Nitric Oxide in the Crustacean Brain:Regulation of Neurogenesis andMorphogenesis in the Developing OlfactoryPathwayJ.L. Benton,* D.C. Sandeman, and B.S. Beltz
Nitric oxide (NO) is a diffusible gas-eous signaling molecule that is pro-duced by nitric oxide synthase (NOS)during the conversion of L-arginine tocitrulline. Once present, NO is mem-brane-permeable and in many sys-tems effects change by activating sol-
uble guanylate cyclase in target cells,resulting in the production of cyclic3,5 guanosine monophosphate (cGMP).cGMP then activates a variety ofdownstream pathways by means of ac-tions, for example, on phosphodiester-ases or protein kinases, ultimatelyevoking cellular responses. There arealso cGMP-independent pathways of
NO action, such as when NO interactswith metal complexes or oxygen spe-cies. However, in the nervous systemof both vertebrate and invertebratespecies, the cGMP-dependent actionsappear to be the most prevalent.
NO has been implicated in manyphysiological processes (e.g., vasodila-tion, muscle contractility, neurotrans-
ABBREVIATIONS AL accessory lobe AMPN anterior medial protocerebral neuropil AnNII antenna II neuropil CB central body CEGcircumesophageal (commissural) ganglion CL cell cluster: 6 8 9 10 15/16 and 17 DC deutocerebral commissure DCN deutocerebralcommissure neuropil DGN dorsal giant neuron LAN lateral antennular neuropil L CL lateral cluster OGT olfactory globular tract OGTNolfactory globular tract neuropil OL olfactory lobe PB protocerebral bridge PMPN posterior medial protocerebral neuropil PZ of CL 10proliferation zone of cluster 10
Neuroscience Program, Wellesley College, Wellesley, MassachusettsGrant sponsor: NIH; Grant number: 1R01 MH67157; Grant sponsor: NSF; Grant number: IBN 0344448; Grant sponsor: The MarenFoundation; Grant sponsor: Mount Desert Island Biological Laboratory.*Correspondence to: Jeanne L. Benton, Neuroscience Program, Wellesley College, Wellesley, MA 02481.E-mail: [email protected]
DOI 10.1002/dvdy.21340Published online 17 October 2007 in Wiley InterScience (www.interscience.wiley.com).
mission) in a variety of organisms.The functions of NO are particularlydiverse in the nervous system, butperhaps most notable is the associa-tion of NO with dynamic processessuch as neuronal migration, differen-tiation, and synapse formation duringdevelopment (Truman et al., 1996;Kuzin et al., 2000; Gibbs, 2003;Bicker, 2005), and neuronal plasticity,synaptic remodeling, and sensory pro-cessing in the mature nervous system(Chen et al., 2004; Collmann et al.,2004; Sunico et al., 2005; Moreno-Lopez and Gonzalez-Forero, 2006).NOS is concentrated in the primaryolfactory centers of vertebrate and in-vertebrate species (Broillet and Fir-estein, 1996; Gelperin et al., 1996;Fuji et al., 2002), and there is strongphysiological evidence for a role of NOin odor processing (Collmann et al.,2004). Nitric oxide mechanisms alsoappear to be important during bothdevelopmental and adult neurogen-esis, where NO can act as an antipro-liferative agent in some systems(Moreno-Lopez et al., 2004; Matarre-dona et al., 2005; Ciani et al., 2006;Romero-Grimaldi et al., 2006; Torro-glosa et al., 2007), while promotingneurogenesis in others (Zhang et al.,2001; Lu et al., 2003; Cayre et al.,2005). These dual roles may be ex-plained by the levels of NO and thetiming of synthesis (Cardenas et al.,2005).
The life-long addition of new neu-rons has been found in the olfactorypathway of vertebrate and inverte-brate organisms (Kempermann, 2005),and of particular interest to our work,in the brains of lobsters and othercrustaceans (Harzsch and Dawirs,1996; Schmidt, 1997; Harzsch et al.,1999; Beltz and Sandeman, 2003).Neurogenesis in the crustacean brainis influenced by a variety of endoge-nous and exogenous factors (Beltz andSandeman, 2003; Sullivan et al.,2007), including serotonin (Bentonand Beltz, 2001a; Beltz et al., 2001).In addition to stimulating neurogen-esis, serotonin promotes the survivaland differentiation of new projectionneurons in the olfactory pathway oflobsters (Sullivan et al., 2000; Beltz etal., 2001; Benton and Beltz, 2001a). Arole for NO in regulating neurogenesiswas suggested by preliminary studiesthat demonstrated the coexistence of
NO with serotonin in identified neu-rons and synaptic regions in the lob-ster brain, and its presence in regionswhere neurons continue to proliferatethroughout life (Benton and Beltz,2001b; Beltz and Sandeman, 2003). Inaddition, several studies in vertebratesystems suggest an interrelationshipbetween serotonin and NO levels(Kaehler et al., 1999; Sinner et al.,2001; Ramos et al., 2002; Tagliaferroet al., 2003).
Other neuronal functions for NO indecapod crustaceans have also beenidentified, including network parti-tioning and motor pattern selection inthe stomatogastric nervous system(Scholz et al., 2001; Christie, 2003;Stein et al., 2005) and retrograde sig-naling in the heart (Scholz et al., 2002;Goy, 2005). A role in central nervoussystem development is suggested by alarge number of NO-sensitive neuronsfound in lobster larvae, and by subse-quent changes in the expression ofNOS and of NO sensitivity (Scholz etal., 1998). Scholz et al. concluded thatthe NO/cGMP pathway participates inthe developmental maturation of neu-ral circuits in the accessory lobes,higher order processing centers in theolfactory pathway. Studies in adultcrayfish suggest that, as in insectsand mammals, NOS is strongly ex-pressed in the olfactory centers of thebrain (Johansson and Carlberg, 1994;Johansson and Mellon, 1998).
Our primary interest is in the devel-opment and maturation of olfactorycenters in the crustacean brain, and inthe life-long production of neurons inthe olfactory pathway. Because NOappears to be important in mecha-nisms underlying both olfaction andneurogenesis in a variety of species,we investigated the role of this mole-cule in the olfactory system in theAmerican lobster, Homarus america-nus. The goal of the present study is todescribe NO-associated pathways inthe embryonic and adult crustaceanbrain, and to define potential roles forNO. We have used immunocytochem-ical techniques to identify and de-scribe putative sources of NO (NOSimmunoreactivity) in the embryonicand adult brain. The NO donor so-dium nitroprusside (SNP) in combina-tion with isobutylmethylxanthine(IBMX; which blocks cGMP turnoverby phosphodiesterases) were used to
increase cGMP levels in NO-targets,and cGMP was then labeled immuno-cytochemically. To reveal possiblefunctional roles of NO in the brain,levels of NO were manipulated usingthe NO donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and the NOSinhibitor NG-Nitro-L-arginine (L-NAA) at various times during embry-onic development; structural andchemical changes in the lobster brainwere then documented. We also askedwhether the rate of neurogenesis inthe embryonic brain is influenced byNO levels. Finally, the lateral flagellaof the antennules of juvenile lobsterscontaining the olfactory receptor neu-rons were ablated, and changes inNOS expression in the olfactory path-way were documented, as were levelsof neurogenesis among the olfactoryprojection neurons.
