Review Nitric oxide as a regulator of neuronal motility and regeneration in the locust embryo Michael Stern *, Gerd Bicker Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, D-30173 Hannover, Germany Contents 1. Introduction ..................................................................................................... 958 2. Nitric oxide/cGMP signaling regulates peripheral neurite outgrowth ........................................................ 960 3. NO/cGMP in neuronal migration in the locust enteric nervous system ...................................................... 962 4. NO/cGMP during central nervous system development................................................................... 962 5. Nitric oxide/cGMP during axonal regeneration in the ventral nerve cord .................................................... 963 6. NO and neuronal development and plasticity in vertebrates .............................................................. 963 7. Conclusions ..................................................................................................... 964 Acknowledgements ............................................................................................... 964 References ...................................................................................................... 964 1. Introduction Nitric oxide (NO) is a gaseous cellular messenger with multiple functions in the vascular, immune, and nervous system. In contrast to classical vesicle-released transmitters, NO is produced in a calcium-dependent manner by the cytoplasmic enzyme NO-synthase (NOS) and diffuses freely across cell membranes into target cells (Fig. 1). There, its main receptor is soluble guanylyl cyclase (sGC), which responds to NO binding by producing the second messenger, cGMP (reviewed by Garthwaite, 2008). In the vertebrate brain, important roles of NO signaling pathways have been implicated in cell prolifera- tion, synaptogenesis, synaptic plasticity, and neurological disease (Boehning and Snyder, 2003; Godfrey and Schwarte, 2003; Packer et al., 2003; Keynes and Garthwaite, 2004). Since the two migratory locust species (Locusta migratoria, Schistocerca gregaria) have been used as key experimental preparations to unravel neuroendocrine mechanisms in insect behavior and development (Norris and Pener, 1965; Pener et al., 1978; Ayali and Pener, 1995), some research labs relied also on these robust animals for the analysis of NO signaling in neuronal function. And indeed, locusts made possible the initial biochemical demonstration for calcium-dependent NO production in the insect brain (Elphick et al., 1993, 1995; Mu ¨ ller and Bicker, 1994). They continued to be prime subjects of studies regarding the involvement of NO in the processing of visual (Elphick et al., Journal of Insect Physiology 56 (2010) 958–965 ARTICLE INFO Article history: Received 23 February 2010 Received in revised form 18 March 2010 Accepted 19 March 2010 Keywords: Growth cone Insect embryo cGMP Protein kinase G Carbon monoxide ABSTRACT Nitric oxide (NO) is known as a gaseous messenger in the nervous system. It plays a role in synaptic plasticity, but also in development and regeneration of nervous systems. We have studied the function of NO and its signaling cascade via cyclic GMP in the locust embryo. Its developing nervous system is well suited for pharmacological manipulations in tissue culture. The components of this signaling pathway are localized by histochemical and immunofluorescence techniques. We have analyzed cellular mechanisms of NO action in three examples: 1. in the peripheral nervous system during antennal pioneer axon outgrowth, 2. in the enteric nervous system during migration of neurons forming the midgut nerve plexus, and 3. in the central nervous system during axonal regeneration of serotonergic neurons after axotomy. In each case, internally released NO or NO-induced cGMP synthesis act as permissive signals for the developmental process. Carbon monoxide (CO), as a second gaseous messenger, modulates enteric neuron migration antagonistic to NO. ß 2010 Elsevier Ltd. All rights reserved. * Corresponding author at: Stiftung Tiera ¨ rztliche Hochschule Hannover, Physio- logisches Institut, Zellbiologie, Bischofsholer Damm 15/102, D-30173 Hannover, Germany, Tel.: +49 511 856 7767; fax: +49 511 856 7687. E-mail address: [email protected](M. Stern). Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.03.031
Transcript
Journal of Insect Physiology 56 (2010) 958–965
Review
Nitric oxide as a regulator of neuronal motility and regeneration in thelocust embryo
