NADPH Diaphorase Histochemistry in theThoracic Ganglia of Locusts, Crickets,and Cockroaches: Species Differences

and the Impact of Fixation

SWIDBERT R. OTT* AND MALCOLM BURROWS

Department of Zoology, University of Cambridge, Downing Street,Cambridge CB2 3EJ, United Kingdom

ABSTRACTThe NADPH diaphorase (NADPHd) reaction is widely used as a histochemical marker for

nitric oxide synthase (NOS). In this study on locusts, crickets, and cockroaches, wedemonstrate 1) that related species can differ considerably in the fixation sensitivity ofputatively NOS-related NADPHd; and 2) that prolonged fixation can induce NADPHd activityin cells that are diaphorase negative under mild fixation regimes. These two phenomenareconcile previous, contradictory reports on the distribution of NADPHd in locusts andcrickets. In locusts, neuronal NADPHd is found exclusively in interneurones. The projectionneuropiles of the exteroceptors contain a dense NADPHd-positive fibre meshwork, butsensory afferents do not stain. In crickets, staining has been reported in sensory afferents, inmotor neurones and dorsal unpaired median (DUM) neurones, and in a non-fibrousdistribution throughout the sensory neuropiles. We demonstrate that this widespread,non-selective staining is induced by strong formaldehyde fixation. Weak fixation resulted in ahighly selective labelling of a few individual interneurones and of a fibre meshwork in theprojection neuropiles of the exteroceptive afferents. Staining was absent in the afferentsthemselves, in motor neurones, and in efferent DUM neurones. Thus, after weak fixation, thestaining pattern closely matched that in the locust. The similar distribution of putativelyNOS-related NADPHd in the thoracic nervous systems of orthopteroid insects suggests aspecies-independent role for nitric oxide in the processing of mechanosensory information.Histopharmacological techniques such as permanganate oxidation, or incubation in the NOSinhibitors methylene blue or dichlorophenolindophenol, did not allow discrimination betweenthe selective and the fixation-induced staining. The species-specific impact of different fixationregimes may necessitate reconsideration of results obtained in other cross-species compari-sons. J. Comp. Neurol 410:387–397, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: nitric oxide synthase; insect nervous system; motor neurone; DUM neurone;

afferent neurone; mechanoreception

The first suggestion that the so-called NADPH diapho-rase (NADPHd) staining reaction (Thomas and Pearse,1964) can serve as a marker for the enzyme nitric oxidesynthase (NOS) came from Hope et al. (1991) and Dawsonet al. (1991). NOS catalyzes the biosynthesis of the diffus-ible messenger molecule nitric oxide (NO) by convertingL-arginine into L-citrulline. Neuronal NOS uses NADPHas a co-substrate, and a cytochrome P-450 reductase-likedomain is responsible for its NADPHd activity (Bredt etal., 1991; Blottner et al., 1995). In the NADPHd reaction,dehydrogenases transfer electrons from NADPH onto anartificial electron acceptor, usually Nitro Blue tetrazolium(NBT), leading to the formation of water-insoluble forma-

zan. The reaction is by no means unique to NOS; at leastsix different mammalian enzymes can reduce NBT in thepresence of NADPH (Stoward et al., 1991). The NADPHdtechnique as a histochemical marker for NOS is based onthe relative insensitivity to aldehyde fixation of the NOS-

Grant sponsor: Balfour Fund studentship (Department of Zoology, Cam-bridge, UK); Grant sponsor: Furst Dietrichstein’sche Stiftung studentship(Austria); Grant sponsor: NIH; Grant number: NS16058.

*Correspondence to: Swidbert R. Ott, Department of Zoology, Universityof Cambridge, Downing Street, Cambridge CB2 3EJ, United Kingdom.

Received 7 December 1998; Revised 1 February 1999; Accepted 26February 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 410:387–397 (1999)

r 1999 WILEY-LISS, INC.

related reductase activity. The specificity of the methoddepends, therefore, on eliminating NOS-independentNADPHd activity while preserving NOS-associatedNADPHd (Masumoto et al., 1993; Buwalda et al., 1995).NADPHd activity alone cannot prove the presence of NOS,but in species in which specific antibodies against NOSenzymes are not yet available, the NADPHd technique isstill indispensable for the screening of potential NOS-containing cells (e.g., Serfozo et al., 1998).

In the ventral nerve cord of the locust, the diaphorasetechnique results in a selective staining of individualneurones including their arborizations in the neuropiles(Muller and Bicker, 1994; Ott and Burrows, 1998). AllNADPHd-positive neurones appear to be interneurones;no staining is present in motor neurones, efferent dorsalunpaired median (DUM) neurones, or afferent neurones.The projection neuropiles of the mechanosensory affer-ents, however, contain a dense NADPHd-positive fibremeshwork, which is derived from collaterals of interseg-mental interneurones (Ott and Burrows, 1998). In con-trast, a study in the cricket Gryllus bimaculatus (Schur-mann et al., 1997) reported NADPHd staining in themechanosensory afferents themselves and in efferent DUMneurones and motor neurones. The neuropiles exhibited anon-fibrous, uniform staining, with a striking absence ofindividually stained neuronal processes.

