developing grasshopper neurons show variable levels of guanylyl cyclase activity on arrival at their...

Developing Grasshopper Neurons ShowVariable Levels of Guanylyl CyclaseActivity on Arrival at Their Targets

ELDON E. BALL* AND JAMES W. TRUMAN

Molecular Evolution and Systematics Group, Research School of Biological Sciences,Australian National University, Canberra, A.C.T. 2601, Australia

ABSTRACTThe ability of certain grasshopper neurons to respond to exogenously applied donors of

nitric oxide (NO) by producing cyclic GMP (cGMP) depends on their developmental state.ODQ, a selective blocker of NO-sensitive guanylyl cyclase, blocks cGMP production at 1025 M,thus confirming the nature of the response. Experiments in which the distal axon is separatedfrom its proximal stump before application of an NO donor show that guanylyl cyclase isdistributed uniformly throughout the neuron. In the locust abdomen, where segments areformed sequentially, the pattern of guanylyl cyclase up-regulation is predictable andsequential from anterior to posterior. There are two patterns of innervation by cGMP-expressing motor neurons. In the first, typified by muscle 187, an innervating neuron begins tobe NO responsive on arrival at its muscle and continues to be so over most of the remainder ofembryonic development, including the formation of motor end plates. In the second, typifiedby a neuron innervating muscle 191, the neuron extends well along the muscle, apparentlylaying down a number of sites of contact with it, before it becomes NO responsive. In bothpatterns, however, NO responsiveness marks the neuron’s transition from growth coneelongation to the production of lateral branches. Individual muscles receive innervation frommultiple motor neurons, some of which express transient NO sensitivity during developmentand others which do not. With the exception of the leg motor neuron SETi, the first motorneuron to reach any muscle is usually not NO responsive. We suggest that cGMP plays a rolein, or reflects, the early stages of communication between a target and specific innervatingneurons. J. Comp. Neurol. 394:1–13, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: nitric oxide; guanylate cyclase; Locusta migratoria; synaptic development; motor

neuron

Cyclic GMP (cGMP) is a versatile second messenger thatis produced through several different transduction path-ways (Goy, 1991; Schmidt et al., 1993). The most promi-nent families of guanylyl cyclases are the receptor cycla-ses, which are membrane bound and respond to signals,such as atrial naturetic peptide, and the soluble guanylylcyclases (sGCs), which are found in the cytoplasm andrespond to nitric oxide (NO) and other readily diffusiblemolecules such as CO. The latter soluble system isespecially well developed in the nervous system, where NOand its sGC are implicated as a retrograde signalingsystem involved in long-term changes in synaptic function(Garthwaite, 1991; Hawkins et al., 1994; Zhuo et al., 1994;Arancio et al., 1996; Son et al., 1996). Because of this rolein synaptic plasticity, there has also been interest in thepossible involvement of this pathway in the initial develop-ment of synaptic contacts (see, e.g., Gally et al., 1990).Although experimental (see, e.g., Wu et al., 1994; Wang et

al., 1995) and circumstantial (see, e.g., Samama et al.,1995; Lizasoain et al., 1996; Truman et al., 1996) evidencefor such a role exists in various systems, the exact pro-cesses that are influenced by this pathway are obscure.The recent finding that externally applied NO causes thecollapse of axonal growth cones (Renteria and Constantine-Paton, 1996) suggests that it may play a role in repulsingor arresting growth cones during development.

Grant sponsor: National Institutes of Health; Grant number: NS-13079.J.W. Truman’s permanent address is Department of Zoology, University

of Washington, Box 351800, Seattle, WA 98195.*Correspondence to: Eldon E. Ball, Molecular Evolution and Systematics

Group, Research School of Biological Sciences, Australian National Univer-sity, P.O. Box 475, Canberra, A.C.T. 2601, Australia.E-mail: [email protected]

Received 13 September 1997; Revised 31 October 1997; Accepted 3November 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 394:1–13 (1998)

r 1998 WILEY-LISS, INC.

Exposure of locust embryos to NO donors results in adramatic induction of cGMP in specific neurons. Thisresponse occurs in sensory neurons, motor neurons, andinterneurons, although, in at least the last two classes,only a subset of the neurons showed this response (Tru-man and Ball, 1994; Truman et al., 1996). For mostneurons, this ability is transient and ends prior to hatch-ing. In this paper, we first describe further analyses of thecellular basis of the cGMP response. We then provide adetailed analysis of the state of neuronal development atthe time that neurons enter into and pass through theirNO-responsive phases by using selected, characterizedneurons and neuromuscular systems of the developinggrasshopper embryo (Goodman et al., 1984; Meier et al.,1991; Xie et al., 1992; Boyan and Ball, 1993). Finally, wediscuss the properties of the neurons that are NO respon-sive vs. those that are not.

MATERIALS AND METHODS

Experimental animals

A laboratory colony of Locusta migratoria was main-tained on a diet of wheat germ and sprouted wheatseedlings. Breeding females were provided access to cupsof moist sand, into which they deposited pods of up to 70eggs. The oviposition cups were removed twice weekly andwere maintained at approximately 30°C. Intact individualegg pods were removed from the cups and kept in Petridishes between layers of moist cheesecloth. The age of theclutch could then be determined accurately by sampling afew embryos. In some cases, the clutch was kept at aconstant temperature and repeatedly sampled to followchanges in responsiveness to the NO donor sodium nitro-prusside (SNP). Staging of embryos was based on thesystem of Bentley et al. (1979), with additional criteria forlater embryos, as listed in Table 1, established by carryinga control clutch through to hatching at 30°C. Many of thecriteria in Table 1 are only adequate for staging to 65%,but some, such as the appearance of the black crescent onthe metathoracic tibia shortly before hatching, providevery precise markers. Not all of the criteria used byBentley et al. (1979) for Schistocerca are in the sametemporal relationship to one another in Locusta, so, when

questions of stage arose, the morphology of the metatho-racic leg was given the greatest weight in determining age.Ages of embryos are expressed as a percent of embryonicdevelopment (%E).