The results of this study provide anoverall view of the changing locationof NOS during embryonic develop-ment and its final distribution in theadult brain, as well as identifying pre-sumptive target areas where cGMPwas up-regulated in response to in-creased levels of NO. Our data suggesta role for NO in (1) establishing thestructural integrity of olfactory glo-meruli during development, (2) synap-togenesis in the accessory lobe, (3) theregulation of neurogenesis, and (4) theneural response to injury.
RESULTS
NOS and cGMP LocalizationStudies
Distribution and sequence ofappearance of NOS in theembryonic and adult brain.
We defined the temporal and spatialpatterns of expression of NOS in theembryonic lobster brain by assessingthe distribution of NOS immunoreac-tivity at several developmentalstages, beginning at E40% (whereE0% is the time of fertilization, andE100% is hatching) and continuinguntil just before hatching (Table 1).These localization patterns can be cor-related with specific developmentalcharacteristics in the brain (Fig. 1). AtE40%, for example, the anlagen of theaccessory lobes (ALs) have justemerged; the olfactory lobes (OLs),whose primordia first appeared at
3048 BENTON ET AL.
E10–E15%, are well established andthe OL glomeruli begin to form atE45% (Helluy and Beltz, 1991; Beltzet al., 1992; Helluy et al., 1993). Mid-embryonic life is characterized by theelaboration and growth of variousbrain structures, until �E85% whenthe animal enters a developmentalplateau (Helluy and Beltz, 1991).Throughout this period, the eyes andbody do not grow even when animalsare reared at constant temperature,and neurogenesis undergoes a precip-itous decline as the neuroblasts die(Harzsch et al., 1999; Beltz and Sand-eman, 2003). While brain growth thencontinues after hatching due to the
elaboration of fibers and addition ofglia, neurogenesis resumes only in re-stricted areas in the olfactory (deuto-cerebral cell clusters 9 and 10) andoptic pathways (Harzsch et al., 1999;Sullivan and Beltz, 2005).
In the embryonic brain, NOS immu-noreactivity is found in the cytoplasmof neuronal cell bodies, as well as infiber tracts and neuropil (synaptic) re-gions. As embryogenesis progresses,NOS immunoreactivity gradually be-comes more intense and the sites oflabeling more widespread, eventuallyincluding structures throughout thebrain by the embryonic plateau(�E85%; Table 1). At E40%, NOS im-
munoreactivity is most pronounced inprotocerebral and tritocerebral re-gions of the brain, although the inten-sity of labeling is relatively faint andvariable (Table 1). In the tritocere-brum, two pairs of cell bodies in clus-ters 15/16 label consistently, but areless intense than at later stages. Inthe protocerebrum, punctate labelingis found in the central body and pro-tocerebral bridge, as well as the ante-rior and posterior median protocere-bral neuropils (AMPN, PMPN). Incontrast, there is an absence of stain-ing in clusters 6, 8, and 9, the frontal(naupliar) eye (Eloffson, 2006), and inlongitudinal medial fiber tracts. How-
TABLE 1. Localization of NOS in the Brains of Adult and Embryonic Lobstersa
Stage of development 3 E40% E60% E68% E75% E85% E95% Adult
aE is percentage embryonic development where 0% is the time of fertilization and 100% is hatching. Three regions within the brainare distinguished: protocerebrum (anterior regions), deutocerebrum (midbrain regions), and tritocerebrum (posterior regions). Theintensity of NOS labeling in neuropil structures, cell clusters, and individual cells and fibers is subjectively rated on a relative scale:(�) � no labeling observed; (� �) � weak and variable; (�) � consistent and strong; (��) � intense. Embryonic NOS labelingreaches it widest distribution throughout the brain and most intense level at E85% (boxed scores), whereas NOS is found primarilyin the olfactory pathway (boxed scores) in the adult brain. The number of brains assessed per stage is indicated at the bottom of eachcolumn. For abbreviations, see list.
NITRIC OXIDE IN THE CRUSTACEAN BRAIN 3049
ever, by E70–E75%, NOS immunore-activity is much more extensive andculminates in maximal labeling atE85%; at that time, intensely labeledstructures include fibers in clusters 6(Table 1; Fig. 2A,B,E) and 8, neuropilin the lateral lobes of the PMPN (Fig.2G), and longitudinal tracts that ex-tend on the dorsal surface of the brainfrom the protocerebrum to the esoph-ageal connectives (Fig. 2F). In the tri-tocerebrum, neurons in clusters 15/16and 17 label lightly by E40%; the con-sistency and intensity of staining in-creases in these regions as develop-ment progresses (Table 1). In cellclusters 15/16 at E85%, several largecells label, and some of these appearas pairs within the cluster (Fig. 2F,arrowheads). At least one fiber emerg-ing from CL 15/16 consistently labelsand projects into the OL (Fig. 2G, ar-row), and ascending NOS-immunore-active fibers from these clusters alsoinnervate the OGTN. There is a dis-tinct decrease in NOS labeling in mostbrain areas, with the exception of thetritocerebrum, during the period justbefore hatching. Notably, NOS label-ing is absent throughout embryonicdevelopment in the lateral antennularand antenna II neuropils (LAN, AnNII) and in the emerging ALs (Table 1).
NOS immunoreactivity in most deu-
tocerebral regions in the embryo ap-pears later relative to protocerebraland tritocerebral areas, although it isin the deutocerebrum that the mostintense staining persists during adultlife (Table 1; Fig. 3). Immunoreactiv-ity in the deutocerebrum becomes con-sistent and intense during the devel-opmental plateau (Table 1, E85%). Atthat time, labeling is observed intracts, cells, and synaptic regions as-sociated with olfactory processing(i.e., the olfactory globular tract[OGT]; Fig. 2C) and its neuropil (theOGTN; Fig. 2C, asterisk), fibers andglomeruli primarily in the cortex ofthe OL (Fig. 2B,C), and fibers emerg-ing from cell cluster 10 (Fig. 2C). Byadult life, staining in these areas isintense and includes labeling in theDC interneurons of cluster 11 (Fig.3A,C, red circle), DCN, especially theanterolateral half (Fig. 3C, red aster-isk) and DC (Fig. 3A,C). Individualfibers from the DC project into the ALand innervate single AL glomeruli(Fig. 3B,Bi). The dorsal giant neuron(DGN), a serotonergic neuron (Fig.3E) that innervates both the OLs andALs, also labels for NOS (Fig. 3D);however, while staining for serotoninis consistently strong, NOS labeling ishighly variable in this neuron.Whether this variability is associated
with time of day, neuronal activity, orbehavior is not known. Glomeruli inthe accessory lobe, which do not labelduring embryonic life, stain intenselyin the adult brain (Fig. 3B arrows, Farrowheads). The intensity of stainingis not evenly distributed throughoutthe AL, but is often confined to a sub-set of glomeruli that can be clustered(Fig. 3B) or distributed throughoutthe AL. The bases of the cylindricalAL cortical glomeruli (Helluy et al.,1996) label consistently (Fig. 3F, ar-row). As in the E85% embryos, theprimary neurites of olfactory projec-tion neurons (cluster 10; Fig. 3G, ar-row) and their branches in the OLsand ALs (Fig. 3G, arrowhead) are in-tensely labeled. The antenna II neuro-pil and LAN also contain faint NOSimmunoreactivity in the adult brain.