To understand the role of NO in mechanosensory process-ing, it is important to establish the similarities, or differ-ences, between the nitrergic systems in different species.This paper demonstrates that for orthopteroid insects, theprincipal discrepancies in the distribution of NADPHdstaining can be resolved into two phenomena: first, speciesdifferences in the fixation sensitivity of putatively NOS-related NADPHd activity, and second, a hitherto unde-scribed form of diaphorase activity which is induced bystrong formaldehyde fixation.

MATERIALS AND METHODS

Experimental animals and tissue preparation

Adult desert locusts (Schistocerca gregaria Forskal;gregarious phase), house crickets (Acheta domesticus L.),field crickets (Gryllus bimaculatus de Geer), and Americancockroaches (Periplaneta americana L.) were obtained

from our departmental colonies. Throughout the resultssection, n values are given in the form of n 5 i[k], where iindicates the number of preparations stained, and k indi-cates the number of animals used.

In initial experiments, the following dissection protocolswere compared. Animals were dissected 1) in physiologicalsaline at room temperature (20–24°C); 2) in ice-cold salineafter cold-immobilization in crushed ice; 3) in ice-coldfixative (see below) after cold immobilization; or 4) inice-cold fixative after perfusion of the cold-immobilizedanimals with ice-cold fixative. No influence of the dissec-tion protocol on the staining pattern was observed, andanimals were dissected using protocol 3 in all later experi-ments. We have not observed any differences in thestaining between male or female animals, or betweenadult animals of different ages.

Fixation

After dissection, ganglia were immediately transferredinto the fixative, consisting of 4% paraformaldehyde in0.1 M phosphate-buffered saline (PBS; pH 7.2) with orwithout 5% sucrose; the presence of sucrose minimized thetime necessary for cryoprotection and thus the loss inactive enzyme. Fixation regimes were varied between 1hour and overnight, on ice, at 4°C, at 10°C, or at roomtemperature, and were followed by cryoprotection in 5% or10% sucrose in PBS for 7–16 hours, at the same tempera-ture as for fixing. Tissue was then embedded in 20%gelatine which was solidified by cooling in the fridge.Gelatine blocks were mounted on microtome chucks, fro-zen (Cryospray 134, Bright Instrument Co., Huntington,England), and sectioned at 10–40 µm on a Slee rotarycryostat. Sections were collected on chrome alum-gelatine-coated slides and air dried. For many experiments, serialsections were mounted onto two or more slides to give setsof adjacent sections. In the following, we will refer to fixationfor 1–2 hours on ice as weak and to overnight fixation at 10°Cas strong; other fixation regimes will be given explicitly.

The effects of weak versus strong fixation were com-pared directly between different thoracic ganglia from thesame animal, and between corresponding ganglia fromdifferent animals processed in parallel. Ganglia were firstfixed for 1–2 hours on ice in paraformaldehyde/PBS contain-ing 5% sucrose. One ganglion was then cryoprotected inplain 10% sucrose/PBS, for 7 hours to overnight, whereasthe other ganglion was subjected to a combined cryoprotec-tion/postfixation in paraformaldehyde/PBS containing 10%sucrose for the same time period. Subsequently bothspecimens were washed in 10% sucrose/PBS and cryosec-tioned as described above. The effect of prolonged fixationwas also tested on adjacent sections: one series of sectionswas directly processed for NADPHd histochemistry asdescribed below; the other series was first postfixed over-night in paraformaldehyde/PBS at 10°C.

NADPH diaphorase histochemistryand image processing

The NADPHd protocol used here is based on the proto-cols of Muller (1994), Elphick et al. (1996), and Ott andBurrows (1998). Sections of fixed tissue were rehydratedand permeabilized in Tris saline (0.1 M Tris buffer, pH 7.4,containing 0.9% NaCl and 0.5% Triton X-100) for 10–15minutes before they were incubated in 0.1 M Tris buffer(pH 8.0) containing 1.0 mM b-NADPH and 0.25 mM NBTfor 30–70 minutes at room temperature. Some prepara-

Abbreviations

CPR cytochrome P-450 oxidoreductaseDPIP 2,6-dichlorophenolindophenolDUM

neurone dorsal unpaired median neuroneHO-2 heme oxygenase-2N5 nerve 5NADH nicotinamide adenine dinucleotide (reduced)NADHd NADH diaphoraseNADPH NAD phosphate (reduced)NADPHd NADPH diaphoraseNBT Nitro Blue tetrazoliumNO nitric oxideNOS NO synthasePBS phosphate-buffered salineR3 root of nerve 3VAC ventral association centre

aVAC anterior VAClVAC lateral VACmVAC medial VACvVAC ventral VAC

VMT ventral median tract

388 S.R. OTT AND M. BURROWS

tions were deliberately overstained for up to 2 hours. Aftertermination of the staining in distilled water, sectionswere washed in Tris saline, mounted in phosphate-buffered glycerol, and viewed and photographed with aZeiss Axiophot compound microscope. Photographic nega-tives or slides were digitized on a Nikon LS-1000 filmscanner (Nikon UK, Kingston upon Thames, Surrey, UK).Negatives were inverted, and all images were scaled andhistogram-corrected, in Adobe Photoshop 4.0 (Adobe Sys-tems, Mountain View, CA).

