Calcium-Activated Release of Nitric Oxide and Cellular Distribution of Nitric Oxide-Synthesizing Neurons in the Nervous System of the Locust

The Journal of Neuroscience, December 1994, 74(12): 7521-7528

Calcium-activated Release of Nitric Oxide and Cellular Distribution of Nitric Oxide-Synthesizing Neurons in the Nervous System of the Locust

Uli Miller and Gerd Bicker lnstitut ftir Neurobiologie, 14195 Berlin, Germany

Nitric oxide (NO) is generated by a Ca2+/calmodulin-acti- of NO synthasehave also been characterized with the sensitive vated NO synthase and diffuses as a short-lived transcellular oxyhemoglobin assay(Feelisch and Noak, 1987; Murphy et al., messenger through the plasma membrane. This study in- 1991; Mayer et al., 1992) in the brain of Drosophila and Apis vestigates the neurochemistry and anatomical distribution (Miiller, 1994), showing that in both speciesNO synthaseac- of NO-releasing cells in the CNS of the locust. Ca*+/cal- tivity and NADPH diaphorase(NADPHd) activity after fixation modulin-activated NO synthase is responsible for fixation- arc caused by identical enzymes. Furthermore, measurements insensitive NADPH diaphorase (NADPHd) activity in cell of NO synthaseactivity in homogenatesof the various neuropils homogenates of the nervous system. Therefore, neurons correlated well with the histochemical staining pattern for expressing NO synthase were detected by NADPHd histo- NADPHd (Mtiller and Buchner, 1993; Miiller, 1994). As in chemistry performed in whole-mounts. The anatomical vertebrates (Dawson et al., 1991; Hope et al., 1991; Vincent screening revealed fewer than 1% NADPHd-positive cells and Kimura, 1992), NO synthasexxpressing cells of insect ner- in the ventral nerve cord, some of which were single poten- vous systemscan be reliably identified by NADPHd histochem- tially identifiable neurons, and groups of cell bodies in sev- istry. eral regions of the cerebral ganglion. A prominent feature Werman (I 966) has formalized the experimental approaches of the histochemical survey in the cerebral ganglion is a needed to establishwhether a given substanceis functioning as group of 45 intensely stained cells innervating the olfactory a neurotransmitter in the nervous system. Briefly, theserequire- neuropil of the antenna1 lobe. ments can be summarized as presence,synthesis, release,in- A basic requirement for identifying NO as a messenger activation, identity of action, and reception. One of the criteria molecule is the Ca2+-dependent release during nerve cell for transmitter identification that in practice is difficult to prove depolarization. With a sensitive photometric assay we dem- is the calcium-dependent release following depolarization of onstrated that dissociated cells from brain areas rich in neurons.Obviously, the identification criteria of Werman (1966) NADPHd-positive neurons release NO after stimulation by were formulated for conventional neurotransmitters releasedin agents elevating cytoplasmic Ca*+ levels and by the excit- a calcium-dependentprocess from synaptic vesicles. Neverthe- atory neurotransmitter acetylcholine. The combined anatom- less,there are clear formal parallels to the generation and re- ical and biochemical experiments therefore provide firm ev- ception of unconventional messengermolecules like NO. After idence that NO is a messenger molecule released in the CNS their Ca’+-activated production catalyzed by NO synthase,the of the locust. Since locust neurons can be readily grown in short-lived NO molecules are thought to diffuse through the primary culture, NO-induced elevations of CGMP levels and plasma membrane as a transcellular messengerand stimulate other signal transduction mechanisms in target cells will also the guanylyl cyclaseofadjacent cells (reviews: Garthwaite, 1991; be amenable to a cellular anirlysis. Bredt and Snyder, 1992). It would therefore be advantageous [Key words: NADPH diaphorase, NO synthase, transmitter to work with a preparation in which the completechain ofevents release, olfactory neuropil, signal transduction] from plasma membrane depolarization to NO releaseonto de- fined target cells can be studied. The