Michael Stern *, Gerd Bicker
Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, D-30173 Hannover, Germany
Nitric oxide (NO) is a gaseous cellular messenger withmultiple functions in the vascular, immune, and nervous system.In contrast to classical vesicle-released transmitters, NO isproduced in a calcium-dependent manner by the cytoplasmicenzyme NO-synthase (NOS) and diffuses freely across cellmembranes into target cells (Fig. 1). There, its main receptor issoluble guanylyl cyclase (sGC), which responds to NO binding byproducing the second messenger, cGMP (reviewed by
0022-1910/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jinsphys.2010.03.031
Garthwaite, 2008). In the vertebrate brain, important roles ofNO signaling pathways have been implicated in cell prolifera-tion, synaptogenesis, synaptic plasticity, and neurologicaldisease (Boehning and Snyder, 2003; Godfrey and Schwarte,2003; Packer et al., 2003; Keynes and Garthwaite, 2004). Sincethe two migratory locust species (Locusta migratoria, Schistocerca
gregaria) have been used as key experimental preparations tounravel neuroendocrine mechanisms in insect behavior anddevelopment (Norris and Pener, 1965; Pener et al., 1978; Ayaliand Pener, 1995), some research labs relied also on these robustanimals for the analysis of NO signaling in neuronal function.And indeed, locusts made possible the initial biochemicaldemonstration for calcium-dependent NO production in theinsect brain (Elphick et al., 1993, 1995; Muller and Bicker, 1994).They continued to be prime subjects of studies regarding theinvolvement of NO in the processing of visual (Elphick et al.,
(RpcGMPS) blocks cellular responses of the cGMP/PKG pathway. The NO donor
sodium nitroprusside (SNP) and the membrane permeable cGMP analogue 8-
bromo-cGMP (8Br-cGMP) can be applied to raise cGMP levels in the target cell.
Carbon monoxide (CO) produced intracellularly by activation of the enzyme heme
oxygenase (HO-2) may regulate sGC activity by competition with NO.
Fig. 2. NO enhances pioneer neuron outgrowth in the locust antenna. (A–D)
Antennal tips of 32% embryos after 24 h in culture stained for a general neuronal
marker (anti-horseradish peroxidase). Two pairs of pioneer neurons (open arrows,
black arrows) and a guidepost cell at the base of the antenna (white arrows) are
labeled. In control experiments (A), pioneer neurites reached the guidepost cell
within 24 h of culture. When NO synthesis was blocked by addition of 500 mM 7NI
(B), or sGC was blocked by 200 mM ODQ (C), axons failed to reach the guidepost cell.
In a rescue experiment this growth inhibition by ODQ was cancelled by addition of
500 mM of the membrane permeant cGMP analogue 8Br-cGMP (D). cGMP
production is revealed in the antennal pioneer neurons after stimulation by the
NO donor SNP in the presence of phosphodiesterase inhibitor, IBMX, and
subsequent immunostaining for cGMP (E).
M. Stern, G. Bicker / Journal of Insect Physiology 56 (2010) 958–965 959
1996; Bicker and Schmachtenberg, 1997; Seidel and Bicker,1997; Schmachtenberg and Bicker, 1999) and olfactory informa-tion (Elphick et al., 1995; Bicker et al., 1997; O’Shea et al., 1998)as well as in the modulation of motor patterns (Rast, 2001;Wenzel et al., 2005; Rand et al., 2008).
Recently, there have been several significant advances in themethodology of histological demonstrations of the NO–cGMPpathway. NO-induced cGMP can be visualized immunocytochemi-cally (De Vente et al., 1987), when living tissue is incubated with anNO donor together with a phosphodiesterase inhibitor which leadsto accumulation of cGMP, which can, after fixation, be stained(Figs. 2–5). This technique has been optimized by the introductionof the allosteric activator of cGC, YC-1 (Friebe and Koesling, 1998;Ott et al., 2004). A simple method for visualization of nitrergicneurons is the NADPH-diaphorase technique (NADPHd) whichexploits the fact that NOS depends on NADPH as a co-factor whichis oxidized during NO production. If fixed tissue is incubated withNADPH and reducible tetrazolium dyes, NOS converts the yellowtetrazolium into an insoluble blue formazan (Figs. 4(B) and 5(G)).Under suitable conditions, NOS is the only NADPH-convertingenzyme which is resistant to fixation. The resolution of thismethod has been greatly improved by optimized fixationtechniques (Ott and Elphick, 2002, 2003). Finally, improvedmonoclonal antibodies against the by-product of NO synthesis,L-citrulline, have enabled visualization of NOS activity at highspatial resolution (Martinelli et al., 2002; Wenzel et al., 2005; Sieglet al., 2009).