Fixation, processing,and immunocytochemistry

Locusta embryos were freed from the eggshell and yolkin phosphate-buffered saline (PBS). The forming gut wasremoved, and the embryos were pinned out flat in disheslined with Sylgard (Dow-Corning, Midland, MI). To induceactivity of the sGC, embryos were stimulated with SNP inthe presence of the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX). After some initial experimentswith different concentrations of these chemicals, which aredescribed in Results, we used standard concentrations of10 mM SNP plus 0.1 mM IBMX. These concentrationsresulted in the expression of cGMP in a maximum numberof neurons.

Following a 15-minute exposure, the SNP and IBMXwere removed, and the embryos were quickly rinsed withPBS followed by fixation in 3.7% formaldehyde in PBS forabout 2 hours at room temperature or overnight at 4°C.cGMP levels were then determined in situ by using theimmunocytochemical methods developed by de Vente et al.(1987). After repeated washes, fixed tissues were preb-locked in 5% normal goat serum (NGS) or 5% normaldonkey serum (NDS) in PBS with 0.3% Triton X-100(PBS-TX) and then incubated with a 1:4,000 dilution of arabbit anti-cGMP or a 1:10,000 dilution of a sheep anti-cGMP antiserum in PBS-TX with 1% NGS or NDS, asappropriate. After 12–36 hours at 4°C, the tissues wererepeatedly washed and then incubated overnight with aperoxidase conjugated goat anti-rabbit immunoglobulin(IgG; Kierkegaard and Perry Laboratories, Gaithersburg,MD) or donkey anti-sheep/goat IgG (Silenus Laboratories,Hawthorn, Victoria, Australia) in PBS-TX with 1% NGS orNDS, as appropriate. After additional rinses, the locationof the antibody complexes was revealed by reaction ofdiaminobenzidine (DAB, Sigma, St. Louis, MO) with glu-cose oxidase (Watson and Burrows, 1981) or H2O2. Thedeveloping solution contained 0.03% nickel chloride toyield a black reaction product.

To show the relation of cGMP-expressing neurons toother tissues, embryos were sometimes double stainedwith a second antibody. The antibodies used were Mes-3,which is specific to developing muscles and to a subset ofneurons (Kotrla and Goodman, 1984), and anti-Fasciclin II(anti-Fas II; Bastiani et al., 1987), which intensely stainscertain bundles of neurons in the grasshopper centralnervous system (CNS) and less intensely stains the periph-eral nervous system. Both are monoclonal antibodies(MAbs) raised in mouse. After processing to reveal cGMP,followed by thorough washing, embryos were incubated inone of the MAbs, thoroughly washed, incubated with aperoxidase-conjugated goat anti-mouse IgG (JacksonImmunoResearch, West Grove, PA), and reacted with DABwithout nickel. For double-labeled embryos destined forconfocal analysis, we used fluorescein isothiocyanate(FITC)-conjugated goat anti-mouse IgG (Jackson Immuno-Research) and Texas Red-conjugated goat anti-rabbit IgG(Jackson ImmunoResearch) as secondary antibodies.

Following either single- or double-staining, embryoswere dehydrated, cleared in methyl salicylate, and mountedin Permount (Fisher Biotech, Orangeburg, NY). Alterna-

TABLE 1. Staging Criteria for Locusta migratoria Embryosas Used in This Paper

60% Dorsal closure occurring, no white fatbody cells moving into midline68% White fatbody cells across dorsal midline, cuticle on legs wrinkled and opaque,

abdominal ganglion A3 just docking with A271% Retinal pigment extending across 40% of eye, pigmentation just coming onto

antenna, segmentation of tarsus apparent, tarsus 35–40% of length of tibia74% Pigmentation has almost reached base of antenna, retinal pigment covers 70%

of eye, double bend in tibia, longitudinal stripe of brown pigmentation alongdorsal femur, first trace of white stripe crossing head and eye

77% Two rows of chevrons on femur now pigmented, tarsal claws apparent, firstfour abdominal segments have white spots uniformly distributed; fromthere, posteriorly, dorsal midline becomes steadily more transparent

80% Dorsal midline now has distinct narrow white stripe83% Area of yellow pigment has appeared at distal end of mesothoracic femur,

white dorsal suture on head now very clear86% White stripe now strong on eye, transparent crescent on outside of femur tip

now clearly apparent due to pigmentation of rest of leg89% Longitudinal brown stripes on tibia very strong, white stripe on eye wider

than white stripe on head92% Brown spots just anterior to eyes have expanded, still no blackening of claws95% Transparent crescents on outside tip of metathoracic femur have now black-

ened, and tarsal claws have turned black97% Egg shell falls to pieces when picked up, mandibular teeth have turned brown,

head capsule turning black100% Hatching

2 E.E. BALL AND J.W. TRUMAN

tively, some embryos were dehydrated through a glycerolseries and photographed in 90% glycerol.

Pharmacology

Filleted grasshopper embryos were exposed to 1H-[1,2,4]-oxadiazolo[4,3-a] quinoxalin-1-one (ODQ; Tocris Cookson,Bristol, United Kingdom), a selective inhibitor of NO-sensitive guanylyl cyclase (Garthwaite et al., 1995). TheODQ was made up as a 1-M stock solution in dimethylsulfoxide (DMSO). A dilution series from 1024 M to 10210 MODQ was then made up in PBS. Control incubationsincluded 0.01% DMSO, which represents the concentra-tion of DMSO in the highest dosage of ODQ. Embryos wereincubated for 30 minutes in the inhibitor before they wereexposed to 10 mM SNP and 0.1 mM IBMX (in thecontinuing presence of the inhibitor). Embryos were stimu-lated for 20 minutes before fixation.