NAD(P)H diaphorase histopharmacology

The co-factor specificity of the enzyme(s) responsible forthe NADPHd activity was tested by substituting b-NADPHwith equimolar amounts of b-NADH in the staining solu-tion. The alkaline phosphatase blocker levamisole wasused to test whether endogenous alkaline phosphatasecontributes to neuronal NADPHd (reviewed in Stoward etal., 1991; Grozdanovic and Gossrau, 1995). Sections werepreincubated for10minutes inTris salinecontaining0.2 mg/mllevamisole and were then stained for NADPHd with0.2 mg/ml levamisole added to the incubation medium.

The (non-specific) NOS inhibitors methylene blue (Luoet al., 1995) and 2,6-dichlorophenolindophenol (DPIP;Klatt et al., 1992; Spessert and Layes, 1994; Sancesario etal., 1996) were applied to test whether they differentiallyaffect the different patterns of NBT reductase activity.Sections were preincubated in Tris saline containing0.1 mM or 1.0 mM inhibitor, and then stained for NADPHdor NADHd in the presence of 0.1 mM or 1.0 mM inhibitor.The Nissl staining caused by methylene blue could bewashed out again in Tris saline. It was observed duringinitial experiments that an NAD(P)H-NBT-methylene bluemixture rapidly changes its colour from the pure bluetypical of dilute methylene blue to a dark violet. Test tubeexperiments were conducted to see whether colour changes,indicative of reactions between the different components,occur spontaneously. Methylene blue (final concentration0.1 mM) was added to 1) Tris saline (control); 2) Tris salinewith 2.0 mM b-NADPH; 3) Tris saline with 0.5 mM NBT;or 4) Tris saline with 1.0 mM b-NADPH and 0.25 mM NBT.The vials were kept either in bright light or in the dark,and resulting color changes were visually compared.

Since NOS-related NADPHd activity has been reportedto be less sensitive to oxidation than is NOS-unrelatedactivity (Grozdanovic et al., 1995; Blottner et al., 1995),sections were preincubated (oxidized) for 10 minutes withpermanganate (0.05–0.5 mM) in plain Tris saline prior toNADPHd staining as above.

RESULTS

Locusts

The pattern of NADPHd staining in locust thoracicganglia following formaldehyde fixation for 2 hours at 4°Chas been described in detail in Ott and Burrows (1998).Similar protocols were used in previous studies in thebrain (Muller and Bicker, 1994; Bicker and Hahnlein,1995; Elphick et al., 1996). Here we summarize briefly theobserved pattern in the thoracic central nervous system.The NADPHd technique selectively stains a comparativelysmall number of local and intersegmental interneurones.The cytoplasmatic staining in cell bodies was homoge-neous, sharp, and dark blue to black in colour (Fig. 1A;arrowheads in Fig. 1C). In the neuropiles, individual

stained processes stood out bluish-black against a clear orpale pink background. The definition of individual neuro-nal processes was similar to that obtained in vertebrates(e.g., cf. figures in Hope et al., 1991; Vincent and Hope,1992). The highest density of fibrous staining occurred inthe ventral association centres (VACs; with the exceptionof the medial VAC, mVAC), where a meshwork of NADPHd-positive processes is formed by collaterals of intersegmen-tal neurones (Fig. 1B, asterisks). No staining was presentin thoracic afferents, motor neurones (Fig. 1C, stars), orefferent DUM neurones. After prolonged incubation in thestaining medium, the dendritic neuropiles and virtuallyall cell bodies display a pale pink ‘‘background,’’ and afaint, granulated, ‘‘Nissl-like’’ soma staining was seen inDUM and motor neurons.

Shortening the duration of the fixation and lowering thetemperature enhanced selectivity but still resulted in thesame selectively stained processes and cell bodies (totaln 5 27 [19]). Fixation (1–2 hours) and cryoprotection on icefurther reduced the ‘‘background’’ and the faint Nissl-staining in DUM and motor neurone cell bodies (n 5 8 [5]).By contrast, strong fixation greatly amplified the ‘‘back-ground’’ staining in all dense neuropiles and neuronal cellbodies (Fig. 1D), but afferent nerve roots and VACs did notstand out particularly (n 5 6 [4]). The general cell bodystaining varied considerably in intensity and colour.Whereas most of the somata were stained pink to ma-genta, a more intense bluish staining was observed inothers, some of which were identified by cell body size andposition as motor neurones (black stars in Fig. 1D,E) andefferent DUM neurones (white stars in Fig. 1 D,F). Thisstaining had a granulated, ‘‘Nissl-like’’ appearance, andless intensity and definition compared with that in weaklyfixed ganglia (compare Fig. 1A with E, F). Strong fixationdid not, however, abolish the selective staining of indi-vidual cell bodies and processes (Ott and Burrows, 1998;asterisks in Fig. 1D–F). After overnight fixation at 22°C,however, the selective staining was almost completelyabsent (n 5 2 [2]). Even after this fixation regime, epithe-lial cells of the tracheae were unstained (cf. results in cricketsand cockroaches). Therefore in the locust, strong formalde-hyde fixation induces widespread additional NADPHdactivity, but the selective staining is still apparent.