In this review, we focus on the role of NO/cGMP duringdevelopment and repair of the locust nervous system. Sinceembryonic development proceeds in whole animal tissue culture,the locust embryo can serve as convenient experimental model tostudy the effects of chemically manipulating NO/cGMP signaling(Fig. 1) on the formation of the nervous system.
Fig. 3. Nitric oxide regulates enteric neuron migration on the embryonic locust midgut. (A–D) NO-induced cGMP immunostaining on the midgut at different embryonic
stages. Leading neurons display filopodia (A and B), are followed by a chain of migrating cells (C), and later leave the migratory routes to form a plexus (D). (E–G) Drawings of
cGMP-stained enteric neurons before (start), or after 24 h in culture (all other drawings), only two out of four migratory routes are drawn. At the beginning of the experiment,
neurons are grouped at the boundary between midgut (yellow) and foregut. Within 24 h, they migrate�300 mm posterior (E). Inhibition of NOS by 7NI retards migration, but
can be rescued by direct stimulation of sCG by protoporphyrin IX (F). Inhibition of sCG by ODQ also inhibits migration, but is rescued by application of 8Br-cGMP (G).
Fig. 4. Development of the NO–cGMP pathway in the brain. (A) Schematic diagram of the brain of a locust nymph just after completing embryogenesis with the major neuropil
areas. (B) Brain of a 90% embryo stained for NADPH-diaphorase. (C) Time table of nitrergic marker expression during development of the locust from embryogenesis to
adulthood. al: antennal lobe, cbl: central body, lower division, cbu: central body, upper division, lam: lamina, lob: lobula, med: medulla, ol dist: distal optic lobe, ol prox:
Pioneer neurons establish the first axonal pathways that arefollowed by later-growing axons using mechanisms of contactguidance. This pathfinding strategy is beautifully exemplified in
locust embryos where the early axonal pathways in the peripheralnervous system are laid down by easily identifiable pioneerneurons growing from the tip of each appendage into the CNS,while distances are still short (Bate, 1976; Bentley and O’Connor,1992). Pathfinding appears to involve selective adhesion of theirgrowth cones to substrate bound guidance cues and guidepost cells
Fig. 5. NO enhances regeneration of serotonergic axons in the ventral nerve cord. (A) Fillet preparation of a 65% locust embryo for tissue culture with exposed CNS. (B) One of
two connectives between abdominal ganglia is crushed (arrowheads) in two positions before 48 h of culture. (C) Serotonin immunostaining after 48 h in culture reveals four
cell bodies in the ganglion, four axons in uncrushed connectives, and a variable number of regenerated axons on the lesion side (arrowheads). (D) Under control conditions,
�40% of the axons regenerate; in the presence of the NO donor NOC-18 (500 mM), more than 60% do. (E) Removing NO by the NO-scavenger carboxy-PTIO reduces
regeneration. This can be partially rescued by application of membrane permeable cGMP. (F) Inhibiting sCG by application of ODQ (200 mM) reduces regeneration, which can
be rescued by co-application of 8Br-cGMP. Data in (D–F) are means from 10 embryos. (G) NADPHd reveals the presence of putative NO producing neurons that this stage. (H)
Confocal image of an abdominal ganglion double-labeled for NO-induced cGMP and serotonin. Many cells respond to NO, including the serotonergic neurons.
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(Bentley and O’Connor, 1992) as well as on two gradients of the cellrecognition molecule sema-2a (Isbister et al., 1999; Legg andO’Connor, 2003).
In the locust antenna, the first neural pathways are establishedin a similar manner by identified pairs of pioneer neurons at theantennal tip. There are two pairs of pioneers (ventral and dorsal,Fig. 2), which send their neurites in separate pathways proximally(Bate, 1976; Ho and Goodman, 1982; Berlot and Goodman, 1984),to pioneer the antennal nerves together with later pioneer neuronsborn in a step-wise manner along the growing antennal bud(Boyan and Williams, 2004).