Image capture and processing

Stained preparations were examined on several differ-ent Zeiss (Thornwood, NY) and Nikon (Tokyo, Japan)microscopes. Images were either photographed on Ekta-chrome (ASA 50 or 64; Kodak, Rochester, NY) or videocap-tured by using Sony DXC-930 or 960 MD video cameras(Tokyo, Japan). Some images originally captured on filmwere later scanned for digitization. Two-dimensional pro-jections of neurons within ganglia or of muscles wereconstructed from stacks of optical sections that wereimported into Adobe Photoshop (version 3.0; Adobe Sys-tems, Mountain View, CA). Stacks were assembled byusing the layering palettes with the out-of-focus elementsdeleted. Optical sections of some double-stained embryoswere also obtained by using a BioRad MRC 600 confocalmicroscope (Richmond, CA). Figures were assembled andlabeled electronically.

RESULTS

The time course over which neurons become sensitive toSNP was determined by challenging embryos from stagedclutches with SNP and IBMX. For stages older than about45%E, exposure of embryos to SNP plus IBMX resulted ina reproducible set of neurons producing levels of cGMPthat we could detect by using immunocytochemistry (Tru-man et al., 1996). To provide the reader with an idea of themagnitude of the response, Figure 1 shows a filletedembryo at about 57%E, a time when peripheral staining isnear its peak.

Effect of NO donors and phosphodiesteraseinhibitors on the number of neurons

exhibiting cGMP expression

Depending on the age of the embryo, in the absence ofSNP stimulation, cGMP immunoreactivity (cGMP-IR) iseither absent or confined to a small and predictable set ofneurons (Fig. 2, spont; see also Fig. 2 of Truman et al.,1996). Treatment with SNP alone consistently reveals arelatively small but repeatable set of neurons (Fig. 2,SNP). Addition of IBMX to the SNP, thus preventing thebreakdown of the cGMP that is formed, greatly increasesthe number of neurons expressing cGMP at levels that canbe readily detected by using immunocytochemistry (Fig. 2,SNP 1 IBMX). The neurons that were stimulated by SNPalone now stain intensely, and many additional neuronsare apparent.At any particular stage of embryonic develop-

ment, neurons varied reproducibly in their response to thistreatment, with some consistently showing strong cGMP-IR, others showing moderate-to-weak levels, and othersshowing no response.

Fig. 1. A: Photomicrograph of a filleted grasshopper embryo at57% of embryonic development (%E), showing cyclic GMP immunore-activity (cGMP-IR) that was induced by incubation with a nitric oxide(NO) donor and an inhibitor of phosphodiesterase. The embryo hasbeen cut down the dorsal midline and gutted, and the body wall hasbeen pinned out to provide maximal access to the nervous system bybathing chemicals. The cGMP-IR was confined to the central nervoussystem (CNS) and the peripheral nervous system. Lines and lettersdown the right of the figure indicate the boundaries between thesubesophageal (S) and thoracic (T) segments and the thoracic (T) andabdominal (A) segments. B: A filleted embryo treated in the same wayas that shown in A except that the anti-cGMP antiserum was beenpreincubated overnight in 1024 M cGMP prior to immunostaining.Scale bar 5 300 µm.

cGMP UP-REGULATION IN DEVELOPING NEURONS 3

Dose-response relationships within the CNS

Embryonic neurons that showed NO sensitivity can bedivided somewhat arbitrarily into high-sensitivity andmoderate-sensitivity classes. Figure 3 shows the responseof A4 ganglia from embryos at about 60%E that wereexposed to successively higher concentrations of SNPalong with a constant concentration of IBMX. Levels ofSNP as low as 1–10 µM caused consistent increases inintracellular cGMP in a small group of interneurons. Thecells in this high-sensitivity class include theAVL interneu-rons (Truman et al., 1996) and other intersegmentalinterneurons with axons that run in the longitudinalconnectives. At these low concentrations, we rarely sawresponses from local interneurons or interneurons project-ing across the commissures (Fig. 3B,C).

There was an abrupt increase in the number of cellsresponding to doses of SNP between 10 µM (see, e.g., Fig.3C) and 100 µM (see, e.g., Fig. 3D). These neurons, whichshowed moderate SNP sensitivity, included interneuronswith axons that had diverse orientations and motor neu-rons along with their peripheral axons. Levels of SNPabove 100 µM SNP further enhanced cGMP expression inthese neurons, but significant numbers of new cells werenot recruited.

Apparently, differences in the sensitivity of neurons toSNP treatment are not a function of their developmentalage. This conclusion is based on a comparison of theresponse of 60%E embryos with the response of olderembryos at almost 80%E. In the latter embryos, interneu-rons with their axons in the longitudinal tracts, includingAVL, were still the most sensitive cells in the CNS (datanot shown).

Biochemical nature of the response

Figure 4 shows the response of ganglia to application ofthe NO donor SNP in the presence of increasing concentra-tions of ODQ, a selective inhibitor of the NO-sensitive sGC(Garthwaite et al., 1995). The number of respondingneurons was reduced at 1027 M ODQ, and, following

exposure to 1025 M ODQ, the response to a standard doseof SNP was virtually eliminated. Consistent with thisfinding, preincubation of the anti-cGMP antiserum with1024 M cGMP prior to applying it to the embryo abolishedthe cGMP immunostaining (Fig. 1B).

