom body (MB). MB neurons extend axons into parallel tracts known as the peduncle and lobes. The temporal order of neuronal birth is reflected in the organization of axons within these tracts. Axons of the youngest neurons, known as core fibers, extend within a single bundle at the center, while those of older neurons fill the outer layers. RPTPs are selectively expressed on the core fibers of the MB. Ptp10D and Ptp69D regulate segregation of the young axons into a single core bundle. Ptp69D signaling is required for axonal extension beyond the peduncle. Lar and Ptp69D are necessary for the axonal branching decisions that create the lobes. Avoidance of the brain midline by extending medial lobe axons involves signaling through Lar. [Copyright &y& Elsevier]

Abstract: Stathmin family phosphoproteins participate in the control of microtubule dynamics and have been proposed to be involved in the control of neuronal differentiation. We examined the biological properties and functions of SCLIP, the stathmin family protein most similar to SCG10, a widely studied and recognized neuronal differentiation marker. SCLIP, like SCG10, is present from the earliest stages of hippocampal neuron differentiation in culture at vesicle-like structures following dynamic microtubules. Its inhibition by RNA interference resulted in increased axonal branching, revealing a novel biological role for SCLIP, distinct from SCG10 whose down-regulation in the same conditions promoted growth cone expansion. The enhanced axonal branching resulted from the formation of collateral lamellar protrusions, with cytoskeleton reorganization typical of normal branching. In addition to revealing a novel function for SCLIP in axonal morphogenesis, our results demonstrate for the first time that stathmin family proteins fulfill different and complementary roles during neuronal differentiation. [Copyright &y& Elsevier]

Abstract: α1-tubulin expression occurs in a neural-specific, temporally regulated, and regeneration-inducible fashion in zebrafish. A GFP reporter driven by the α1-tubulin promoter in transgenic zebrafish acts as a stable, in vivo molecular tag that follows neuronal development from birth/specification through postmitotic differentiation to axonal outgrowth and synaptogenesis. We exploited this transgenic system in a reporter expression-dependent (morphology-independent) mutagenesis screen to identify disruptions in genetic loci essential for neuronogenesis and axon elaboration, which would manifest as visually appreciable perturbations in GFP fluorescence. Thirty-two such recessive mutations were obtained, a subset of which was screened through a secondary RNA quantification-based assay to eliminate housekeeping gene defects. Three representative loci, when characterized in detail, were found to exhibit missteps in discrete, sequential stages of embryonic neuronal development. Mutation in sookshma panneurally diminishes the neural precursor pool by affecting cell proliferation in the developing embryo while patterning along the neuraxis remains unperturbed. Disruption of drishti on the other hand ameliorates the mitotic neural population by affecting cell cycle exit of progenitors and stalling their progression to the postmitotic neuronal stage, without impairing subsequent cell fate determination or differentiation. Finally, dhruva is required during neuronal differentiation for axonal branching and terminal innervation in spinal motoaxons and the retinotectal projection. Molecular identification of these loci and analysis of the remaining mutational repertoire will offer unique insights into the genetic inputs that go on to make a mature, differentiated neuron. [Copyright &y& Elsevier]

Axonal branching is an important process for establishing the final pattern of connections between a neuron and its target cells. Cortical connections between upper-layer cells in the neocortex have provided insights into the cellular mechanisms by which electrical activity regulates neural connectivity, including branch formation. Recent evidence further indicates that spontaneous firing and synaptic transmission contribute to axonal branching of cortical neurons through postsynaptic activation. [ABSTRACT FROM AUTHOR]

Summary: The nucleus “k”, in the reticular core of the rabbit caudal pons, is divided into a large medial (composed of dorsal k1 and ventral k2) and a small lateral (k3) subdivision. In this study, the nucleus “k” subdivisions were examined in the rabbit with respect to projections to the cortex of rostral (rPML; face-forelimb region) and caudal (cPML; hindlimb region) paramedian lobule of the cerebellum. The retrograde fluorescent labeling method with Fast Blue (FB) and Diamidino Yellow (DY) was used. Numerous single FB or DY labeled neurons were found in defined regions of all nucleus “k” subdivisions bilaterally, with an ipsilateral preponderance. The distribution of these neurons indicated that afferents originating from different nucleus “k” subdivisions terminated in overlapping regions within the rPML and the cPML rather than in separate domains. Apart from this, double FB + DY labeled neurons (n = 104) were intermingled within a common region of single labeling, but exclusively on the ipsilateral side. Such neurons occupied predominantly the central and lateral regions of the caudal two thirds of the k1 subdivision, and were scattered in the caudal half of k2 as well as throughout the entire rostrocaudal extent of the k3 subdivision. The size of labeled perikarya varied from 20 to 40 ?m in diameter. The number of neurons with branching axons was considerably lower than those with single projections to the rPML and the cPML. It amounted to about 3% in k1 and k3, and 2% in the k2 subdivision. However, this population may form an intralobular link between two somatotopically non-corresponding PML regions. The present study provides a morphological basis for further investigations for comparison with other species using both anatomical and electrophysiological methods, also with respect to other connections of the nucleus “k”. [Copyright &y& Elsevier]

The double fluorescent retrograde technique was employed to examine the distribution of the inferior olive (IO) neurones projecting to the cortex of the rostral and caudal parts of the paramedian lobule (PML) in the rabbit cerebellum, known to be the face-forelimb and hindlimb receiving areas, respectively. Moreover, this technique was also used to investigate the possibility that IO projections reaching these two somatotopically non-homologous PML regions are collaterals of the same axones. No other reports have addressed this question. After non-overlapping unilateral injections of the cytoplasmic tracer fast blue (FB) and the nuclear dye diamidino yellow (DY) into the rostral and caudal PML, respectively, numerous single FB- or DY-labelled cells were found in the defined regions of the contralateral IO. These regions showed considerable overlap, apart from the dorsal accessory olive where a clear spatial separation of labelled cell groups was observed. Furthermore, double FB + DY-labelled neurones (n = 310) were seen in the medial accessory olive, the dorsal and ventral laminas of the principal olive, in the dorsomedial cell column and the β nucleus. It suggests that IO neurones may branch to supply the two functionally different PML regions and in this way participate in the mechanisms of forelimb-hindlimb coordination.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The neocortex is composed of a characteristic layered structure, which is a basis of extrinsic and intrinsic cortical connections. In recent years the cellular and molecular mechanisms, which are responsible for the formation of lamina-specific connections, have been explored by extensive molecular and in vitro studies. This article attempts to address what cell–cell interactions are required for axonal targeting and what molecules regulate cellular events, focusing upon the development of the thalamocortical projection. [Copyright &y& Elsevier]

Vasoactive intestinal polypeptide (VIP)-immunoreactive cells in the primary visual cortex of the rat were classified on the basis of ramification pattern of cell processes. The distribution of cells over cortical layers, and proportions of cell classes relative to total cell numbers were evaluated by means of quantitative methods. Two main types of VIP-positive neurons, the bipolar and the multipolar were distinguished constituting 76% and 24% of the VIP populations, respectively. The axons of vertically oriented bipolars were observed to ramify within a column around the descending dendrite. By contrast, multipolar cells have a non-oriented ramification pattern. The two overlapping axonal systems form the VIP-innervation of the rat visual cortex. [ABSTRACT FROM AUTHOR]