Crickets

The staining observed in Gryllus bimaculatus (n 5 9 [8])and Acheta domesticus (n5 16 [13]) was very similar, andtherefore the following description applies equally to bothspecies unless explicitly stated otherwise. A pattern asdescribed by Schurmann et al. (1997) was observed after2 hours of fixation at 10°C, but the staining intensity waslow. Extending fixation overnight induced the same pat-tern at a much higher intensity (Fig. 2A–E; n 5 8 [7]). Allsynaptic neuropiles and cell bodies displayed a pink ‘‘back-ground’’ staining. A more intense, purple to blue stainingwas present 1) in the VACs (Fig. 2A–E); 2) in many afferentnerve roots of the paired lateral nerves (white arrows inFig. 2A,B; R3 in Fig. 2C,E); 3) in certain fibre tracts such asthe lateral ventral tracts (white arrows in Fig. 2D); and 4)in certain cell bodies; most prominent among the latterwere the efferent DUM neurones (stars in Fig. 2D,G). Thestaining of cell bodies had a granulated, ‘‘Nissl-like’’ appear-ance similar to that in strongly fixed locust ganglia. Therewas no selective labelling of individual neuronal processes,and no fibrous staining was detectable in the VACs. In

COMPARATIVE NADPH DIAPHORASE HISTOCHEMISTRY 389

Fig. 1. NADPH diaphorase staining in the locust metathoracicganglion after weak fixation (10°C for 1 hour; A–C) and strong fixation(10°C overnight; D–F). This ganglion consists of the fused neuromeresof the metathoracic and the first three abdominal segments. For thenomenclature of NADPHd-positive neurones, see Ott and Burrows(1998). Transverse sections, dorsal is top. A–C: Weak fixation produceshighly selective, dense, and homogeneous staining in a few interneuro-nal cell bodies [A, a dorsal unpaired median (DUM)-like type 3A2

interneurone in the abdominal neuromere 2 of the metathoracicganglion; C, two type 2 interneurones in the metathoracic neuromere,indicated by arrowheads], and in individual processes in the neuro-piles (A, B). A dense meshwork of stained fibres is present in the

projection neuropiles of the mechanosensory afferents (ventral associa-tion centres, VACs; asterisks in B). Somata of motor neurones areunstained (black stars in C). D–F: Strong fixation induces strongbackground staining in the neuropiles, as well as general staining ofsomata that varies considerably between different neurones. The cellbodies of motor neurones (black stars in D and E) and efferent DUMneurones (white stars in D and F) are amongst the most stronglystained. They contain granulated NADPHd-positive material (E, F).The selective staining of individual fibres, however, is still present(asterisks). For anatomical abbreviations, see list. Scale bars 5 50 µmin A, C, E and F; 100 µm in B and D.

390 S.R. OTT AND M. BURROWS

Fig. 2. NADPH diaphorase staining in cricket thoracic gangliaafter strong fixation (overnight at 10°C; A–E and G) and weak fixation(1–2 hours on ice; F and H). Prothoracic ganglion except D, mesotho-racic ganglion. Transverse sections, dorsal is top. C and D are fromAcheta, all others from Gryllus. A–E: Strong fixation induces generalstaining in all dense neuropiles. Staining is stronger, however, in theprojection neuropiles of the mechanosensory afferents (ventral associa-tion centres, VACs) and in afferents entering the VACs via lateralnerve roots (white arrows in A and B; R3 in C and E) and vialongitudinal fibre tracts (white arrows in D). No individual fibres areseen in the VACs (E). F: Weak fixation results in selective staining of a

meshwork of fine fibres and boutons in the VACs. Afferents in R3 arenot stained, and the background staining in the dense neuropiles isgreatly diminished. G: Strong fixation induces staining in the cellbodies of efferent DUM neurones (stars). H: No such staining is seenafter weak fixation (stars), although this section was deliberatelyoverstained. Arrowheads, a pair of heavily stained somata consis-tently found in the prothoracic ganglion after weak fixation, but neverseen after strong fixation. In Gryllus, tracheae show up strongly afterstrong fixation (A, B, E and G, black arrows); in Acheta, tracheae arealways unstained (C, D). For anatomical abbreviations see list. Scalebars 5 100 µm.

Gryllus (n 5 4 [4]), but not in Acheta (n 5 4 [3]), strong bluestaining was also present in tracheal epithelial cells, tosuch an extent that all structures resembling neuronalprocesses in Fig. 2A,B,E, and G (black arrows) are tra-cheae.