Localization of guanylyl cyclasewithin the cell

Motor axons extend well into the periphery in the bodywall of the embryonic grasshopper (Fig. 1). This made itpossible to use a simple cutting experiment to test whetherguanylyl cyclase is distributed throughout the neuron oronly in the cell body (Fig. 5). Embryos were openedmiddorsally and pinned out flat in a small Petri dish linedwith Sylgard (Dow-Corning). A cut was made in the bodywall parallel to the CNS by using a chip of razor blade,thus severing motor axons running to muscles in thedorsal body wall. The other side of the embryo was leftintact as a control. Application of SNP, fixation, andantibody application were then carried out as describedabove. The detached distal segments of the nerves showedcGMP responses similar to those shown by their contralat-eral homologues on the intact side of the embryo (Fig. 5).

Development of the capability to expresscGMP studied by using identified neurons

Neurons responding to SNP were first evident at about42–43%E, early in katatrepsis (blastokinesis). Typically,among the first neurons to become responsive was a T3motor neuron leaving the ganglion via N3. At the time thisneuron first stains, its axon extends into the periphery anddisappears down the developing leg. Within 8–12 hours(2–3%E) many additional thoracic neurons, including theidentified motor neurons aCC and RP2, begin to respond toSNP treatment (Fig. 6A). During this initial period, theneurons in T3 and T2 lead their homologues in T1 in theiracquisition of sensitivity to SNP, as is also true for otheraspects of morphological and biochemical development(see, e.g., Bastiani et al., 1992).

A segmental delay is especially evident among theserially homologous neurons in the abdomen (Fig. 7A,B),because the abdominal segments (except for the terminalsegment) are formed sequentially from anterior to poste-rior. This sequence is reflected in the order in whichsegmentally homologous neurons begin any developmen-tal process, such as sending out their axons (Goodman etal., 1982; Ho et al., 1983). Although we typically saw asegmental progression in response, a given neuron occa-sionally began to express cGMP out of the normal sequen-tial order, as is the case for the RP2 motor neuronindicated by the arrow in Figure 7C. Within a givenabdominal segment, the earliest motor neurons to becomeNO responsive were RP2 and aCC, which innervate dorsallongitudinal muscles, and two motor neurons, VML1 andan unidentified partner, which have axons that projectdown the connectives and out the intersegmental nerve toinnervate the ventromedial longitudinal muscles, 187 and188 (Fig. 6B; Truman et al., 1996).

Changes in the axonal arbor of VML1 and its partnerthrough the period when they are NO sensitive are shownin Figures 8C–E and 9. When these neurons first respondto SNP, their growth cones are club-shaped and arepositioned over their target muscles (Figs. 8C, 9A). Shortlythereafter, the growth cones begin to branch repeatedlyand spread over the surface of the muscle. The first

Fig. 2. Altered expression of cyclic GMP immunoreactivity (cGMP-IR) in abdominal ganglia A4 and A5 following pharmacological treat-ments. In the absence of chemical stimulation, the ganglia show nocGMP expression (spont). With exposure to 10 mM sodium nitroprus-side (SNP) for 15 minutes prior to fixation, many neurons, includingsome that frequently express spontaneously, now show cGMP expres-sion. In the presence of SNP and 0.1 mM isobutylmethylxanthine(IBMX; SNP 1 IBMX), cGMP is apparent in many more neurons.Scale bar 5 50 µm.


bifurcation (Fig. 9B,C, asterisk) results in branches thatsupply muscles 187 and 188, respectively. The axonsremain NO responsive during the period when higherorder branches are being made (Figs. 8D,E, 9C–G) and as

end plates are being established (Fig. 9H). The response ofthe neurons to NO then fades as the time for hatchingapproaches (Fig. 9I).

The motor neurons aCC and RP2 innervate dorsalmuscles 183 and 182 (Fig. 8A). The appearance of NOsensitivity in these neurons, likewise, occurs at a pointcoinciding with a change in growth cone behavior—fromextension of a simple compact growth cone to a complexstructure that branches as it spreads over its targetmuscle (Fig. 8B).

When the unidentified motor neuron depicted in Figures8F,G and 10 first becomes responsive to SNP, its axon hasalready made multiple contacts along the length of itstarget, muscle 191, and it may have a distal contact on anadjacent muscle (184). Consequently, its NO sensitivitydoes not coincide with the first contact with a target.Rather, this sensitivity appears at a time when its axonshifts from longitudinal extension to the start of lateralbranch growth from the multiple contact sites.

Central changes after entryinto the NO-sensitive phase

The time of onset of NO sensitivity marks an alterationnot only in the pattern of growth at the axon terminal butalso of the central arbors. Figure 11 shows segmentalprogressions for both RP2 and aCC, illustrating how thecentral arbors of these cells change after NO sensitivityappears. When both neurons become NO sensitive, their

Fig. 5. Cutting experiments demonstrate that guanylyl cyclase ispresent throughout the cell. Here, the severed tips of axons (smallarrows), which are located distal to a cut (large arrow) made beforeapplication of sodium nitroprusside (SNP), express cyclic GMP (cGMP)equally with their still-connected ipsilateral and contralateral homo-logues. Scale bar 5 200 µm.

Fig. 3. A–F: Photomicrographs of the fourth abdominal ganglionshowing the cyclic GMP (cGMP) response of 57%E embryos that wereexposed for 15 minutes to different concentrations of sodium nitroprus-side (SNP) starting at 1027 M. All treatments were also in the presenceof 0.1 M isobutylmethylxanthine (IBMX). At 1026 M, SNP acts toincrease expression in cells that were already expressing and to bring

a few more cells above threshold. Note also the first sign of expressingfibers in the connectives. Such successive recruitment continuesacross the figure with rising concentrations of SNP. There is aparticularly noticeable rise between 1025 M SNP and 1024 M. By 1023

M, the number of expressing neurons appears to have peaked. Scalebar 5 100 µm.