Developmental neurobiologists are accustomed to the conceptof neurite outgrowth as being guided by the extracellulardistribution of attractive and repellent guidance cues. Theseguidance cues comprise secreted or cell surface bound families ofproteins that are ligands of specific receptor types on themembrane of motile growth cones (Song and Poo, 2001; Dickson,
2002; Chilton, 2006). Within this conceptual framework, immu-nocytochemical evidence for a role of the gaseous messenger NO ingrowth cone behavior of pioneer neurons was somewhat surpris-ing. Outgrowing pioneer neurons at the tip of the antennasynthesize cGMP in response to exogenous NO treatment(Fig. 2(E)). Possible sources of NO are epithelial cells that facethe basal lamina in the embryonic antenna, which transiently stainfor NADPHd during a period ranging from about 32% to 35% ofembryonic development, exactly during the phase of pioneerneuron outgrowth (Seidel and Bicker, 2000).
Using an embryo culture system, it could be shown thatpharmacological inhibition of endogenous NO-synthase and sGCactivity results in a perturbation of the pioneering pathways fromthe tip of the antenna (Fig. 2). Since the pharmacologicaldisruption of pioneering pathways can be rescued by supple-menting the whole embryo culture with membrane permeantcGMP or with a NO-independent activator of sGC (Fig. 1)
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unspecific side effects of the enzyme blockers are unlikely (Seideland Bicker, 2000). Thus, embryonic pioneer neuron outgrowthconstitutes an accessible in vivo system in which the role of NO/cGMP signaling during pathfinding can be studied at the level ofidentified nerve cells.
3. NO/cGMP in neuronal migration in the locust entericnervous system
Since molecular guidance cues of axonal outgrowth are alsoused for directed movements of neuronal cell bodies (Song andPoo, 2001) our laboratory (Haase and Bicker, 2003), and othersbefore (Wright et al., 1998), hypothesized that NO/cGMP signalingmight influence migration of embryonic insect neurons. Theformation of the insect stomatogastric or enteric nervous system(ENS) provides a well-established model to study the cell biology ofneuronal migration (Hartenstein, 1997). The midgut plexus (MG)neurons of the locust embryo are born in a neurogenic zone on theforegut, forming a packet of postmitotic but immature neurons atthe foregut–midgut boundary (Ganfornina et al., 1996). Subse-quently, they undergo a rapid phase of migration, during which theneurons cross the foregut–midgut boundary and move in fourmigratory pathways on the midgut surface (Fig. 3). At thecompletion of migration, the MG neurons invade the spacebetween the four migratory pathways and extend terminalsynaptic branches on the midgut musculature.
Locust MG neurons exhibit inducible cGMP-IR throughout thephase of migration (Fig. 3(A)–(D)) and neurite branching (Haaseand Bicker, 2003). When the midgut plexus acquires its matureconfiguration, the cGMP-IR decreases, indicating a precise coin-cidence of sGC activity with periods of neuronal motility andoutgrowth. NADPHd histochemistry indicated labeling in theembryonic midgut epithelium (Haase and Bicker, 2003) andWestern blots probed with an antibody against a conservedsequence revealed the expression of NOS enzyme in midgut tissue(Knipp and Bicker, 2009a).
To establish a causal role of NO/cGMP signaling in the directedmigration of the MG neurons we used again pharmacologicalmanipulations in whole embryo culture (Haase and Bicker, 2003;Knipp and Bicker, 2009a). Blocking of endogenous NO synthesis bythe NOS inhibitor 7NI (Fig. 1) retards migration of the MG neurons.Since incubation with the extracellular NO-scavenger hemoglobin(Fig. 1) blocked migration, a transcellular NO signal from themidgut epithelium seems to regulate MG neuron motility (Knippand Bicker, 2009a).
Treatment with ODQ, a specific inhibitor of sGC, also preventsthe MG neuron migration in a dose-dependent manner. In embryostreated with the specific PKG inhibitor RPcGMPS, MG neuronmigration is significantly reduced. This effect suggests that cGMPmight influence migration via activating PKG.
