BIG-2 mediates olfactory axon convergence to target glomeruli

Olfactory sensory neurons expressing a given odorant receptor converge axons onto a few topographically fixed glomeruli in the olfactory bulb, leading to establishment of the odor map. Here, we report that BIG-2/contactin-4, an axonal glycoprotein belonging to the immunoglobulin superfamily, is expressed in a subpopulation of mouse olfactory sensory neurons. A mosaic pattern of glomerular arrangement is observed with strongly BIG-2-positive, weakly positive, and negative axon terminals in the olfactory bulb, which is overlapping but not identical with those of Kirrel2 and ephrin-A5. There is a close correlation between the BIG-2 expression level and the odorant receptor choice in individual sensory neurons. In BIG-2-deficient mice, olfactory sensory neurons expressing a given odorant receptor frequently innervate multiple glomeruli at ectopic locations. These results suggest that BIG-2 is one of the axon guidance molecules crucial for the formation and maintenance of functional odor map in the olfactory bulb.     

Semaphorin-1a controls receptor neuron-specific axonal convergence in the primary olfactory center of Drosophila

In the olfactory system of Drosophila, 50 functional classes of sensory receptor neurons (ORNs) project in a highly organized fashion into the CNS, where they sort out from one another and converge into distinct synaptic glomeruli. We identified the transmembrane molecule Semaphorin-1a (Sema-1a) as an essential component to ensure glomerulus-specific axon segregation. Removal of sema-1a in ORNs does not affect the pathfinding toward their target area but disrupts local axonal convergence into a single glomerulus, resulting in two distinct targeting phenotypes: axons either intermingle with adjacent ORN classes or segregate according to their odorant receptor identity into ectopic sites. Differential Sema-1a expression can be detected among neighboring glomeruli, and mosaic analyses show that sema-1a functions nonautonomously in ORN axon sorting. These findings provide insights into the mechanism by which afferent interactions lead to synaptic specificity in the olfactory system.     

RasGRF1 mediates brain-derived neurotrophic factor-induced axonal growth in primary cultured cortical neurons

The appropriate development and regulation of neuronal morphology are important to establish functional neuronal circuits and enable higher brain function of the central nervous system. R-Ras, a member of the Ras family of small GTPases, plays crucial roles in the regulation of axonal morphology, including outgrowth, branching, and guidance. GTP-bound activated R-Ras reorganizes actin filaments and microtubules through interactions with its downstream effectors, leading to the precise control of axonal morphology. However, little is known about the upstream regulatory mechanisms for R-Ras activation in neurons. In this study, we found that brain-derived neurotrophic factor (BDNF) has a positive effect on endogenous R-Ras activation and promotes R-Ras-mediated axonal growth. RNA interference knockdown and overexpression experiments revealed that RasGRF1, a guanine nucleotide exchange factor (GEF) for R-Ras, is involved in BDNF-induced R-Ras activation and the promotion of axonal growth. Phosphorylation of RasGRF1 by protein kinase A at Ser916/898 is needed for the full activation of its GEF activity and to facilitate Ras signaling. We observed that BDNF treatment markedly increased this phosphorylation. Our results suggest that BDNF is one of the critical extrinsic regulators for R-Ras activation, and that RasGRF1 is an intrinsic key mediator for BDNF-induced R-Ras activation and R-Ras-mediated axonal morphological regulation.     

A divergent pattern of sensory axonal projections is rendered convergent by second-order neurons in the accessory olfactory bulb

The mammalian vomeronasal system is specialized in pheromone detection. The neural circuitry of the accessory olfactory bulb (AOB) provides an anatomical substrate for the coding of pheromone information. Here, we describe the axonal projection pattern of vomeronasal sensory neurons to the AOB and the dendritic connectivity pattern of second-order neurons. Genetically traced sensory neurons expressing a given gene of the V2R class of vomeronasal receptors project their axons to six to ten glomeruli distributed in globally conserved areas of the AOB, a theme similar to V1R-expressing neurons. Surprisingly, second-order neurons tend to project their dendrites to glomeruli innervated by axons of sensory neurons expressing the same V1R or the same V2R gene. Convergence of receptor type information in the olfactory bulb may represent a common design in olfactory systems.     