Distribution of cGMP-likeimmunoreactivity.
In an effort to visualize cGMP-depen-dent targets of the NOS-immunoreac-tive cells, antibodies were used to lo-calize cGMP immunocytochemically.However, the only sites labeled forcGMP in the embryonic brain are asubset of cells that compose the fron-tal eye (Fig. 4Ai) in the protocere-brum. To increase the likelihood of
Fig. 1. A time line of brain development in Homarus, emphasizing the developmental sequence in the olfactory pathway. Numbers indicatepercentage development, from fertilization (0%) to hatching (100% development); juvenile and adult stages complete the range. Three assays relatedto the nitric oxide (NO) pathway were used: immunocytochemical localization of nitric oxide synthase (NOS) and cGMP (A); immunocytochemicallabeling for synapsin and serotonin to monitor morphogenic changes (B); 5-bromo-2�-deoxyuridine (BrdU) labeling to assess the numbers of cells inS phase (C). To accomplish these studies, NO and cyclic 3,5 guanosine monophosphate (cGMP) levels also were manipulated using pharmacologicalagents (sodium nitroprusside [SNP], S-Nitroso-N-acetyl-D,L-penicillamine [SNAP], NG-Nitro-L-arginine [L-NAA], isobutylmethylxanthine [IBMX]) andby antennular ablations. The timing of the immunocytochemical and pharmacological experiments are noted below the timeline at the developmentalstage when they were performed.
3050 BENTON ET AL.
identifying cGMP-dependent NO tar-gets in embryos, levels of NO andcGMP were up-regulated in vitro us-ing IBMX and SNP. When IBMX andSNP are combined in the incubation
medium to maximize cGMP levels,cell bodies in clusters 11 and 15/16(Fig. 4Aiii) label, as well as severalfiber tracts: (1) a commissure caudalto the PMPN that projects between
the eyestalks (Fig. 4Aiii, asterisk); (2)fibers from the eyestalk that end incell cluster 8 (Fig. 4Aiii); (3) a tractthat arborizes within the CB (seenalso in the adult brain, Fig. 4Bii); (4) aset of cGMP-labeled fibers extendingmedially between cluster 6 and trito-cerebral cell clusters 15/16 (Fig. 4Aiii,arrow); (5) a fan-shaped collection oflabeled fibers in the deutocerebrum,just medial to cluster 11 (Fig. 4Aiii,double arrow); elements from this re-gion project in the connectives to thecommissural ganglia (Fig. 4Aiii; alsoin adults see Biii). The AMPN, PMPN,and LAN also label for cGMP in theembryonic brain. There is no cGMPlabeling in the olfactory and accessorylobes, DC, and OGT in embryos.
There is no cGMP labeling in theadult brain in the absence of SNP andIBMX. However, when adult brainsare incubated in these pharmacologi-cal agents, cGMP labeling is found inwhat appear to be (1) derivatives ofthe frontal eye (Fig. 4Bi), (2) AMPN(Fig. 4Bii), (3) PMPN, (4) LAN, (5) fi-bers projecting between cluster 6 andthe tritocerebrum, and (6) cell bodiesin clusters 15/16 (Fig. 4Biii). Cells incluster 11 that labeled in embryonicbrains do not label in the adult, sug-gesting that this staining is related toa developmental function.
DevelopmentalManipulations
From our localization studies, weknow that NOS is found in the outputpathway of those cluster 10 neuronsthat reside closest to the accessorylobe. This particular area of cluster 10houses the proliferation zone wherenew cluster 10 neurons are bornthroughout life (Harzsch et al., 1999).NOS also is found in OL and AL glo-meruli at specific times in develop-ment and in the midbrains of adultlobsters, suggesting that NOS plays arole in the developing olfactory path-way. We therefore tested the influenceof embryonic NO levels on neurogen-esis and glomerular formation andstabilization. Specifically, we askedwhether the rate of neurogenesis re-sponds differently to NO manipula-tions at various stages of embryonicdevelopment. Changes in NO levelswere timed to coincide with particulardevelopmental events in the lobster
Fig. 2. Distribution of NOS immunoreactivity in the embryonic brain at E85%. In all figures,preparations labeled with a single antibody are shown in black and white images; preparationslabeled with multiple antibodies where more than one color channel is displayed, are illustrated incolor. B–G: Stacked confocal images of whole-mounted brains that were double-labeled withanti-NOS antibody (green) and anti-synapsin antibody (red). F: NOS immunostaining alone. A:Schematic brain diagram, compressed in the dorsal–ventral plane, indicates neuropil regions andcell clusters that label for NOS (light blue shading) with major NOS-labeled tracts (green lines).Three horizontal sections at different depths in the brain illustrate details on the dorsal surface (F),more ventrally (G), and mid-level in the brain (B). B: Mid-level horizontal section of the brain, lateralprotocerebrum (eyestalks), and circumesophageal ganglia (CEGs) show the extent of NOS labelingat E85%. C: In the lateral deutocerebrum, labeling is seen in fibers of the CL 10 cells that innervatethe OL and AL and project to the protocerebrum in the OGT. A NOS-immunoreactive fiber(arrowhead) from cells in CL 15/16 (not shown) is also found in this region. The asterisk denotes theOGTN. D: An enlarged view of the CEG; cells and fibers descend from CL 15/16 into these ganglia.E: Ascending fibers form dense fine branches that innervate CL 6 (arrow). The base of the frontaleye is also labeled (arrowhead). F: On the dorsal surface of the brain, longitudinal, medial fibersextend across the entire brain with ladder-like branches at intervals (arrows). These branchesproject to the PB, AMPN, PMPN, and anteriorly to the esophagus. Several of the longitudinalprocesses appear to emerge from cell bodies in CL 15/16. The bilateral pairs of very dorsal cells (CL15/16; arrowheads) label throughout embryonic and larval (Scholz et al., 1998) life. G: In this ventralsection, large cells are seen in CL 15/16 (large arrowhead), some of which send fibers to the OL(arrow). The small arrow points to NOS immunoreactive fibers in L CL. For abbreviations, see list.Scale bars � 100 �m in A–C, 50 �m in D,E, 100 �m in F, 50 �m in G.
NITRIC OXIDE IN THE CRUSTACEAN BRAIN 3051
brain, such as the period when olfac-tory glomeruli form, or the earlygrowth of the AL (see Fig. 1; Helluy etal., 1993). The production of new neu-rons was assayed using 5-bromo-2�-deoxyuridine (BrdU) labeling meth-ods. Morphological changes in thebrain were assessed using immunocy-tochemical labeling for synapsinand/or serotonin.