The disruption of MG neuron migration caused by inhibiting NOproduction or cGMP synthesis can be rescued by exogenousapplication of membrane permeant cGMP and pharmacologicalstimulation of sGC (Fig. 3(F) and (G)), suggesting that in vivo acertain level of cGMP is necessary for MG neuron migration. Therescue experiments show clearly that NO/cGMP signaling isessential for the regulation of neuronal migration in the developingENS of the locust. Since pharmacological inhibition of NOS or sGCcauses no significant misrouting of the MG neurons, there is noevidence for a directional guidance function of NO. Thus, growthcone motility and guidance are separate processes. Moreover, thefact that a simple, spatially homogeneous bath application of NOdonors and cGMP to the culture medium can rescue the defect inmigration argues against a role of NO as a guidance factor fordirected cell migration of the MG neurons. Rather, the appearanceof inducible sGC activity in the MG neurons just at the onset of
migration suggests that NO/cGMP signaling might be required forthe initiation and maintenance of migratory behavior.
In animals carbon monoxide (CO) is produced by hemeoxygenase enzymes (HO-2) as a by-product during the cleavageof heme (Boehning and Snyder, 2003). This gaseous messenger isalso thought to be a member of the atypical signaling molecules inthe nervous system (Boehning and Snyder, 2003). In the locustembryo, the enteric neurons exhibit a transient immunoreactivityto the HO-2 enzyme, the constitutive isoform that generates CO inneural tissue, while migrating on the midgut (Knipp and Bicker,2009a). Pharmacological inhibition of HO-2 by zinc-protopor-phyrin IX or zinc deuteroporphyrin-2,4-bis-glycol enhances in again-of-function experiment midgut neuron migration (Knipp andBicker, 2009a). However, the transduction pathway of the COsignal is not yet known. Since both messengers can bind to sGC, butCO is less efficient than NO to stimulate cGMP formation, weproposed a molecular competition mechanism for the regulation ofcGMP concentration and motility of the enteric neurons (Knippand Bicker, 2009a). In this scenario, abundant CO would bind tosGC which in turn produces much less cGMP than it would in thesole presence of NO.
Pharmacological manipulations of MG neuron migration affectthe outgrowth of other neurites innervating the gut, e.g.serotonergic fibers originating in the frontal ganglion on theforegut. The growth cones of these neurites closely follow the pathof migrating MG neurons and are slowed down by inhibitors of theNO/cGMP pathway, although they do not display cGMP-immu-noreactivity themselves (Stern et al., 2007). This confirms the truepioneer character of the MG neurons and emphasizes theimportance of NO/cGMP signaling for the proper formation ofthe enteric nervous system.
To obtain a complete understanding how intracellular cyclicnucleotide signal transduction in migrating neurons is affected bythe extracellular environment of the midgut, it will be essential toidentify the molecular guidance cues. Analysis of time lapse videosof migrating midgut neurons together with an immunofluores-cence study of midgut cellular architecture implies a contributionof the midgut musculature to enteric neuron guidance. Addition-ally, during midgut plexus formation a fasciculating signal forenteric neuron neurites appears to be provided by the cell adhesionmolecule fasciclin I (Knipp and Bicker, 2009b).
4. NO/cGMP during central nervous system development
Having established the influence of NO/cGMP on axon out-growth and neuronal migration in the peripheral nervous system,we were interested in its possible role for CNS development. To thisend, we followed the developmental time course of the NO–cGMPsystem during locust embryogenesis in whole mount nervoussystems and brain sections (Seidel and Bicker, 2002; Stern et al.,2010).
A small number of single NADPHd-positive cells can be found inthe brain and ventral nerve cord at early stages (before 50% ofembryonic development). However, NADPHd-staining appearslater in many major brain neuropil areas. Some neuropils, e.g. themushroom bodies, acquire NADPH-diaphorase staining onlypostembryonically and citrulline-staining, which may indicateconstitutive NOS activity in these structures, first appears longafter the establishment of their anatomical connectivity (Fig. 4).This indicates that NO is unlikely to control the initial formation ofthese neuropils under normal developmental conditions, but doesnot rule out involvement in later stages of brain development, suchas synapse formation and maturation, when ample sources of NOare present. Enhanced NO-induced cGMP staining is seen indeveloping motor neurons of locust and silverfish embryos duringsynapse formation (Truman et al., 1996) and in larval motor
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neurons of Drosophila (Wildemann and Bicker, 1999). In theantennal lobe of the moth, Manduca, cGMP production is enhancedduring the phase of synapse formation and has been discussed as aregulator of synaptogenesis in this animal (Schachtner et al., 1999).