SHP-2 mediates target-regulated axonal termination and NGF-dependent neurite growth in sympathetic neurons

The tyrosine phosphatase SHP-2 has been implicated in a variety of signaling pathways, including those mediated by neurotrophins in neurons. To examine the role of SHP-2 in the development of sympathetic neurons, we inhibited the function of SHP-2 in transgenic mice by overexpressing a catalytically inactive SHP-2 mutant under the control of the human dopamine beta-hydroxylase promoter. Expression of mutant SHP-2 did not influence the survival, axon initiation, or pathfinding abilities of the sympathetic neurons. However, mutant SHP-2 expression resulted in an overproduction of sympathetic fibers in sympathetic target organs. This was due to interference with SHP-2 function, as overexpression of wild type SHP-2 had no such effect. In vitro, NGF-dependent neurite growth was inhibited in neurons expressing mutant SHP-2 but not in those expressing wild type SHP-2. Mutant (but not wt) SHP-2 expression also inhibited NGF-stimulated ERK activation. The NGF-dependent survival pathway was less affected than the neurite growth pathway. Our results suggest that NGF-regulated axon growth signals, and to a lesser degree survival signals, are mediated through a SHP-2-dependent pathway in sympathetic neurons. The increased sympathetic innervation in target tissues of neurons expressing mutant SHP-2 may result from interference with normal "stop" signals dependent on signaling by gradients of NGF.     

Retinoic acid receptor and CNGA2 channel signaling are part of a regulatory feedback loop controlling axonal convergence and survival of olfactory sensory neurons

Little is known about the identities and functions of extracellular signaling molecules that work in concert with neuronal activity to regulate refinement and maintenance of the mouse olfactory sensory map. We show that expression of a dominant negative retinoic acid receptor (RAR) in olfactory sensory neurons (OSNs) increased the number of glomeruli that incorrectly contained OSN axons expressing different odorant receptors. This phenotype became apparent postnatally, coincided with increased cell death, and was preceded by increased Neuropilin-1 and reduced Kirrel-2 expressions. Kirrel-2-mediated cell adhesion influences odorant receptor-specific axonal convergence and is regulated by odorant receptor signaling via the olfactory cyclic nucleotide-gated (CNG) ion channel. Accordingly, we found that inhibited RAR function correlated with reduced CNG channel expression. Naris occlusion experiments and analysis of CNG channel-deficient mice further indicated that RAR-regulated CNG channel levels influenced the intrinsic neuronal activity required for cell survival in the absence of odor stimulation. Finally, we showed that CNG channel activity regulated expression of the retinoic acid-degrading enzyme Cyp26B1. Combined, these results identify a novel homeostatic feedback mechanism involving retinoic acid metabolism and CNG channel activity, which influences glomerular homogeneity and maintenance of precisely connected OSNs.     

Modification of synapse formation of accessory olfactory bulb neurons by coculture with vomeronasal neurons

Previously, a coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal (VN) neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of VN neurons on the development of AOB neurons was examined in a coculture system. Spine density was quantitatively measured for various culture periods of 21, 28, 36, and 42 days in vitro. The densities of dendritic spines were lower in the coculture than in single culture for all periods in vitro. Synapse formation on spines was analyzed immunocytochemically using an anti-synaptophysin antibody. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. These changes were not observed in the coculture in which there was no physical contact between AOB neurons and VN neurons. These observations suggest that synapse formation on the spines of AOB neurons is modified by physical contact with VN neurons.     

Extracellular signal-regulated kinase 1/2 mediates survival, but not axon regeneration, of adult injured central nervous system neurons in vivo

Neurotrophins play important roles in the response of adult neurons to injury. The intracellular signaling mechanisms used by neurotrophins to regulate survival and axon growth in the mature CNS in vivo are not well understood. The goal of this study was to define the role of the extracellular signal-regulated kinases 1/2 (Erk1/2) pathway in the survival and axon regeneration of adult rat retinal ganglion cells (RGCs), a prototypical central neuron population. We used recombinant adeno-associated virus (AAV) to selectively transduce RGCs with genes encoding constitutively active or wild-type mitogen-activated protein kinase kinase 1 (MEK1), the upstream activator of Erk1/2. In combination with anterograde and retrograde tracing techniques, we monitored neuronal survival and axon regeneration in vivo. MEK1 gene delivery led to robust and selective transgene expression in multiple RGC compartments including cell bodies, dendrites, axons and targets in the brain. Furthermore, MEK1 activation induced in vivo phosphorylation of Erk1/2 in RGC bodies and axons. Quantitative analysis of cell survival demonstrated that Erk1/2 activation promoted robust RGC neuroprotection after optic nerve injury. In contrast, stimulation of the Erk1/2 pathway was not sufficient to induce RGC axon growth beyond the lesion site. We conclude that the Erk1/2 pathway plays a key role in the survival of axotomized mammalian RGCs in vivo, and that activation of other signaling components is required for axon regeneration in the growth inhibitory CNS environment.     