Regulation of NO levels andneurogenesis.
SNAP and L-NAA were used to up-and down-regulate NO levels, respec-tively, at several embryonic stagesand corresponding levels of neurogen-esis assessed in cluster 10 duringthose periods. Incubation in SNAP for3 days at E40% decreases neurogen-esis in cluster 10 relative to controlembryos incubated in saline alone; L-NAA treatment for 3 days increasesthe rate of neurogenesis (Fig. 5A).This finding was also true in older em-bryos incubated in L-NAA for 2 weeksat E75–E85%, where L-NAA treat-ment caused an increase in the num-bers of BrdU-labeled cells in cluster 10(Fig. 5B), again indicating that NO isan inhibitor of neurogenesis in thissystem.
Longer incubations in these com-pounds were attempted to examinethe influence of NO on neurogenesisduring specific developmental events.For instance, embryos were incubatedin SNAP and L-NAA from E45–E70%,which includes the period when olfac-tory glomeruli begin to form. Thequestion here was whether the forma-tion of the olfactory glomeruli wouldbe delayed, advanced, or disrupted inany way by changes in NO levels.However, the embryos were particu-larly sensitive to prolonged treatmentwith SNAP and L-NAA during thisearly period, and animals began to de-teriorate and die within a few days ofthe beginning of treatment, evenwhen SNAP and L-NAA levels werereduced.
Influence of NO levels onmorphogenesis in the olfactorypathway.
When NO levels are manipulated dur-ing E75–E85%, there are several ma-jor morphological changes in thebrain, mostly in the deutocerebrum.
Down-regulation of NO with L-NAA re-sults in clear and significant changes inthe OLs and ALs: the olfactory glomer-uli, which begin to develop at E40% andincrease in number until the end of lar-val life (Helluy et al., 1996), lose theircharacteristic form (Fig. 6). We used an-tibodies raised against synapsin, a pro-tein found in synapses that is importantin the process of transmitter mobiliza-tion (Rosahl et al., 1995), to explore the
effect of NO down-regulation on themorphogenesis of the crustacean olfac-tory glomeruli because we suspect thatthe period of synaptogenesis coincideswith glomerular formation (Oland andTolbert, 1996; see also Fig. 7).
The lobster olfactory lobes first labelfor synapsin during the mid-embryonicperiod when glomeruli begin to appear(E40–E45%; Helluy et al., 1996). How-ever, when L-NAA treatment is ap-
Fig. 3. Confocal images of vibratome sections showing NOS, serotonin, and synapsin immuno-reactivity in the adult Homarus brain. A: Schematic diagram indicates neuropil regions and cellclusters that label for NOS (light blue) with most intense NOS-labeled regions in a darker blue. Thered circle (DC neurons: also in C), asterisk (DCN; also in C), and the proliferation zone (PZ) of CL10 in the diagram indicate brain regions that correspond to labeled tissue shown in the images.B,Bi: A small number of AL glomeruli show NOS immunoreactivity (arrows). Higher magnification ofa NOS-labeled process from a DC neuron ending in a glomerulus is shown in Bi. C: The DC fibertract, the DC cell bodies (in red circle), and the lateral region of the DCN (asterisk) label for NOS.D,E: In a double-labeled section, NOS (D, green) and serotonin (E, red) colocalize in the DGN(arrows in D,E). F: The morphological characteristics of the OGTN (red arrowhead) are highlightedwhen colabeled for NOS (green) and synapsin (red). The OGTN (red arrowhead; double-labeled,yellow), AL glomeruli (white arrow heads), and base of the AL cortex (arrow) are labeled in thisimage. G: Fibers from the CL 10 neurons in the proliferation zone (arrow) innervating the OL and AL(arrowheads) and scattered cell bodies throughout CL 10 (Gi) are labeled. For abbreviations, seelist. Scale bars � 3 mm in A; 100 �m in B,Bi,C,F,G, 50 �m in D,E.
3052 BENTON ET AL.
plied at E75–E85%, the tissue conden-sations associated with glomeruli inthe olfactory lobes disappear, al-though an evenly distributed punctatesynapsin labeling persists throughoutthe OL (Fig. 6B).
Glomerular formation in the acces-sory lobes occurs much later than inthe olfactory lobes, during larval, notembryonic life (Helluy et al., 1996).Nevertheless, as in the olfactory lobe,the onset of synapsin labeling in the
accessory lobes also coincides with thefirst evidence of glomerular formationduring the transition from the first tothe second larval stage, and is intenseby the fourth postembryonic stage(Fig. 7A–D). Unexpectedly, however,ALs in embryos treated with L-NAAat E85% to reduce NO levels exhibitprecocious and intense labeling forsynapsin (Fig. 8Bii).
Synapsin labeling in the OLs andALs of untreated E85% embryosshows that labeling in the OLs is nor-mally approximately 3 times more in-tense than in the ALs (Fig. 8Ai). Fol-lowing a 2-week L-NAA treatment(E75–E85%), the intensity of synapsinlabeling in the AL is similar to, oreven greater, than that in the OL (Fig.8Bi). It appears that lowered NO lev-els accelerate some aspects of acces-sory lobe development, but not glo-merular formation.
Down-regulation of NO with L-NAAtreatment for 2 weeks (at E75–E85% ), also increases the intensity ofserotonin immunolabeling in theDGN, and increases the numbers ofserotonin-immunoreactive cells around
Fig. 4.
Fig. 4. Localization of cyclic 3,5 guanosinemonophosphate (cGMP) in the embryonic(E85%) and adult brain when NO is up-regu-lated with SNP and IBMX. cGMP antibody isrevealed in green in the double-labeled images.The colabel, synapsin (red), delineates brainstructures. A,B: Schematic diagrams of embry-onic (A) and adult (B) stages indicating neuropilregions and cell clusters that label for NOS (lightblue). The red lines in A and B represent longi-tudinal fibers and fibers that cross the brain atthe level of the PB and CB. Some fibers lie inthe protocerebral tracts. Ai: By E85%, threecomponents of the frontal eye (arrowhead) la-bel. A second cluster of smaller photoreceptorcells also label (arrow). Aii: cGMP antibodyphotoreceptor cells and AMPN (punctate label-ing, arrowheads). The asterisk marks the baseof the naupliar eye. Aiii: Medial tracts (arrow) inthe embryo (in the same location as NOS-la-beled fibers at this same stage), as well as acommissure that projects to the eyestalks (as-terisk), label for cGMP. Also stained are cells inCL 11 (small bilateral arrowheads) and 15/16(large arrowhead). The double arrowheadmarks the deutocerebral fan-shaped collectionof fibers whose origin has not been identified.Bi: In the adult brain, photoreceptor cells labelin the protocerebrum; the three large compo-nents of the frontal eye do not. Bii: In fibers inthe CB, AMPN and CL 6 label. Biii: Labeledcells in CL 15/16 have fibers that extend into theconnectives. For abbreviations, see list. Scalebars � 80 �m in Ai,Aiii, 50 �m in Aii, 40 �m inBi, 50 �m in Bii,Biii.