The pattern of staining was dramatically different whenfixation (1–2 hours) and cryoprotection were carried out onice (weak fixation; Figs. 2F,H and 3A,C,E,G,I; n 5 12 [10]).Selective staining was now observed in individual cellbodies and processes not seen after strong fixation, whereasthe ‘‘background’’ in the dense neuropiles was reduced orabolished. Staining was no longer present in efferent DUMneurones (stars in Figs. 2H and 3G), motor neurones (Fig.3E, stars), or afferents (R3 in Fig. 2F; arrowhead in Fig.3C). Tracheal cells in Gryllus were now unstained (Fig.2F,H; n 5 4 [3]). The number of NADPHd-positive cellbodies revealed with this protocol was much lower than inthe locust. For example, the prothoracic ganglion con-tained a single pair of heavily stained somata (30 µm) atits posterior end (Fig. 2H), and a cluster of approximately10 faintly to medium stained small (10 µm) cell bodies atthe anterior end (not shown). A pair of heavily stained cellbodies was present in each of the two abdominal neuro-meres fused into the metathoracic ganglion (Fig. 3I).Throughout the projection neuropiles of the mechanosen-sory exteroceptors (VACs, but not mVAC) a dense mesh-work of fine NADPHd-positive fibres and strongly stainedboutons was present (Figs. 2F, 3A,C,E). A few stainedaxons ran longitudinally through the thoracic ganglia inthe ventral median tracts (VMTs; not shown). They mightcorrespond to the diaphorase-positive axons seen in thehomologous tract in the locust. In the latter species,collaterals of these axons appear to be the main source forthe meshwork in the VACs. It is tempting to conclude thatan analogous scheme applies to the origin of the diaphorase-positive meshwork in the cricket VACs. Indeed, as in thelocust, collaterals of the intersegmental axons in the VMTsentered the VAC (not shown). The very fine diameter ofthese fibres, however, makes it difficult to decide whetherthey constitute the prime source of diaphorase-positivefibres in the VACs.

There are two possibilities for why the selective stainingwas not detectable in strongly fixed tissue, namely,1) masking of the selective staining by fixation-inducedstaining; or 2) inhibition of the selective staining byfixation. The following observation shows that prolongedexposure to the fixative does indeed diminish the selectivestaining. Certain cell bodies displayed very heavy stainingafter weak fixation (Figs. 2H, 3I) that could not be maskedby the much weaker staining induced by strong fixation;they were, however, not discernible in strongly fixedtissue, indicating that the enzyme responsible for theselective staining is susceptible to strong fixation. Selec-tive staining was more robust to fixation in Acheta than inGryllus, and it was possible to obtain a superposition ofboth patterns in the same preparation by fixing a ganglionovernight on ice or at 4°C (n 5 5 [4]).

Comparison between differently processed ganglia fromthe same animal, or from different animals processed inparallel, shows that the fixation regime determines whichof the two patterns of staining is obtained (total n 5 25 [21]).Furthermore, the effects of prolonged fixation on wholeganglia could be mimicked by postfixation of sectionedmaterial that prior to sectioning had been fixed on ice for 1hour only. The left-hand column in Figure 3 summarizes

the main features of selective staining obtained after shortfixation on ice: fibrous staining in the VACs (Fig. 3A,C,E),absence of staining in afferents (Fig. 3C, arrowhead),motor neurones (Fig. 3E, stars), and efferent DUM cells(Fig. 3G, stars), and heavy staining in a few cell bodies andneurites (Fig. 3I, arrows and arrowheads). The right-handcolumn shows adjacent sections that have been postfixedovernight. The selective staining was no longer seen, and adifferent pattern was induced corresponding to that givenin Schurmann et al. (1997): non-fibrous staining in theVACs and ‘‘background’’ in all other dense neuropiles (Fig.3B,D,F,J), staining in afferents (Fig. 3D, arrowhead),motor neurones (Fig. 3F, stars), and efferent DUM cells(Fig. 3H, stars). Cell bodies that stained heavily withoutpostfixation displayed very faint staining in adjacentsections which were post-fixed, demonstrating that theselective staining is not simply masked by the increasedbackground (arrows in Fig. 3I,J). The pattern of staininginduced by on-slide postfixation was capricious, and in twoof the four experiments the only effect was the completeabolition of the selective staining.

Cockroaches

After strong fixation, a general pink staining of non-fibrous appearance was present throughout all denseneuropiles (not shown; n 5 2 [2]). The VACs and someafferent nerve roots were darker and more bluish in colour,although this differential effect was less pronounced thanin crickets. Neuronal cell bodies also exhibited a generalpink shading after overnight fixation, but motor neuronesand efferent DUM neurones did not stand out. Afterfixation at 10°C for 2 hours, background was significantlyreduced, and very weak fibrous staining was seen in theVACs. No staining was present in efferent DUM neuronesor motor neurones (n 5 3 [2]). Fixation on ice for 1 hoursuppressed all background staining, but no stained fibreswere seen (n 5 2 [2]). Selective fibrous staining occurred inthe VACs after fixing for 2 hours on ice (n 5 3 [3]). Thestaining peaked after fixation for 4 hours on ice (Fig. 4;n 5 2 [2]) but decreased with stronger fixation regimes(n 5 3 [2]). The individual boutons were strongly stainedbut because they are very fine the overall appearance ofthe VAC staining was much fainter than in locusts orcrickets. A single pair of intersegmental axons was seenrunning through the thoracic nerve cord in the VMTs.