GABA-mediated inhibition of primary olfactory receptor neurons

Applying GABA (1 microM-1 mM) to the soma of cultured lobster olfactory receptor neurons evokes an inward current (V(m) = -60 mV) accompanied by an increase in membrane conductance, with a half-effect of 487 microM GABA. The current-voltage relationship of this current is linear between -100 and 100 mV and reverses polarity at the equilibrium potential for Cl(-). The current is blocked by picrotoxin and bicuculline methiodide, and is evoked by trans-aminocrotonic acid, isoguvacine, muscimol, imidazole-4-acetic acid, and 3-amino-1-propanesulfonic acid, but not by the GABA(C)-receptor agonist cis-4-aminocrotonic acid and the GABA(B)-receptor agonist 3-aminopropylphosphonic. Applying GABA to the soma of the cells in situ reversibly suppresses the spontaneous discharge and substantially decreases the odor-evoked discharge. The effects of GABA on the cell soma in situ are antagonized by both picrotoxin and bicuculline methiodide. Taken together with evidence that GABA directly activates a chloride channel in outside-out patches excised from the soma of these neurons, we conclude that lobster olfactory receptor neurons express an ionotropic GABA receptor that can potentially regulate the output of these cells. Copyright Copyright 1999 S. Karger AG, Basel     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.     

Odorant representations are modulated by intra- but not interglomerular presynaptic inhibition of olfactory sensory neurons

Input to the central nervous system from olfactory sensory neurons (OSNs) is modulated presynaptically. We investigated the functional organization of this inhibition and its role in odor coding by imaging neurotransmitter release from OSNs in slices and in vivo in mice expressing synaptopHluorin, an optical indicator of vesicle exocytosis. Release from OSNs was strongly suppressed by heterosynaptic, intraglomerular inhibition. In contrast, inhibitory connections between glomeruli mediated only weak lateral inhibition of OSN inputs in slices and did not do so in response to odorant stimulation in vivo. Blocking presynaptic inhibition in vivo increased the amplitude of odorant-evoked input to glomeruli but had little effect on spatial patterns of glomerular input. Thus, intraglomerular inhibition limits the strength of olfactory input to the CNS, whereas interglomerular inhibition plays little or no role. This organization allows for control of input sensitivity while maintaining the spatial maps of glomerular activity thought to encode odorant identity.     

Ectopic trkA expression mediates a NGF survival response in NGF- independent sensory neurons but not in parasympathetic neurons

We have investigated the role of trkA, the tyrosine kinase NGF receptor, in mediating the survival response of embryonic neurons to NGF. Embryonic trigeminal mesencephalic (TMN) neurons, which normally survive in the presence of brain-derived neurotrophic factor (BDNF) but not NGF, become NGF-responsive when microinjected with an expression vector containing trkA cDNA. In contrast, microinjection of ciliary neurotrophic factor (CNTF)-dependent embryonic ciliary neurons with the same construct does not result in the acquisition of NGF responsiveness by these neurons despite de novo expression of trkA mRNA and protein. The failure of trkA to result in an NGF-promoted survival response in ciliary neurons is not due to absence of the low-affinity NGF receptor, p75, in these neurons. Quantitative RT/PCR and immunocytochemistry showed that TMN and ciliary neurons both express p75 mRNA and protein. These findings not only provide the first direct experimental demonstration of trkA mediating a physiological response in an appropriate cell type, namely NGF-promoted survival of embryonic neurons, but indicate that not all neurons are able to respond to a trkA-mediated signal transduction event.     

Normal levels of Rac1 are important for dendritic but not axonal development in hippocampal neurons

Background information. Rho family GTPases are required for cytoskeletal reorganization and are considered important for the maturation of neurons. Among these proteins, Rac1 is known to play a crucial role in the regulation of actin dynamics, and a number of studies indicate the involvement of this protein in different steps of vertebrate neuronal maturation. There are two distinct Rac proteins expressed in neurons, namely the ubiquitous Rac1 and the neuron‐specific Rac3. The specific functions of each of these GTPases during early neuronal development are largely unknown. Results. The combination of the knockout of Rac3 with Rac1 down‐regulation by siRNA (small interfering RNA) has been used to show that down‐regulation of Rac1 affects dendritic development in mouse hippocampal neurons, without affecting axons. F‐actin levels are strongly decreased in neuronal growth cones following down‐regulation of Rac1, and time‐lapse analysis indicated that the reduction of Rac1 levels decreases growth‐cone dynamics. Conclusions. These results show that normal levels of endogenous Rac1 activity are critical for early dendritic development, whereas dendritic outgrowth is not affected in hippocampal neurons from Rac3‐null mice. On the other hand, early axonal development appears normal after Rac1 down‐regulation. Our findings also suggest that the initial establishment of neuronal polarity is not affected by Rac1 down‐regulation.     

Parenchymal,but not leukocyte,TNF receptor 2 mediates T cell-dependent hepatitis in mice

TNF-alpha is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type --> tnfr2 degrees and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-kappaB activation and cytokine production in the livers of both wild-type and tnfr2 degrees mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2 degrees, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.     