Fig. 4. Effect of [1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), aselective inhibitor of NO-sensitive guanylyl cyclase, on the ability ofsodium nitroprusside (SNP) and isobutylmethylxanthine (IBMX) toevoke a cyclic GMP (cGMP) response. The ODQ is dissolved in

dimethyl sulfoxide (DMSO), hence, the DMSO control. At 1027 MODQ, the extent of the cGMP response is noticeably reduced from thatof the DMSO control; at 1026 M, it is greatly reduced; and, at 1025 M, itis essentially gone. Scale bar 5 50 µm.


primary process is unadorned, with occasional thickeningsor twiglets that identify sites of future growth. Shortlythereafter, central branches start to be extended.

When interneurons begin to become NO responsive, theneuropil is generally already crowded with responding

processes from numerous cells, and it is difficult to resolvethe details of entire neurons. One interneuron (Int) thatwas favorably arranged for study is depicted in Figure 11.At the time that it is first NO sensitive, it has laid down thebasic scaffold of its neuritic arbor but appears to havegrowth cones extending in several different directions (Fig.11, arrows). It then rapidly elaborates side branches fromvarious parts of this primary arbor.

Although the central and peripheral parts of a neuronbecome NO responsive at the same time, the variousregions can differ with regard to when they lose thissensitivity. For example, for the motor neuron RP2, theaxonal arbor maintains its NO sensitivity until about85%E (Truman et al., 1996). The cell body of RP2, bycontrast, no longer shows a cGMP response after about60%E (data not shown).

Temporal and spatial aspectsof the NO-sensitive neurons in relation

to motor innervation

The relationship of NO-sensitive axons to other motorneurons innervating the body wall musculature was deter-mined by double staining SNP-treated embryos for cGMPand also with an antibody to grasshopper Fas II, whichlightly stains the entire peripheral nervous system. It isevident from Figure 8H,I that the Fas II staining indicatedthat the NO-sensitive axons are not the first axons toarrive at their targets in the abdomen. In the intersegmen-tal nerve of the grasshopper, a pair of motor neurons, U1and U2, precede RP2 and aCC in their growth to theperiphery (Goodman et al., 1984; du Lac et al., 1986) andprobably constitute the Fas II-staining axons present onthe dorsal muscles prior to the arrival of the two NO-responsive neurons. Likewise, for muscles 187 and 188, anarbor from an earlier arriving neuron (or neurons) isalready in place at the time that VLM1 and its associatemake contact with the muscle and begin to spread over it(Fig. 9). In general, examination of Fas II and cGMPstaining for the body wall muscles shows that the firstwave of axons arriving at these muscles do not give adetectable cGMP response. It is only some of the laterarrivals to a given muscle that are NO-responsive.

The intensively studied neurons innervating the exten-sor tibiae (ETi) muscle of the metathoracic leg are afavorable system for examining the relationship betweencGMP expression and other variables. Both the fast (FETi)and the slow (SETi) extensor tibiae motor neurons expressan antigen that is recognized by the Mes-3 MAb (Kotrlaand Goodman, 1984), and this allows their cell bodies to berecognized unambiguously throughout much of embryogen-esis. Figure 12 demonstrates that SETi showed an early,transient phase of NO sensitivity. In contrast, FETi, whichis easily recognizable by its size and position, failed toshow cGMP-IR after NO treatment at every time that welooked throughout embryogenesis. Thus, SETi shows aperiod of NO sensitivity that FETi appears to lack.

DISCUSSION

Anti-cGMP antibody as a tool for studyingneuronal development

Under some circumstances, the anti-cGMP antibody canbe very useful for detailed studies on the development ofthose neurons that express it. It reveals fine details, such

Fig. 6. Camera lucida drawings from cyclic GMP (cGMP)-stainedembryos showing the first motor neurons that become nitric oxide(NO)-responsive. A: In the thorax, the first motor neurons to becomeresponsive are aCC and RP2, which join one another in the interseg-mental nerve and extend dorsally up the body wall to innervate thedorsal longitudinal muscles, and a leg motor neuron. B: A highermagnification view of the early NO-responsive motor neurons in theabdomen, including aCC, RP2, and the ventromedial longitudinal(VML)1 motor neuron that innervates the A2 homologue of muscle187. The cell body of the other responsive motor neuron that inner-vates this muscle could not be identified with certainty.


as filopodia, and shows the farthest reaches of the cell justas well as the cell body. Its three major limitations are thatnot all neurons express cGMP, that expression in oneneuron can be obscured by expression in others, and thatthe full extent of the neuron is only revealed early in theinterval between the arrival of a growth cone at its targetand the establishment of synapses on that target. How-ever, in those situations where these limitations are not aproblem, this method can produce data much faster andmore easily than filling single neurons (see, e.g., Fig. 11).

Biochemical nature of the response

The previously presented evidence for the biochemicalnature of the immunostaining that we observed was, first,that the immunostaining was induced or enhanced bytreating embryos with NO donors, agents that are knownto elevate cGMP levels in vivo; second, the specificity of theanti-cGMP antibody that we were using has been repeat-edly established. Preabsorption of the antibody with cGMPabolishes immunoreactivity, whereas preabsorption withcAMP or GMP causes little or no reduction in immunoreac-tivity in either vertebrate preparations (de Vente et al.,1987) or insect preparations (Ewer et al., 1994). We haverepeated these preabsorption experiments on grasshopperembryos with the same results (Fig. 1B). Moreover, theresponse to NO donors is markedly enhanced by blockingphosphodiesterase activity (Fig. 2). The experiments withODQ (Fig. 4) further confirm that the response to appliedSNP, which we are visualizing, results from the activationof a NO-sensitive sGC.