Fig. 3. Distribution of NADPH diaphorase activity in the metatho-racic ganglion of the cricket Acheta after weak fixation (1 hour on ice;left column), and the effect of on-slide postfixation (overnight at 10°C;right column), demonstrated on adjacent transverse sections; Nomar-ski DIC, dorsal is top. A, C: After weak fixation on ice, a meshwork ofstained fibres and boutons is present in aVAC, vVAC, and lVAC.Afferents are unstained (C, arrowhead). B, D: In adjacent sectionsthat have been postfixed, fibrous staining is abolished and generalnonfibrous staining is induced in the VACs and to a lesser extent in allother dendritic neuropiles. Afferents show blurred staining (D, arrow-head). E: Motor neurone somata (stars) are unstained after weakfixation. F: Postfixation induces shades of staining in the motorneurone somata (stars). G: DUM cell bodies (stars) are unstained afterweak fixation. H: Postfixation induces strong granular staining inDUM cell bodies (stars). I: After weak fixation, a pair of heavily stainedneurones (arrows) and their neurites (arrowheads) show up againstclear background in the second abdominal neuromere of the metatho-racic ganglion. J: Postfixation greatly reduces the cell body staining(arrows) and renders the neurites indistinguishable from background;note that now other cell bodies display staining of comparableintensity. For anatomical abbreviations see list. Scale bars 5 100 µm.

392 S.R. OTT AND M. BURROWS

Figure 3

Tracheal staining was present with all fixation protocols(arrows in Fig. 4; n 5 15 [13]). Its intensity showed strongpositive correlation with fixation time, so that heavytracheal staining was observed after overnight fixation.Whatever the fixation conditions, no selectively labelledneuronal cell bodies were seen in the thoracic ganglia.

NADH diaphorase histochemistry

In further experiments we attempted a histopharmaco-logical characterization of the enzymes responsible for thedifferent types of NADPHd staining. The co-factor specific-ity was investigated in locusts and crickets by substitutingb-NADH for b-NADPH in the staining solution(n 5 13 [12]). This caused widespread and heavy stainingthat developed rapidly in both weakly (not shown;n 5 10 [9]) and strongly (Fig. 5A,C; n 5 3 [3]) fixedpreparations. This rapid development made it difficult tojudge whether the enzyme responsible for the selectiveNADPHd staining can also use b-NADH, but comparingalternate sections of briefly fixed locust ganglia stained forNADPHd and NADHd, respectively, suggests it does not.The highest density of NADHd-positive material occurredin the dendritic neuropiles (white stars in Fig. 5A,C) and inthe cytoplasm of neuronal cell bodies. Axons containeddistinctly stained subcellular structures embedded in clearcytoplasm—most likely mitochondria (Stoward et al., 1991;Johnson, 1991). In the soma cytoplasm, both mitochondriaand material of Nissl-like appearance seem to contributeto the staining. The latter was especially conspicuous inthe somata of efferent DUM neurones (arrowheads in Fig.5A) and certain motor neurones, a pattern reminiscent offixation-induced NADPHd (arrowheads in Fig. 5B). Afurther correlation between the distribution of NADHd(Fig. 5A,C) and that of fixation-induced NADPHd (Fig.5B,D) was observed in the neuropiles, where both types ofstaining occurred in high densities in dendritic areas(white stars in Fig. 5), whereas axon tracts were weaklystained (black stars in Fig. 5).

Spherical or elongated subcellular structures resem-bling mitochondria were also stained in some NADPHdpreparations. They were usually absent after brief fixation

on ice, but no other correlation with the fixation conditionscould be established. They showed up primarily on theupper surface of the sections, and/or in tissue that is closeto the surface of the (intact) nervous system, such as inperipheral nerves and connectives, and the anteriormostand posteriormost portion of the ganglion. In the lateralnerves, chains of mitochondria in the axons can be mis-taken for beaded NADPHd-positive fibres.

NAD(P)H enzyme histopharmacology

In vertebrates, several histopharmacological procedureshave been reported to discriminate between NOS-relatedand -unrelated NADPHd activity, including permanganateoxidation (Grozdanovic et al., 1995; Blottner et al., 1995)and incubation with the NOS inhibitors DPIP (Klatt et al.,1992; Spessert and Layes, 1994; Sancesario et al., 1996)and methylene blue (Luo et al., 1995). The effects ofpermanganate oxidation on the selective and the fixation-induced NADPHd staining were tested in strongly fixedlocust tissue, in which both types of staining co-exist(n 5 3 [2]), and in weakly fixed locust tissue (n 5 7 [6]).After pretreatment with 0.5 mM permanganate, selectivestaining was clearly present, although somewhat attenu-ated in intensity, whereas the fixation-induced stainingwas no longer observed. This result would have beenexpected even if there were no differential effect, since thefixation-induced staining is weaker and would thereforebe thresholded out first. Indeed, prolonged incubation inthe staining solution re-established the pattern seen in theabsence of oxidation, indicating a similar reduction in theselective and the fixation-induced NADPHd staining.