Drosophila olfactory response rhythms require clock genes but not pigment dispersing factor or lateral neurons

Odors elicit a number of behavioral responses, including attraction and repulsion in Drosophila. In this study, the authors used a T-maze apparatus to show that wild-type Drosophila melanogaster exhibit a robust circadian rhythm in the olfactory attractive and repulsive responses. These responses were lower during the day and began to rise at early night, peaking at about the middle of the night and then declining thereafter. They were also independent of locomotor activity. The olfactory response rhythms were lost in period or timeless mutant flies (per0, tim0), indicating that clock genes control circadian rhythms of olfactory behavior. The rhythms in olfactory response persisted in the absence of the pigment-dispersing factor neuropeptide or the central pacemaker lateral neurons known to drive circadian patterns of locomotion and eclosion. These results indicate that the circadian rhythms in olfactory behavior in Drosophila are driven by pacemakers that do not control the rest-activity cycle and are likely in the antennae.     

Role of macrophages as modulators but not as autonomous accessory cells in primary antibody response

The role of macrophages (M phi) in the in vitro primary antibody response of murine lymphocytes to sheep erythrocytes was investigated. Peritoneal M phi were activated to express Ia antigens either in vitro or in vivo. Nonactivated Ia- M phi were also examined. We observed that only Ia- M phi but also Ia+ M phi failed to trigger the antibody response, in contrast with splenic dendritic cells (DC) which served as potent and autonomous accessory cells, but that M phi modulated the level of response which was dependent primarily on the DC content of culture. The modulation appeared to incline to suppression rather than enhancement, when M phi were allowed to remain throughout the culture period for 4 days. A highly enhancing capacity of M phi, however, could be revealed by removing M phi 2 days after the initiation of culture, indicating that M phi exerted their suppressive effect more strongly in the late phase than in the early phase of in vitro antibody response. The modulatory activity seemed higher in Ia+ M phi than in Ia- M phi.     

Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons

Olfactory bulb ensheathing glia (OEG) have attracted special attention during the last few years because of their unique properties in promoting regeneration of adult mammalian central nervous system (CNS) components. However the molecular and cellular characteristics responsible for this capacity remain to be revealed. Such studies are presently hindered by the lack of a plentiful source of homogenous OEG. Thus the availability of immortalized OEG lines maintaining the regenerative characteristics of the primary cultures would represent an unlimited source of OEG for use not only in biochemical analyses of neuroregenerative mechanisms but also to characterize their regenerative properties in models in culture and in vivo. We have immortalized primary rat OEG using the SV40 large T antigen expressed from a constitutive cellular promotor, and report here the isolation and characterization of clonal lines. These OEG clonal lines were comparable to primary OEG and Schwann cells in the promotion of axonal regeneration of mature rat retinal ganglion neurons (RGN) but, significantly, this culture assay system more closely reflects the in vivo reparative properties of OEG on transected nerves than other assays of neuritogenesis in that it revealed OEG cells to promote the growth of a larger number of long axons than Schwann cells. Using this assay we were able to grade our OEG lines for their neuroregenerative capacity, opening the possibility of identifying molecules with correlative expression levels in these cells. Our preliminary characterization revealed that the expression level of a classical OEG marker, the p75-NGF receptor, does not correlate with neuroregenerative capacity.     

Xanthine oxidase mediates cytokine-induced,but not hormone-induced bone resorption

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have been implicated as mediators of osteoclastic bone resorption. Xanthine oxidase (XO) a ubiquitous enzyme is widely known for its production of these ROS. We therefore evaluated the potential of XO as a source of ROS in cytokine-and hormone-induced bone resorption. XO activity in rat calvarial osteoblasts was found to be significantly elevated upon stimulation by the cytokines, TNFalpha and IL-1beta. These cytokines also caused a dose related increase in bone resorption of mouse calvariae, which was significantly inhibited by catalase (10 IU/ml). Allopurinol, the competitive inhibitor of XO, also caused a dose related (1-50 microM) inhibition of TNFalpha (20 ng/ml) and (0.01-10 microM) IL-1beta (50 IU/ml)-induced bone resorption, respectively. PTH- and 1,25-(OH)2 Vitamin D3-induced bone resorption could also be inhibited by catalase (100 IU/ml) but was unaffected by allopurinol, indicating that another mediator, other than XO, is required for hormone-induced bone resorption. These results demonstrate, that modulation of the redox balance in the bone microenvironment, which contains XO, can affect the bone resorbing process. Therefore, XO may play a pivotal role in cytokine-induced bone resorption and, if manipulated appropriately, could show a therapeutic benefit in inflammatory bone disorders such as RA.