NITRIC OXIDE IN THE CRUSTACEAN BRAIN 3053
the DGN in cell cluster 11. These cellshave been noted in earlier studies(Benton and Beltz, 2001a), but do notlabel reliably for serotonin in lobstersreared in laboratory conditions. Sero-tonin labeling within the olfactory andaccessory lobes, however, decreases inintensity after L-NAA treatment.
NO and Response to Injury
Antennule ablation: increases inNOS labeling in the OGT.
The data reported above have resultedfrom pharmacological treatments oflobster embryos with NOS inhibitorsand NO donors. How might suchchanges in NO levels occur in the nor-mal events and physiological changesduring a lobster’s life? One insightinto how NO levels may be altered in
vivo comes from studies where NOSlabeling in the brain was assessedover a month-long period followingunilateral antennule ablation. Dam-age or loss of the antennules, whichcontain the cell bodies and axons ofthe primary olfactory receptor neu-rons, can occur under natural condi-tions in these animals, and it is knownthat following antennule ablation in
juvenile crayfish, the initial responseis a reduction in the volume of theipsilateral olfactory lobe due to thedeath of olfactory afferents. This sizereduction is also associated with anincrease in the numbers of apoptoticprofiles among the local (cluster 11)and projection (cluster 10) neurons,resulting in first an overall decreasein numbers of interneurons in the ol-
Fig. 5. Manipulation of NO levels influencesembryonic neurogenesis in cluster 10. A:Counts of 5-bromo-2�-deoxyuridine (BrdU)-la-beled CL 10 cells for embryos at E40% incu-bated in SNAP and those incubated in L-NAAfor 36 hr are compared with BrdU-labeled cellcounts from embryos incubated in untreatedlobster saline. Analysis of variance analysis ofdata and further t-tests reveal a significant dif-ference among the three groups (n � 6 embry-os/group; P � 0.05). B: Treatment of lobsterembryos with L-NAA for 2 weeks beginning atstage E75%, resulted in a 33% increase inBrdU-labeled soma counts compared with em-bryos incubated in untreated saline. Studentt-test reveals a significant difference betweenthe two groups (n � 9 embryos/group; P �0.001). For abbreviations, see list. Fig. 6. Stacked confocal images show that OL
glomeruli, which had begun to form �E45%,are deconstructed following long-term NO inhi-bition during embryogenesis. Embryos at E75%were treated with L-NAA in saline for 2 weeksand dissections were then completed at E85%,followed by immunocytochemistry for synap-sin. A: Control embryos (E85%) labeled for syn-apsin show the morphology of typical OL glo-meruli (arrows) at this stage. B: L-NAAtreatment of lobster embryos results in amarked decrease or complete loss of OL glo-merular structure. Synapsin labeling is punctateand diffuse. For abbreviations, see list. Scalebars � 100 �m.
Fig. 7. Stacked confocal images illustrate thedevelopmental sequence of immunocytochem-ical labeling for synapsin in the accessory lobeduring normal development (late embryonicthrough larval life shown). A: At E60%, there isno staining for synapsin in the AL (arrow), al-though the OL shows distinct labeling for thisprotein; however, labeling with antiserotoninantibody clearly outlines the morphology ofboth the OL and AL (Ai; arrow). B: At E85%,there is faint background-level labeling for syn-apsin (arrow) in the AL. The outline of the AL isdifficult to see and glomerular structures are notpresent. C: The AL in a postembryonic stage IVlobster is labeled for synapsin (arrow) andshows the glomerular morphology that is typi-cal at this stage. Scale bars � 50 �m in A,C,200 �m in B.
3054 BENTON ET AL.
factory pathway, and then later intime an increase that re-establishesthe original number of interneurons(Sandeman et al., 1998).
We labeled NOS in the brains ofjuvenile lobsters at intervals followingunilateral antennule ablation. Fourhours after ablation, the OGT andcells in clusters 9, 10, and 11 exhibitan increase in the intensity of NOSlabeling; 24 hr after ablation, the gen-eral level of staining for NOS is evenmore intense, such that on the ablatedside the AL and cluster 10 neurons areuniformly labeled (Fig. 9B). NOS la-beling in the OGT in unablated juve-nile animals is only detectable as itemerges from cluster 10 (as also isseen in the brains of embryos andadults; Figs. 2C, 3G), but is not visiblein the more medial parts of the brain(Table 1). However, at 48 hr after uni-lateral ablation the OGT labels onboth sides of the brain, consistent with
the bilateral projection of the cluster10 axons in this tract (Fig. 9C, arrow).
Antennular ablation influenceslevels of neurogenesis in juvenilelobsters.
Our studies in embryos where NO lev-els were manipulated and BrdU incor-poration in cluster 10 cells was as-sessed, demonstrated that increasedlevels of NO result in decreased levelsof neurogenesis (Fig. 5). Therefore, theincrease in NOS labeling in the olfac-tory pathway following antennularablation would be expected to corre-late with decreased levels of neuro-genesis among the cluster 10 neurons.To test this possibility, we ablated thelateral flagella of both antennules injuvenile lobsters and 1 week later in-cubated these animals in BrdU. Wefound a significant decrease (P �0.0001) in the numbers of BrdU-la-
beled profiles in the cluster 10 prolif-eration zones of the antennule-ablated animals. This result is consis-tent with the idea that NOS levelsincrease after antennular ablation inthe olfactory pathway, and, either di-rectly or indirectly, suppress neuro-genesis among the olfactory projectionneurons.
DISCUSSION
NOS and cGMP Localization
The distribution of NOS immunoreac-tivity in the lobster brain is distinctivefor each embryonic stage that hasbeen examined. The sequence of label-ing and its spatial distribution sug-gest a requirement for NO early indevelopment in the protocerebrumand tritocerebrum, and in the deuto-cerebrum later in embryonic develop-ment. Although intense NOS labelingin the deutocerebrum is a relativelylate embryonic event, it is in theseareas that NOS persists throughoutjuvenile and adult life. The intensestaining of deutocerebral regions asso-ciated with the olfactory pathway isconsistent with reports that NOS isconcentrated in the primary olfactorycenters in many species (Broillet andFirestein, 1996; Gelperin et al., 1996).
The onset of intense and wide-spread labeling in the brain at �E85%coincides with the embryonic develop-mental plateau (Helluy and Beltz,1991). This period is characterized bya lack of growth in both the eye indexand the cephalothoracic length, and isequivalent to stage D0 of the metan-aupliar molt cycle that occurs duringembryonic life. Molt cycles in postem-bryonic crustaceans are under hor-monal (ecdysteroid) control, and it islikely that embryonic molts are regu-lated in a similar way. NO levels inthe embryo may, therefore, be regu-lated by circulating hormones that co-ordinate the expansion of NOS expres-sion with the ongoing molt cycle andimpending eclosion. Evidence from in-sect systems also suggests that ecdys-teroids are involved in regulating neu-rogenesis by means of an NOsignaling pathway (Champlin andTruman, 2000).