Pathfinding and target recognitionby developing neurons

Intercellular communication obviously plays a promi-nent role in the development of a neuron. During axonaloutgrowth, a neuron’s growth cone encounters a constantlychanging set of chemical stimuli, some that have attractiveor repulsive action over a distance and others that remainlocalized at a surface (Goodman and Shatz, 1993; Good-man, 1996). These navigational cues appear to be noninter-active signals, in that the navigating growth cone is notknown to elicit an active response from the cells over andamong which it crawls. This noninteractive mode, how-ever, must change when the neuron arrives at its destina-tion, and bidirectional communication is likely involved inestablishing the final pattern of contacts between a neuronand its target (Martin and Kandel, 1996; Sheng, 1996). Inthe case of muscle innervation, it is also likely that thereare interactions between a given neuron and other motorneurons as well as between the neuron and its targetmuscle. Activity-dependent mechanisms that refine synap-tic contacts in regions of the nervous system, such as thevisual cortex (Goodman and Shatz, 1993), represent themost extreme form of this interactive mode.

The nature of the dialogue that occurs between a neuronand its targets during the initial phases of contact is notyet well understood, although progress in this area is rapid(Budnik, 1996; Keshishian et al., 1996; Martin and Kan-del, 1996; Sheng, 1996). Due to its role as a retrogrademessenger that is involved in synaptic modifications in

Fig. 7. The ability to produce cyclic GMP (cGMP) in response to asodium nitroprusside (SNP) stimulus appears sequentially down theabdominal ventral nerve cord. A: In the central nervous system (CNS),this is apparent from the sequential turn on of neuron cell bodies. B: Inthe periphery, staining axons also typically appear sequentially (aster-

isks), reflecting the anterior-posterior order of initiation of axonoutgrowth and subsequent arrival at peripheral targets. C: Occasion-ally, a peripheral axon begins to express out of sequence, as shownhere for RP2 (arrow). Scale bars 5 100 µm in A,C, 200 µm in B.


Figure 8


mature nervous systems, NO has been suggested as acandidate for signaling between target and neuron duringdevelopment (Gally et al., 1990). Correlative evidencesupporting a developmental role for NO and its targetenzyme, sGC, comes from developmental modulation ofNO synthase (NOS) and sGC in developing brains (Wil-liams et al., 1994; Samama et al., 1995; Lizasoain et al.,1996). Experimental evidence for a developmental role forNO comes from the chick visual cortex, in which blockingNOS activity results in the maintenance of improperprojections (Wu et al., 1994). Also, in vitro, NO bringsabout growth cone collapse (Renteria and Constantine-Paton, 1996). In Drosophila, pharmacological inhibition ofNOS results in retinal axons that grow beyond theirnormal targets in the optic lobes (Gibbs and Truman,1998).

Many neurons in the developing CNS of the locust showa window during which they express NO sensitivity. Theanterior/posterior segmental delay in locust embryos al-lows one to determine precisely the state of the neurons atthe time that they make this transition by comparingneurons in different abdominal segments. Our observa-tions on motor neurons (see, e.g., Fig. 9) clearly show that aneuron acquires sGC activity after it arrives at its targetbut prior to the time that it begins to spread over it. Byusing the techniques employed in this study, we cannotvisualize the neuron prior to the time that it becomes NOresponsive. Consequently, we do not know whether there isa substantial wait between the time that the growth conearrives at the target and the appearance of NO responsive-ness. One might expect that bidirectional communicationwould be important during the late phases of neuron-target interaction, as synapses are established and ma-ture. Our data indicate that such exchanges might actu-ally be occurring starting with the earliest phases ofneuron-target interaction.

Although they are potentially involved with target inter-action, the sGC and cGMP responses are likely not in-

volved in target recognition per se. This conclusion comesfrom our observations on a motor axon that innervates thetergosternal muscles (Figs. 8F,G, 10). This motor axonappears to make a linear set of contacts with its targetmuscles before it switches on its NO sensitivity (Fig. 8F).Hence, its initial target recognition must occur success-fully in the absence of this capacity. The appearance of sGCactivity is then associated with the change in growthpattern from axon extension to branching growth (Figs.8G, 10). Similarly, the slow extensor tibiae motor neuron(SETi) first arrives at its target muscle at 41%E (Myers etal., 1990) but does not start showing NO sensitivity untilat least 45%E.

Experimental systems for examining neuronal growthhave relied heavily on neurons that send a single axon overa rather complex landscape. For such neurons, it is easy todetermine when the target has been reached, but thisdetermination is much more complicated for interneuronsthat have complex central arbors. When we first seeinterneurons become responsive to SNP, they have pro-duced a basic skeleton of their central arbor, but it isdevoid of extensive branches and elaborations. Taking alesson from motor neurons, we expect that the growth thatoccurs to establish this scaffold may be through pathfind-ing mechanisms, whereas its subsequent branching andelaboration will involve bidirectional communication withother cells.

For motor neurons, the appearance of NO sensitivity isassociated with changes occurring throughout the cell. Thestart of elaboration of the axon terminal also coincideswith the beginning of elaboration of central arbors (Fig.11). For the motor neurons that we examined, we saw littleevidence of central branches as the cells enter into theirNO-responsive phase. It seems likely that the cGMPstaining is showing us a full representation of the cell atthis time, because neurons at successive stages of growthshow a gradual extension of growing branches (see, e.g.,Fig. 11).

Although we have used NO generators to activate a sGCin locust embryos, we do not know the nature of the actualsignaling molecule, especially because nicotinamide ad-enine dinucleotide phosphate (NADPH) histochemistryfails to reveal diaphorase activity in the body wall of thelocust (Truman et al., 1996). In favor of a NO hypothesis,however, is the finding that, in Drosophila, inhibition ofNOS results in the failure of retinal axons to makeconnections in their proper layers in the optic ganglia(Gibbs and Truman, 1998). Thus, it is still possible thatthere is a form of NOS that does not have diaphoraseactivity. Another possibility is that sGC is also known to besensitive to other small signaling molecules, such ascarbon monoxide (although, under some conditions, this isinhibitory; Ingi et al., 1996) and to metabolites of arachi-donic acid (Hawkins et al., 1994).