Irrespective of the fixation procedure, the presence of0.1 mM DPIP during NADPHd histochemistry causedwidespread background staining which was most pro-nounced in dendritic neuropiles and neuronal cell bodies(n 5 3 [1]). The selective NADPHd staining after weakfixation was reduced in the presence of DPIP, but cellbodies and processes that show up strongly for NADPHd incontrol sections were still discernible against the back-ground staining. At a DPIP concentration of 1.0 mM, allNADPHd staining was abolished (n 5 2 [2]). Surprisingly,NADHd staining was also reduced in the presence of0.1 mM DPIP (n 5 1 [1]) and completely abolished in thepresence of 1.0 mM DPIP (n 5 2 [2]). Similarly, methyleneblue affected NADPHd (n 5 7 [5]) and NADHd (n 5 4 [4])staining equally. Both were strongly reduced in the pres-ence of 0.1 mM methylene blue (n 5 5 [4]), and completelysuppressed in the presence of 1.0 mM methylene blue(n 5 6 [5]). When 0.1 mM methylene blue and 2.0 mMb-NADPH were mixed in Tris saline in a test tube, underbright light conditions the colour gradually faded overapproximately 20 minutes until the solution was almostcolourless. Fading also occurred in the dark, but was lesspronounced. Since reduced methylene blue is colourlessthis result suggests that a redox reaction occurs betweenmethylene blue and NADPH in the absence of NOS.Mixtures of methylene blue and NBT showed no colourchanges indicative of reactions between the two compo-nents. Mixtures containing 1.0 mM b-NADPH, 0.25 mMNBT, and 0.1 mM methylene blue in Tris saline rapidlyturned dark violet both in the light and in the dark.Levamisole (0.2 mg/ml) was tested in strongly fixed Gryl-lus ganglia (n 5 2 [2]) where it did not affect the staining

Fig. 4. NADPH diaphorase staining in the cockroach metathoracicventral association centre (VAC) after 4 hours of fixation on ice. A veryfine meshwork of fibres and boutons is present in the VAC. Trachealcells are also stained (arrows). Transverse section, dorsal is top. Scalebar 5 100 µm.

394 S.R. OTT AND M. BURROWS

pattern, indicating that endogenous phosphatase does notcontibute to the NADPHd staining.

DISCUSSION

The correlation between NOS-like immunoreactivityand NADPHd, and the impact of different fixation regimeson the NADPHd staining pattern, have been extensivelyinvestigated in recent years (e.g., Kishimoto et al., 1993;Cooke et al., 1994; Spessert and Layes, 1994; Spessert etal., 1994; Buwalda et al., 1995; Elphick et al., 1995;Sancesario et al., 1996; Wang et al., 1997; Spessert andClaassen, 1998). A mismatch, however, between NOSimmunohistochemistry and NADPHd enzyme histochem-istry cannot easily be resolved by dismissing the latter asless specific; diaphorase staining may also show genuineNOS isoforms that escape detection by the antibody. Thepresent study demonstrates 1) that species differ consider-ably in the fixation sensitivity of putatively NOS-relatedNADPHd; and 2) that prolonged fixation induces NADPHdactivity in cells that are diaphorase negative under mildfixation regimes. These two phenomena reconcile previ-

ous, contradictory reports on the distribution of NADPHdin two related insects, the locust and the cricket.

Resolving species differences

In the thoracic nerve cord of the locust, selectiveNADPHd staining occurs in morphologically identifiableinterneurones but not in afferents, motor neurones, orefferent DUM neurones (Muller and Bicker, 1994; Ott andBurrows, 1998; this study). The high density of interneuro-nal fibres staining for NADPHd in the tactile projectionneuropiles suggests a role for NO in the processing ofmechanosensory information (Ott and Burrows, 1998). Inanother orthopteran species, the cricket Gryllus bimacula-tus, however, Schurmann et al. (1997) reported staining inafferents, motor neurones, and efferent DUM neurones,and a non-fibrous staining in the sensory neuropiles. Thepresent study demonstrates that this pattern is induced byprolonged formaldehyde fixation. After brief fixation onice, a very different pattern is obtained in two species ofcrickets. Then only a few interneuronal cell bodies, inter-segmental axons, and a dense, fibrous meshwork in thetactile projection neuropiles are stained selectively, while

Fig. 5. Partial correlation between NADH diaphorase (A, C) andfixation-induced NADPH diaphorase staining (B, D) in the cricketAcheta, demonstrated in adjacent transverse sections. Prothoracicganglion; A and B are from the posteriormost region of the ganglion.Dorsal is top. After strong fixation, cell bodies of DUM neurones(arrowheads) show granulated, ‘‘Nissl-like’’ diaphorase staining in the

presence of both NADH (A) and NADPH (B). In the neuropiles, NADHand NADPH also show a corresponding distribution, with densestaining in the dendritic areas (white stars) and absence of staining inaxon tracts (black stars). The fixation-induced NADPHd staining inafferents (R3 in D), however, has no correlate in NADHd activity (R3 inC). Scale bars 5 50 µm.

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afferents, motor neurones, and efferent DUM neurones areall unstained. This pattern corresponds well to that seen inthe locust after mild formaldehyde fixation. In locusts, asin crickets, prolonged fixation induces widespread stainingthat is not seen after mild fixation. In locusts, however, theselective staining of individual somata and processes isquite robust to prolonged fixation, whereas in crickets it ismore easily abolished. This suggests that NOS, which ispresumably responsible for the sharp, selective staining ofindividual neurones in both locusts and crickets, displaysconsiderable species differences in the fixation sensitivityof the NADPH reductase domain.