NOS is not found in the accessorylobe, a higher order processing area inthe deutocerebrum, until postembry-onic stages. The accessory lobes un-
Fig. 8. Aii: Synapsin labeling is not present in the accessory lobes in control embryos (see alsoFig. 7A). Bii: However, after L-NAA treatment (down-regulation of NO) of E85% embryos, there isintense synapsin labeling in the AL. Ai, Bi: Graphs show the relative intensity levels of synapsinlabeling in the OL and AL in embryonic brains at E85%; Control, n � 10 (Ai), NOS-inhibited, n � 10(BI). Bi: Semiquantitative measurements demonstrate that the intensity of synapsin staining hasindeed increased following L-NAA treatment and is comparable to the intensity of labeling in theOL. Each graph represents the average mean intensity values of the two lobes � the SD throughthe z-axis of a stacked confocal image. Designated regions of interest in the confocal stackedimages of the control (Ai) and NOS-inhibited (Bi) brains were analyzed with Leica software toproduce the data for the graphs. The arrows point to the OGTN, which labels in both conditions.For abbreviations, see list.
NITRIC OXIDE IN THE CRUSTACEAN BRAIN 3055
dergo a pronounced increase in sizeduring larval and early postlarval de-velopment (Helluy et al., 1995). Inadult lobsters and crayfish, the ALsare composed of discrete glomerulithat are segregated into a cortex thatis primarily concerned with integrat-ing olfactory inputs, and a medullaryregion that processes visual andmechanosensory information (Sande-man et al., 1995; Sullivan and Beltz,2005). In contrast to the embryonicformation of glomeruli in the olfactorylobes, glomerular formation in the ac-cessory lobes is delayed until mid-lar-val life (Helluy et al., 1993, 1995). Wehave proposed (Helluy et al., 1996)that glomerular formation in the ac-cessory lobes may be contingent onpatterns of activity in visual, mech-anosensory, and olfactory interneu-rons projecting to this area. Such ac-tivity may depend upon primarysensory processing areas as they re-spond to the first environmental inputafter hatching. If so, the coincidence ofNOS labeling in the postembryonic ac-cessory lobe glomeruli (Scholz et al.,1998) may be related to the relation-ships between these inputs and theformation and maturation of the ac-cessory lobe glomeruli. Intense label-ing of a selection of accessory lobe glo-meruli is a feature of the adult brain,although which glomeruli and howmany stain is highly variable. Thereappear to be at least three sources ofNOS found in the glomeruli (1) DCneurons that project to accessory lobeglomeruli; (2) fibers of olfactory projec-tion neurons, which are intensely la-beled as they emerge from cluster 10and project into the AL; and (3) theDGNs, which project ipsilaterally tothe OLs and ALs and have fibers thatinnervate each and every glomerulusin these two regions (Sandeman andSandeman, 1987; Benton and Beltz,2001a). We have found that serotoninand NOS are colocalized in the DGNs.However, while the serotonin labelingis routinely intense, NOS labeling ishighly variable, suggesting an inter-mittent expression of this enzyme.
The presence of cGMP labeling fol-lowing treatment with an NO donorand IBMX suggests that, in many partsof the lobster brain, NO acts by meansof a cGMP-mediated pathway. Howeverin the olfactory pathway, the absence ofcGMP labeling in the olfactory and ac-
cessory lobes suggests that NO action inthese areas is accomplished by means ofa different signaling pathway. InManduca sexta, NO is found in the ol-factory receptor axons that innervatethe primary olfactory area, the anten-nal lobes (Gibson and Nighorn, 2000;Gibson et al., 2001). However, as in lob-sters, cGMP production in moths is notinduced in the antennal lobes by NOdonors in combination with IBMX. Ev-idence in M. sexta suggests that solubleguanylate cyclase does not serve as theNO receptor in the antennal lobes, butthat ADP-ribosylation may serve as theeffector pathway instead (Gibson et al.,2001). Alternative downstream effec-tors of NO signaling in the lobster olfac-tory pathway have not yet been ex-plored.
These studies, in combination withthose of Scholz et al. (1998) on lobster
larvae, suggest at least three specificroles for NO in the lobster brain: (1)interacting with the serotonergic sys-tem, (2) regulating levels of neurogen-esis, and (3) directing specific morpho-genetic changes. In addition, we wouldpropose that NO has more general de-velopmental roles, because NOS is lo-calized in different brain regions at dif-ferent developmental stages, and thatthe intensity of labeling waxes andwanes from one period to another.
Links between NO,Serotonin, and Neurogenesis
The increases in soma labeling inten-sities and altered morphologies of se-rotonergic cells following NOS down-regulation by L-NAA, as well as thecolocalization of NO with serotonin inthe DGNs, suggest that the nitrergic
Fig. 9. Levels of NOS in the olfactory pathway of juvenile lobsters increase after antennular ablation,while neurogenesis in cluster 10 decreases. A: Intense NOS labeling is found in the AL and OGTN inbrains of juvenile lobsters with intact antennules. B: Twenty-four hours following unilateral ablation ofthe lateral antennular flagellum, intense NOS immunoreactivity is observed in the AL, OL, cell bodiesand fibers of CL 10 neurons, and some cell bodies in cluster 11. C: By 48 hr following ablation, NOSstaining has extended bilaterally in the OGT (arrow). D: In experiments where the lateral flagella of bothantennules were ablated, corresponding changes in 5-bromo-2�-deoxyuridine (BrdU) incorporation inCL 10 were documented at 7 days after ablation. These counts revealed a 30% decrease in BrdU-labeled cells in the antennular-ablated lobsters compared with the unablated controls. Student t-testsreveal significant differences (P � � 0.0001) between the control and antennular-ablated groups. Forabbreviations, see list. Scale bars � 100 �m in A,B; 50 �m in C.
3056 BENTON ET AL.
and serotonergic systems in the lob-ster brain are linked. A close relation-ship between these two systems hasbeen noted before by Gibson et al.(2001), who observed that dendrites ofan identified serotonergic neurongrew beyond their normal range fol-lowing a reduction in NO levels. NOalso regulates the release of serotoninfrom the hypothalamus in rats(Kaehler et al., 1999). Interactions inthe opposite direction also have beendocumented by Ramos et al. (2002)and Tagliaferro et al. (2003), whofound that serotonin depletion causedalterations in the nitrergic system inrats. Evidence from several systems,therefore, supports the conclusionthat there is a strong interaction be-tween nitrergic and serotonergic sys-tems, and that this relationship maybe bidirectional.