NO sensitivity and the architectureof the developing nervous system

Our classification of developing neurons into NO-sensitive and NO-insensitive classes depends on the abil-ity of the cell to produce levels of cGMP that can bedetected by the immunocytochemical methods that weemployed. In observations on hundreds of embryos ofLocusta migratoria, neurons consistently fell into one classor the other. Recent observations on embryos of anotherlocust, Schistocerca americana, show more NO-responsive

Fig. 8. Double staining the body wall with antibodies to cyclic GMP(cGMP; black) and the Mes-3 monoclonal antibody (MAb), which bindsto developing muscles, or the Fasciclin II (Fas II) MAb, which binds toperipheral nerves and axon bundles in the connectives, provides abetter idea of the environment into which the motor axons are grow-ing. Several parts of this figure are montages of multiple focal planesusing Photoshop. A: Low-magnification view of a portion of the bodywall showing the major muscles discussed in the text, which have beennumbered. B: cGMP-expressing axons of aCC and RP2 branch overthe more ventral of the dorsal longitudinal muscle (DLM) group.C: The club-shaped growth cone of VML1 is nitric oxide (NO)responsive and is located on muscle 187 (brown). D: Axons of VML1and its partner branch over muscle 187. E: At 80–85%E, both axonsstill express cGMP and show extensive branching over their muscle.F: The axon innervating muscles 191 and 193 shortly after it acquiresits NO sensitivity, as indicated by its cGMP staining. It behavessomewhat differently than cGMP-expressing neurons innervatingother muscles, in that it extends along the muscle, apparently makingcontact at a number of points before it becomes NO responsive.G: Later, a lateral branch emerges from each of these points of contactto innervate the muscle. H: Fas II staining shows the extensiveperipheral innervation (arrows; serially homologous nerves are markedby the same style of arrows) that is already present when the firstcGMP-expressing axons appear in the periphery. I: This compositefrom a through-focus series allows us to follow the cGMP-expressingaxon of the RP2 motor neuron from the cell body into the periphery(white arrows). Note that muscles 187 and 188 are already innervatedby Fas II-staining neurons (black arrow) prior to the time that thegrowth cone of VML1 becomes NO-responsive. Scale bars 5 100 µm inA,H, 50 µm B,E,G,I, 10 µm in C, 20 µm in D, 25 µm in F.


Fig. 9. Development of the innervation of muscle 187 by cGMP-expressing neurons. These photomicrographs span the interval fromthe beginning of the peripheral cyclic GMP (cGMP) response insegment A4 at about 50%E (A) to the fade out of cGMP expression atapproximately 90%E (I). Rather than spacing the photomicrographsequally through time, we have attempted to show significant changesin the pattern of innervation. Because of this, A–E span only from50%E to 60%E, whereas F–I span the remaining interval to 90%E. A–I

were all constructed from stacks of optical sections that were com-bined by using Photoshop. Stained nerves crossing the field have beenobscured purposely in order not to interfere with visualization of theinnervation of muscle 187. Two prominent branch points are indicatedin successive frames by the asterisk and the arrow. The body wall isexpanding throughout this period. Scale bars show the extent ofgrowth at the two ends of the series. Scale bars 5 25 µm in A, 50 µmin I.


neurons than we see in the corresponding stages of Lo-custa. Accordingly, some of the neurons that fail to show adetectable response in Locusta show a weak-to-moderatecGMP response in Schistocerca. The best example of thisspecies difference is seen for SETi and FETi: In Locusta,we see a moderate cGMP response from SETi and noresponse from FETi; whereas, in Schistocerca, SETi showsa very strong response, and FETi produces weak-to-

moderate levels of cGMP (Ball and Truman, unpublishedobservations). This comparison with Schistocerca makes itunclear whether the difference between NO-responsiveand NO-nonresponsive cells in Locusta is based on qualita-tive or quantitative differences in the levels of guanylylcyclase that they possess. Importantly, however, the over-all time during embryogenesis of the onset of NO sensitiv-ity in the CNS corresponds well between the two species.

Within the class of NO-responsive cells in Locusta, theinterneurons show an interesting pattern of NO sensitiv-ity. Those interneurons with axons that project in thelongitudinal connectives are sensitive to much lower con-centrations of NO compared with those with axons thatproject across the commissures. The significance of thisrelationship is presently unknown.

An intriguing aspect of the organization of the motorsystem is that given muscles are innervated by multipleneurons that have different NO sensitivities. For the bodywall muscles, most are innervated by several neurons, andmany of these neurons branch to innervate several muscles.For example, the median dorsal internal muscles areinnervated by seven (Kutsch and Heckmann, 1995) oreight (Tyrer, 1971) motor neurons, each of which appar-ently sends a branch to each bundle. Similarly, Pfluger andWatson (1988) showed that two dorsal unpaired median(DUM) neurons, DUM1 and DUM2, between them, inner-vated nine body wall muscles.

A clear example that the neurons innervating a singlemuscle have different NO response properties is evidentfor innervation of the extensor tibiae muscle by SETi andFETi. Because FETi is a large, readily recognizable cell,especially when it is stained by using the Mes-3 MAb, weare confident that, in Locusta, it does not exhibit a

Fig. 10. Camera lucida drawings showing the development of theinnervation of muscle 191 by cyclic GMP (cGMP)-expressing neurons.A–D: Illustrations of the developing innervation of the muscle over theinterval from approximately 50%E to 60%E. In A, the axon has run thelength of the muscle and has arrived at the distal end before cGMP isup-regulated. Along the way, it appears to have established points ofcontact with the muscle, and, once cGMP is up-regulated, branches arequickly sent out from these points (B). E: The axon at 90%E has grownconsiderably, but now only its major branches are visible. Over thenext 10% of development, immunoreactivity disappears.