Diaphorase histopharmacology

The histopharmacological procedures used in this studydid not allow safe discrimination between NOS-relatedand -unrelated NADPHd activity. The nonselective NOS-inhibitor DPIP (Klatt et al., 1992) at 0.1 mM concentrationcaused a partial inhibition of the selective staining butinduced strong background staining; at 1.0 mM, all stain-ing was abolished—findings that are in agreement withobservations in the rat choroid plexus (Sancesario et al.,1996). DPIP also inhibited NADPHd staining induced byprolonged fixation and NADHd staining; apparently itinterferes non-selectively with different diaphorase reac-tions. Studies that have used DPIP at high (1.0 mM)concentration as evidence for the NOS specificity of thediaphorase staining (e.g., Leake and Moroz, 1996) mighttherefore need reconsideration. Similar to DPIP, methy-lene blue inhibited both NADPHd and NADHd staining ina concentration-dependent manner. Methylene blue hasbeen shown to inhibit the biosynthesis of NO by ratcerebellar NOS in a radiochemical assay, and also theNADPHd histochemical reaction in rat brain frozen sec-tions (Luo et al., 1995). A direct inhibition of NOS bymethylene blue was proposed, via a shunting of singleelectrons from the cytochrome P-450 reductase-like do-main of NOS. Our test tube experiments suggest that aredox reaction between methylene blue and the enzymecofactor NADPH may contribute to the inhibitory effect.

Nature of the fixation-induceddiaphorase activity

We encountered two types of fixation-induced NADPHdactivity: 1) increased background and 2) a more pro-nounced staining in certain cell bodies, including DUMcells and motor neurones (in crickets and locusts), in theVACs and their associated afferent axon bundles (mostprominently in crickets), and in tracheal cells (in Gryllusand in the cockroach). None of the phenomena could berelated to endogenous phosphatase activity (reviewed inStoward et al., 1991; Grozdanovic and Gossrau, 1995)since they persisted in the presence of levamisole. Atpresent it is unclear what causes the fixation-inducedstaining, but it seems that more than one factor is respon-sible. We found some correlation between the distributionof NADHd activity and that of fixation-induced NADPHd(Fig. 5). Material with a Nissl substance-like appearancedisplays both types of staining, especially in DUM andmotor neurone cell bodies. Fixation-induced NADPHd-staining and dark NADHd staining also coincide in thedendritic neuropiles. Therefore the possibility arises thatprolonged fixation alters the cosubstrate specifity of someNADH dehydrogenase(s). While this would account for thefixation-induced NADPHd-staining in cell bodies and dense

neuropiles, it fails to explain fixation-induced staining inafferent fibre tracts in crickets, which contained littleNADHd-positive material (R3 in Fig. 5C,D). Moreover, itcannot explain why, in crickets, the VACs stand out amongthe dense neuropiles in fixation-induced NADPHd but notin NADHd activity.

The enzyme cytochrome P-450 oxidoreductase (CPR) isfrequently discussed as a source of NOS-unrelatedNADPHd activity. Although vertebrate CPR has beenreported to be fixation sensitive (Masumoto et al., 1993)—making it less plausible as a source of fixation-inducedNADPHd—histochemical evidence is contradictory (e.g.,Kishimoto et al., 1993). CPR appears to be the provider ofelectrons for heme oxygenase-2 (HO-2), at least in thevertebrate brain where the two enzymes are stronglycolocalized (Verma et al., 1993). NOS-unrelated NADPHdmay therefore be associated with HO-2. Strikingly, neu-rones displaying fixation-induced NADPHd staining (mo-tor and DUM neurones and mechanosensory afferents)coincide with those containing guanylyl cyclase, the targetenzyme of both NO and carbon monoxide (CO) signalling(S.R. Ott, I.W. Jones, and M.R. Elphick, in preparation).

In the locust there is good evidence that the selectivestaining of individual neurones seen after a standardNADPHd protocol corresponds to neuronal NOS (Mullerand Bicker, 1994; Elphick et al., 1995). In the presentstudy we have also shown, however, that the method canbe very capricious in certain species, where putativelyNOS-related staining is more easily abolished, and adifferent type of staining more readily induced. The pres-ent findings suggest that the distribution of neuronal NOSin the thoracic nervous system is in principle similar inlocusts, crickets, and cockroaches. A main common featureis the absence of NOS in the exteroceptive afferentsthemselves and the presence of a nitrergic fibre meshworkof interneuronal origin in their projection neuropiles. Thisconspicuous NADPHd-positive innervation of the VACssuggests a species-independent role for NO signalling inthe mechanosensory pathways of orthopteroid insects.

ACKNOWLEDGMENTS

We thank Drs. Berthold Hedwig, Thomas Friedel, Mi-chael Gebhardt, Tom Matheson, Mark Wildman, andOliver Morris (all Department of Zoology, Cambridge, UK)for their helpful comments on earlier versions of themanuscript. This study was supported by studentships toS.R.O. from the Balfour Fund (Department of Zoology,Cambridge, UK) and the Furst Dietrichstein’sche Stiftung(Austria), and by an NIH grant (NS16058) to M.B.

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