As a result of the apparent interde-pendence of the nitrergic and seroto-nergic systems, it is difficult to sepa-rate the direct effects of NO frompotential indirect influences of NO bymeans of a serotonin-mediated path-way. For example, we believe that theDGN is one source of serotonin thatstimulates neurogenesis (Beltz et al.,2001; Sullivan et al., 2007). We alsohave demonstrated in the presentstudy that decreased levels of NO inembryos are associated with increasedneurogenesis. Decreased levels of NO(through NOS inhibition) causes a sig-nificant increase in serotonin labelingin the soma of the DGN. Therefore, isthe increased level of neurogenesisseen after NOS inhibition due to adirect effect of NO on the machineryproducing new neurons, or is this ef-fect due to an increase in serotoninlevels that are responsible for regulat-ing neurogenesis? If NO and serotoninact independently to alter neurogen-esis, these two neuroactive com-pounds may provide a push–pullmechanism for up- and down-regulat-ing the speed of the cell cycle and con-sequent neuronal production. Regard-less of the specific mechanism, thecolocalization of NOS and serotonin inthe DGN leads to the possibility thatthe DGN can alter the rate of neuro-genesis, depending upon the relativeconcentrations of the two substancesand the timing of their release.
In contrast to our data that supporta role for NO in suppressing neuro-
genesis, Cayre et al. (2005) have foundthat NO has a stimulatory effect onmushroom body neuroblast prolifera-tion. Furthermore, they show thatneural activity regulates NO produc-tion, as does environmental enrich-ment. Their data, therefore, also sug-gest a key role for NO in neuronalproliferation, but the direction of theinfluence is opposite to what has beenobserved in lobsters and most verte-brates, where increases in NO tend tosuppress neuronal proliferation (Ma-tarredona et al., 2005; Ciani et al.,2006; Romero-Grimaldi et al., 2006).
NO and Morphogenesis
Two observations suggest that nitricoxide is involved in morphogenesis inthe deutocerebrum, and specifically inthe olfactory pathway, during embry-onic life. Down-regulating NO in mid-to late embryonic life results in a dis-solution of the olfactory glomeruli thathad begun to form at E45%. Concom-itantly, synapsin labeling, which nor-mally would not occur until larval lifein the accessory lobes, appears preco-ciously in these regions. Both resultssuggest that NO may be involved inthe formation of glomeruli and syn-apses, and in the coordination of thesetwo processes. These data are remi-niscent of the studies of Gibson et al.(2001), where NOS is expressed in theaxons of the olfactory receptor neu-rons projecting to all antennal lobeglomeruli. In M. sexta, normal glo-merularization depends upon the in-growth of the olfactory receptor axonsthat form protoglomeruli, an eventthat is followed by migration of gliathat encircle the protoglomeruli.When NO levels are reduced using acompetitive inhibitor of NOS (L-NAME) during the period of active in-growth of the sensory axons to the an-tennal lobes, glomerular developmentis abnormal; this finding appears to bedue to the failure of neuropil-associ-ated glial cells in the antennal lobe tomigrate, suggesting that NO in thereceptor cells triggers glial cell migra-tion. NO also appears to limit the ar-borization of serotonergic neurons inthe antennal neuropil (Gibson et al.,2001).
Our finding that NOS inhibitionduring mid- to late embryonic life re-sults in the dissolution of emerging
olfactory lobe glomeruli suggests onceagain that NO is involved in signalingthat underlies morphogenesis in theseprimary olfactory processing areas, al-though the cellular mechanisms un-derlying this effect are not known. Itis possible that NO regulation of sero-tonin levels and arborization of sero-tonergic cells may contribute to theseinfluences. However, in prior studieswhere serotonin levels were reducedfor extended periods in lobster em-bryos, olfactory glomeruli formed atthe expected time and appeared to benormal for that developmental stagein terms of number, size, and generalorganization as assessed at the light-microscopic level (Benton et al., 1997).It follows, therefore, that the changein glomerular morphology induced byL-NAA treatment in embryos is theresult of the alteration in NO levelsdirectly, or indirectly by downstreameffectors, but is not mediated by a se-rotonergic pathway.
The appearance of strong synapsinlabeling in the accessory lobe follow-ing NOS inhibition also suggests anintimate connection between NO andsynaptogenesis. During the normaldevelopment of the lobster, the lateembryonic period is characterized by adevelopmental plateau during whichgrowth ceases. The plateau period canbe of variable length, and it is thoughtthat environmental stimuli are re-sponsible for triggering the end of theplateau period and resumption ofgrowth and development. During thisplateau, neurogenesis slows or stops(Harzsch et al., 1999) and does notresume until after hatching and thenonly in restricted regions in the brain.That NOS expression intensifies dur-ing the plateau period suggests func-tions for NOS in the maturation ofcircuits that is presumably occurringduring this time. That NOS inhibitionaccelerates the timing of synapsin im-munoreactivity in the accessory lobeindicates that normal NOS signalingmay suppress aspects of synaptic de-velopment, which normally occur dur-ing larval life and are coordinatedwith the formation of glomeruli in thisarea.
Overall, the influences of NOS onglomerular development in the olfac-tory lobe, synapsin expression in theaccessory lobe and neurogenesis sug-gest that NO is important in coordi-
NITRIC OXIDE IN THE CRUSTACEAN BRAIN 3057
nating these processes. Normal levelsof NO in the deutocerebrum duringthe late embryonic plateau period pre-sumably suppress neurogenesis aswell as synapsin appearance in theaccessory lobes, while permitting orpromoting the development of the ol-factory glomeruli. These types of influ-ences for NO fit into an existing largeliterature of similar developmental in-fluences in a wide range of organisms.
EXPERIMENTALPROCEDURES
Animals
Lobster eggs (Homarus americanus)were obtained from the New EnglandAquarium Lobster Rearing Facility(Boston, MA), adult lobsters from alocal fish market. At Wellesley Col-lege, lobsters of all stages were main-tained in recirculating artificial sea-water at 14°C in a 12/12 light/darkcycle. Throughout these studies, em-bryos were staged using the Perkinseye index and other morphological cri-teria, where E0% is the time of fertil-ization and E100% is hatching (Helluyand Beltz, 1991).
ImmunocytochemicalProtocols
Brains and nerve cords were dissectedin cold lobster saline (462 mM NaCl,15.96 mM KCl, 26 mM CaCl2, 8 mMMgCl, 11.11 mM glucose, and 10 mMHepes, pH 7.4). Preparations werefixed in either 4% paraformaldeyhyde(PFA) for 12–24 hr, or for NOS label-ing in ice-cold 90% methanol/10% for-malin fixative (Sigma) for 15 min(similar to Ott and Elphick, 2002). Af-ter fixation and then rinsing in 0.1 Mphosphate buffer � 0.3% Triton(PBTx), standard immunocytochemi-cal methods were used to localize sev-eral antigens. Brains from adult lob-sters were processed through thesame steps as embryonic and juveniletissue whole-mounts, except that afterfixation and rinsing they were embed-ded in 6% Noble Agar and 100 �msections produced by a Vibratome.