Immunoreactivity to NO-induced cGMP usually precedes theestablishment of NOS expression and putative activity (Fig. 4(C)). Itcan be detected in the brain from 38% on, however just in a smallnumber of neurons. In the ventral nerve cord, the cGMP responsehas been reported to begin a little later, after 55% (Truman et al.,1996), but enhancement of the staining by co-activation of sGC byYC-1 reveals a few cGMP-positive neurons in the ventral nervecord as early as about 40%. The number of positive cells reaches thefull complement of adult cells at 80%. This earlier presence of theNO-receptor may simply serve to prepare the receptive cells andensure NO-responsiveness when the ligand will finally be present.Alternatively, it may play a role when NO is released underconditions when normal development is disturbed, for instancebecause of hypoxia, during which NO signaling plays a role(Armstrong et al., 2009), or mechanical damage.
5. Nitric oxide/cGMP during axonal regeneration in the ventralnerve cord
We investigated such a possible role of NO in a CNSregeneration paradigm. To this end, we needed to demonstratethat CNS regeneration is possible in the locust at all. Despiteseveral reports on this phenomenon in other insects such ascrickets (Roederer and Cohen, 1983) or cockroaches (Spira et al.,1987), locust CNS regeneration had not yet been shown. On thecontrary, since the early report by Edwards (1969), there lingeredthe notion of poor regenerative capacities in the locust. Morerecently, however, it has been shown that at least the peripheralnervous system of grasshoppers is able to regenerate (Lakes-Harlan and Pfahlert, 1995) as it is in crickets (Chiba and Murphey,1991) and cockroaches (Stern et al., 1997). A few years ago, a studyof axonal regeneration in the adult locust ventral nerve cord couldconfirm the general possibility of regeneration also in the locustCNS (Patschke et al., 2004). To study the possible role of NO on CNSregeneration, we developed a culture system for the 65% embryo inwhich the CNS is exposed to drugs and the connectives betweenabdominal ganglia can be crushed (Fig. 5(A) and (B)). In suchpreparations, single axons of multisegmental interneurons, asrevealed by anti-serotonin immunostaining (Fig. 5(C)), at firstwithdraw their neurite stump after lesion, but grow back into theadjacent ganglion within a few days (Stern and Bicker, 2008).Within two days postcrush,�40% of the axons grow past the lesionsite. This regeneration rate is enhanced in the presence of NOdonors and reduced by inhibitors of the NO–cGMP pathway(Fig. 5(D)–(F)). Rescue experiments with membrane permeablecGMP-analogues confirmed the involvement of the cGMP pathwayas in the peripheral nervous system. At this stage, a large number ofneurons display NO-induced cGMP-immunoreactivity (Trumanet al., 1996; Stern and Bicker, 2008) and double-labeling confirmsthat the regenerating serotonergic neurons are among them(Fig. 5(H)).
Not only externally added NO donors, but also NO from internalsources can enhance regeneration as shown by the reduced growthof severed axons in the presence of NO-scavengers. Axons ofnitrergic neurons damaged by the crush procedure are a likelysource of internal NO. Whereas even in 75% embryos only a smalland variable fraction of the NADPHd-positive axons displaycitrulline-immunoreactivity, strong reliable staining is observedin crushed connectives as early as 60% (Stern et al., 2010). There isno citrulline accumulation in untreated embryos of this stage, or onthe untreated side of embryos with nerves crushed one side,indicating quiescence, or at least significantly lower NOS activity in
these axons under undisturbed conditions. NOS activity is calcium-dependent in the locust CNS (Elphick et al., 1995; Muller andBicker, 1994). It has been shown that internal calcium concentra-tions are dramatically raised in severed axons (e.g. Ziv and Spira,1995), which is a necessary prerequisite for the initiation of axonalregeneration (Rehder et al., 1992). Such an increase in intracellularcalcium could trigger NOS activity in crushed nitrergic neuronsleading to enhanced regenerative outgrowth of other axons.