Fig. 11. Development of motor neurons RP2 and aCC and anunidentified interneuron (Int), as established by examining succes-sively younger abdominal segments in a single embryo stained forcyclic GMP (cGMP). The axons of both RP2 and aCC start out

relatively unadorned, but they quickly put out branches and filopodia.The interneuron has at least three growth cones (arrows), but we knownothing about their normal targets.


NO-sensitive phase during its ontogeny. SETi, by contrast,does show a transient NO sensitivity. Thus, this muscle isinnervated by two excitatory motor neurons, one that isNO-responsive and the other with NO sensitivity that isgreatly reduced or lacking. For the muscles of the bodywall, we do not have markers that allow us to identify all ofthe excitatory motor neurons innervating a given muscle.Also, knowledge of the development of body wall innerva-tion is fragmentary, consisting largely of the order ofdeparture from the ganglion of some of the first motorneurons to leave (Goodman et al., 1984; du Lac et al.,1986). Although these neurons (e.g., U1, U2, aCC, andRP2) are ‘‘identified’’ in the sense that they occupy aconstant position in the ganglion and show a consistentmorphology when filled with dye, they have not in generalbeen traced to their target muscles or related to matureneurons of known adult morphology and physiology. Never-theless, at the time that we observed the first motor axonsstart to show NO responsiveness and spread over theirtarget muscles, anti-Fas II staining (Fig. 8H,I) showedthat essentially all of the developing muscles already hadother motor neurons in advanced stages of spreading overthem. Hence, the first motor neurons to arrive at a bodywall muscle do not become NO responsive as they begin tospread over their target. Later arriving neurons to thesemuscles, by contrast, are typically the ones that becomeNO responsive.

From these observations, it appears that individualmuscles may use different retrograde signaling strategiesto communicate with the multiple motor neurons thatinnervate them. The nature of other possible signalingsystems is unknown, but having a variety of ways ofcommunicating with incoming neurons likely prevents‘‘cross talk,’’ thereby insuring that signals meant for oneneuron are not misinterpreted by another. We did findcases, however, in which two motor neurons innervating

the same muscle both respond strongly to NO. For ex-ample, VML1 and its partner both innervate muscles 187and 188. These neurons start spreading over the muscle atthe same time, and they also stay together in their growthover the muscle (Fig. 9).

Although we do not know of exceptions for the body wallmusculature, it is not a universal finding that the ‘‘pio-neer’’ neuron to first arrive at a muscle is NO insensitive.SETi is a true pioneer neuron, in the sense that it does notfollow other neurons to the ETi muscle (Ho and Goodman,1982), and it is the first neuron to reach that muscle(Kotrla and Goodman, 1984; Myers et al., 1990). By usingthe Mes-3 antibody, we can unequivocally identify SETiand FETi, and we find that SETi only begins to expresscGMP at about 45%E, well after it has reached its muscle.One possible explanation for the delay is that SETi’sgrowth cone may have to reach a more distal portion of themuscle before cGMP is up-regulated. FETi, by contrast, isthe second excitatory motor neuron to reach ETi, and, inLocusta, it consistently fails to respond to SNP in all stagesthat we have tested. Therefore, SETi is a clear exception tothe rule that seems to hold in the abdomen that theNO-sensitive neurons are not the first to arrive at amuscle.

Along with the order of arrival, another possibility isthat the variation among neurons in their NO sensitivityreflects the physiological roles that the neurons will even-tually assume. O’Shea et al. (1988) recognized five func-tional classes of skeletal motor neurons: 1) fast excitatory/glutaminergic, 2) type I slow excitatory/glutaminergic butnonproctolinergic, 3) type II slow excitatory/glutaminergicand proctolinergic, 4) inhibitory/g-aminobutyric acid (GA-BA)ergic, and 5) DUM/octopaminergic. FETi and SETi areamong the few identified embryonic neurons that havealso been characterized physiologically in the adult locust.FETi is a fast excitatory motor neuron, and SETi is a typeII slow excitatory motor neuron. This physiological differ-ence between SETi and FETi brings up the possibility thatthe presence or absence of NO-sensitivity might reflectdifferences in the types of synaptic connections that arebeing made. Association of NO sensitivity with the forma-tion of a particular type of synaptic connection is attrac-tive, because almost all of the body wall muscles appear toreceive at least one axon that is NO sensitive. Resolution ofthis issue, however, requires more information concerningthe mature phenotypes, including the physiology, of themotor neurons that do and do not respond to NO byproduction of cGMP.

Initial attempts to directly test blockers of NO synthesisby injecting them into the egg produced a general retarda-tion of development rather than any specific neural effect.Rather than persist with such experiments, we haveturned to Drosophila, in which genetic as well as pharma-cological investigations are possible (Gibbs and Truman,1998).

ACKNOWLEDGMENTS

We thank Jan de Vente for the anti-cGMP antibody;Corey Goodman for the anti-Fas II and Mes-3 antibodies;Ian Morgan, Pat Miethke, and John Wellard for chemicalsand advice; John Edwards for hospitality and the use of hismicroscope; the ANU Electron Microscopy Unit for use offacilities; and Peter Braeunig for comments on the paper.

Fig. 12. Paired confocal images of a metathoracic ganglion doublestained with the Mes-3 and anti-cyclic GMP (cGMP) antibodies at63%E. The Mes-3 antibody stains two large motor neurons, the fastextensor tibiae motor neuron (FETi) and the slow extensor tibiaemotor neuron (SETi), both of which innervate the extensor tibiaemuscle in the femur of the metathoracic leg. At this stage of develop-ment, SETi (arrows) responds to SNP and IBMX by production ofcGMP, whereas FETi (arrowheads) does not.


This work was supported in part by a grant to J.W.T. fromthe National Institutes of Health (NS-13079).

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