Primary antibodies diluted in PBTxwere applied to tissues for a minimumof 16 hr at 4°C. Preparations weredouble-labeled with (a to c): a. Rabbitanti-uNOS (1:200; universal NOS, Af-
finity BioReagents, Golden, CO; No.PA1-039) and mouse anti-synapsin (1:50, anti-Drosophila synapsin, SYN-ORF1, provided by E. Buchner); b.Rabbit anti-cGMP (1:400; provided byJ. De Vente, EURON, Maastricht, TheNetherlands) and mouse anti-synap-sin (1:50; anti-Drosophila synapsin);c. Rabbit anti-serotonin (1:1,000; Im-munoStar, Hudson, WI; No. 20080)and anti-BrdU (1:50; Becton Dickin-son, Biosciences Pharmingen, SanJose, CA; No. 347580 for the ablationstudies). After primary antibody incu-bations and rinses in PBTx, the appro-priate secondary antibodies (combina-tions of goat anti-rabbit and goat anti-mouse antibodies conjugated to Alexa488 or Alex 594 fluorophors diluted1:50 in PBTx; Molecular Probes) wereapplied overnight at 4°C. Subse-quently, brains were rinsed in phos-phate buffer, mounted with Gelmount(Biomeda), and viewed by laser scan-ning confocal microscopy.
cGMP
To localize NO-sensitive cGMP sitesin tissues, dissected brains were incu-bated in lobster saline with the NOdonor SNP (3 10�2 M, Axxora LLCSan Diego, CA; No. ALX-400-001) andan inhibitor of phosphodiesterases,IBMX (5 10�4 M, Sigma; No.17018)or IBMX alone at 10°C for 15 min. Thereaction was stopped by placing thetissue in 4% PFA. Tissues were subse-quently processed immunocytochemi-cally using rabbit anti-cGMP (deVente et al., 1987; Scholz et al., 1998).In controls, SNP and IBMX were omit-ted from the incubation medium.
NOS localization experiments wereconducted at six time points duringembryonic development (E40%, E60%,E68%, E75%, E85%, and E95%; see nvalues on Table 1) and in the adultbrain. cGMP was evaluated at E40%and E85% and in the adult brain.
Regulation of EndogenousNO Levels to TestNeurogenesis
Three groups of embryos at E40%were treated for 36 hr with: (1) theNOS inhibitor L-NAA (@ 1�5 M inlobster saline, Axxora, NG-Nitro-L-ar-ginine ALX-105-001; final n � � 6/as-say); (2) the NO-donor S-Nitroso-N-
acetyl-D,L-penicillamine (SNAP @ 1 10�6 M in lobster saline, Axxora, No.ALX-420-003; final n � � 6/assay); or(3) served as controls and were incu-bated in lobster saline alone (n � �
6/assay). Treatments were refreshedtwice daily to compensate for the half-life properties of the inhibitors anddonors when in solution. At the end ofthe treatment periods embryos werestaged, dissected, and processed im-munocytochemically as described forcolocalization or BrdU labeling.
A second NO regulation experimentthat used more advanced embryos(E75%) also was conducted. Treat-ments with L-NAA or SNAP contin-ued for 2 weeks with final dissectionsat E85%. Controls were incubated inlobster saline. Although this experi-ment also tested several different con-centrations of SNAP, the embryostreated with this NO-donor experi-enced high mortality levels; hence,this treatment was discontinued, anddata are not included.
BrdU Methods
Cells in S phase were identified by invivo incorporation of the substitutenucleoside BrdU (Sigma; No.B5002).Eggs (@E40% or E85%) were incu-bated in saline, or saline with SNAP(10�6M in lobster saline, 14°C) or L-NAA (10�5 M in lobster saline, 14°C)for 36 hr or 2 weeks, respectively.Brains were then dissected, rinsed inphosphate buffer, treated with 2 NHCl, rinsed with PBTx, and incubatedfor 2.5 hr in mouse anti-BrdU anti-body conjugated to Alexa 488 (1:20;Invitrogen Carlsbad, CA, No. A21303;n � 9). Brains were mounted as pre-viously described.
BrdU-labeled specimens were as-sessed using a Leica TCS SP confocalmicroscope. Optical sections weretaken at intervals of 0.5 �m or 1 �mand saved as three-dimensionalstacks. BrdU-labeled cell profiles incluster 10 in each optical section inthe stacked series were traced onto atransparent sheet attached to themonitor and then counted. Data arepresented as mean counts � SD. Com-parisons between control and treat-ment groups were performed by usingStudent’s t-test or an analysis of vari-ance as appropriate. A value of P �
3058 BENTON ET AL.
0.05 was considered statistically sig-nificant.
Semiquantitative Assessmentof Synapsin Levels
To define the magnitude of change insynapsin labeling in the olfactory (OL)and accessory (AL) lobes in responseto NOS inhibition, the intensity ofsynapsin labeling in these regions wasassessed semiquantitatively. Whole-mounted brains from control and L-NAA–treated embryos (E75–E85%;see above) that had been labeled im-munocytochemically for synapsinwere scanned with a Leica TCS SPconfocal microscope after setting thelaser intensities to fixed levels, andthe images were analyzed using Leicasoftware (Heidelberg, GmbH). Thebrightness of regions of interest (ROI),defined as 10-�m circles throughoutthe OLs and ALs, were determinedusing the stack profile analysis tool.Four ROIs per lobe were assigned tothe most densely labeled areas of boththe olfactory and accessory lobes. Af-ter the analysis tool was applied, onecurve per ROI through the stack ofimages for each of the ROIs was dis-played in a two-dimensional graphshowing the median intensity of eachROI throughout the stacked image.The calculated values range from 0 to255, corresponding to the intensitydistribution statistics of the confocalimage for every pixel in the ROI. Thedata also were imported to Excel soft-ware, where the mean average inten-sity for the ALs and the OLs in allbrains analyzed were computed withstandard deviations and graphed overthe z-axis, which was defined by 1-�msections throughout the depth of eachOL and AL (see Fig. 8).
Antennular Ablations andBrdU Assays
To test whether NOS localization isaltered by damage, the lateral anten-nular flagella of juvenile lobsters (sev-enth stage) were ablated unilaterally,and bilaterally to assess levels ofBrdU incorporation in cluster 10.Brains were dissected and assayedimmunocytochemically for NOS at 4,24, and 48 hr after unilateral ablation.To examine levels of neurogenesis incluster 10, lobsters were incubated in
BrdU (2 mg/ml sea water; n � 5) for 4hr, 7 days after the ablations. Unab-lated animals of the same size wereused as controls (n � 5). Standard im-munocytochemical methods were usedfor the detection of the BrdU (seeabove).
ACKNOWLEDGMENTSWe dedicate this paper to the memoryof Stephen Benton, whose creativityand dedication to science continue toinspire us. We thank J. De Vente andE. Buchner for kindly providing anti-bodies; M. Goy and J. Sullivan forhelpful discussions; Y. Kim, C.Kirkhart, Rosa Lafer-Sosa, and L.Murphy for piloting the antennularablation experiments; M. Tlusty andA. Kim of the New England Aquariumfor lobster rearing; and P. Carey andV. Quinan for technical assistance.
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