An additional source of nitric oxide might be hemocytes, a largeproportion of which display NADPHd (Stern et al., 2010).Hemocytes and microglia appear to be important for regulationof CNS regeneration by NO in a different invertebrate, the leech. Inthis animal, NO is produced by cells of the glia sheath of a crushedventral connective, which leads to directed migration of microgliato the lesion site (Chen et al., 2000) involving the cGMP pathway(Duan et al., 2003). Regeneration is then facilitated by theaccumulated microglia via an unknown mechanism. In the locust,however, there is no evidence for cell migration towards the lesionsite. Regeneration appears to be promoted more directly byincreasing cGMP levels in the axotomized cells themselves. Such adirect effect of NO and cGMP on neuronal regeneration is also seenin the goldfish optic nerve (Koriyama et al., 2009), whereas in othervertebrate models, NO has either an inhibitory effect on nerveregeneration or a positive effect via promotion of Walleriandegeneration which in turn facilitates subsequent regeneration(Keilhoff et al., 2002; Moreno-Lopez, 2010).
6. NO and neuronal development and plasticity in vertebrates
Control of neuronal migration by NO and cGMP may not byrestricted to insects, although functional studies are still ratherscarce. There is histological evidence for the involvement of NO inmigration in the cortex (Santacana et al., 1998; Currie et al., 2006).The neuronal precursor cells of the mammalian rostral migratorystream appear to be targets of NO signaling (Gutierrez-Mecinas et al.,2007). In human neuronal precursor cells, migration is positivelyregulated by NO and cGMP in vitro (Tegenge and Bicker, 2009).
Like in the insect PNS and CNS, axon outgrowth andregeneration can be enhanced by NO/cGMP signaling in thevertebrate nervous system. Koriyama et al. (2009) have shown thatregeneration of the severed optic nerve of the goldfish issignificantly enhanced by NO and cGMP, but reduced when nNOSexpression is inhibited. Normal outgrowth and dendritic branchingdepends on intact NO/cGMP signaling in chick embryonic motorneurons (Xiong et al., 2007). On the other hand, NO signalinginhibits regeneration and synapse formation in rat motor neurons(Sunico et al., 2005). NO/cGMP signaling can modulate theattractive or repellent effects of the guiding factor semaphorin3A during cortical axon and dendrite outgrowth (Polleux et al.,2000). In the rat hippocampus, NO induces synapse formation andmultiple innervation of synaptic spines (Nikonenko et al., 2008).
Synaptic plasticity may be considered a developmental processthat is ongoing throughout embryonic and postembryonic life. NO/cGMP is involved in a particular form of synaptic plasticity, long termpotentiation (LTP) in the hippocampus (reviewed by Hawkins et al.,1998). In this area, LTP depends on both a low tonic level of NO,provided by endothelial NOS in the blood vessels, and a higher phasiclevel of NO provided by neuronal NOS which is activated via Ca2+
through NMDA-receptors in postsynaptic neurons (Hopper andGarthwaite, 2006). From there, it diffuses rapidly into presynapticterminals where it affects the neurotransmitter release machinery(Arancio et al., 1996). In the insect CNS, NO has been shown to act in aretrograde fashion at the photoreceptor synapse (Schmachtenbergand Bicker, 1999). Using synaptic vesicle imaging techniques, it hasbeen shown that presynaptic transmitter release can be controlledby NO and cGMP at the neuromuscular junction of Drosophila
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(Wildemann and Bicker, 1999) as well as in human model neurons inculture (Tegenge et al., 2009).
7. Conclusions
In vivo culture preparations of the developing nervous systems(central, peripheral, and enteric) of locusts provide useful modelpreparations for the study of neuronal outgrowth and motility.Their main advantages are their robustness and easy handling intissue culture with a readily accessible nervous system ascompared to mammalian in vivo systems, and the fact that theyrepresent intact nervous systems within their natural environmentas compared to cultures of dispersed cells or tissue explants. Theseculture systems have provided the means to reveal the effects ofthe NO/cGMP signaling pathway on axon outgrowth andregeneration and on cell migration. Since many molecular signaltransduction cascades are strikingly conserved among vertebrateand invertebrate animals, it is not surprising that similar functionsof nitric oxide on these developmental processes can also beobserved during mammalian brain formation.
Acknowledgements
Our research was funded by the Deutsche Forschungsge-meinschaft, grant number BI 262/10-5. We thank Dr. Sabine Knippfor helpful comments on the manuscript.
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