objective of our study was There is increasing evidence that insect nervous systems use to identify candidate insect neuronsthat would allow the dem- nitric oxide (NO) as a chemical messenger.Biochemical assays onstration of a Ca2+-stimulated NO release. have demonstratedthe presenceofa Ca’ +/calmodulin-activated The nervous system of the locust is built on a clearly seg- NO synthaseand NO-activated guanylyl cyclasein homogenates mented plan containing many neuronsthat can be reliably iden- oflocust brain (Elphick et al., 1993). The biochemicalproperties tified from one animal to another. Since adult and larval locust neurons can be readily kept in primary cell culture (Giles and Usherwood, 1985; Kirchhof and Bicker, 1992) it should also Received Jan. 26, 1994; revised May 24, 1994; accepted June 1, 1994. be possibleto investigate the cellular responsesto NO exposure We thank Hans-Jochen Pfliiger for providing us with the experimental animals, in a controlled environment. Hence, we have analyzed the ner- Christine Jaeckel for technical assistance, and Malcolm Burrows for comments and help with the manuscript. This work was supported by a grant from the vous system of the locust, a commonly used neurobiological Deutsche Forschungsgemeinschaft to the Forschergruppe Lemen, Gedxchtnis und preparation, for NADPHd activity. Neuromodulation in Arthroooden. and a Heisenbere Fellowshio to G.B. Correspondence should be addrkssed to Drs. Uli Miiller and’Gerd Bicker, In- This article addressesthree issues.First, we provide a survey stitut fir Neurobiologie, KGnigin-Luise-Strasse 28-30, 14195 Berlin, Germany. ofthe anatomicaldistribution ofNO-producing cells in the brain Copyright 0 1994 Society for Neuroscience 0270-6474/94/147521-08$05.00/O and segmentalventral ganglia of the locust by NADPHd his- 7522 Mijller and Bicker * Nitric Oxide Release by Locust Neurons tochemistry. The histochemical staining revealed groups of neu- NADPH diaphorase assay. For quantification of the fixation-insen- rons in the brain and single identifiable neurons in the ventral sitive NADPH diaphorase activity that accounts for the staining in the whole-mounts, the following procedure was applied. Equal amounts of nerve cord that are candidates for releasing NO. Second, we protein of cell homogenates (al, br, tg; 30 ~1 each) was bound to nitro- show that certain dissociated neurons express the NADPHd cellulose (NC) filters (5 x 5 mm). After washing with 50 mM Tris-HCl, phenotype. NADPd and NO synthase activity show a ratio of uH 7.7. containina 0.1 M NaCl. the NC filter was incubated in the one in cell homogenates from various parts of the nervous sys- presence or absence of 4% parafo’rmaldehyde for 10 min. After another tem, indicating the identity of the enzyme. Finally, we dem- extensive washing, the NADPH diaphorase activity on the NC filter was determined by incubation with 50 mM Tris-HCl, pH 7.7, 0.1 mM onstrate that dissociated cells obtained from brain areas rich in nitro blue tetrazolium, and 0.1 % Triton X-100 at 30°C in the presence NADPHd-positive neurons release NO after stimulation by or absence of 100 PM /3-NADPH for 24 hr. After termination of the agents that elevate cytoplasmic CaZ+ levels. The combined an- reaction the NC filters were mounted and the formazan product was atomical and biochemical experiments provide firm evidence determined by scanning the NC filters with a color scanner (UMAX UC840) at 585 nm. All procedures, with the exception of the staining that NO is a messenger molecule acting in the CNS of the locust. reaction, were carried out at 4°C. For quantitative determination of the Furthermore, the presence of NO synthase and NO-stimulated NADPH diaphorase activity, conditions were adjusted to keep the re- guanylyl cyclase in brain homogenates (Elphick et al., 1993) and action in the linear range. our demonstration of a calcium-activated NO release from dis- Quantitation of NO synthase activity in viable cells. NO production sociated neurons indicate that the nervous system of the locust of dissociated cells was measured with the oxyhemoglobin assay similar to procedures described by others for cell lines (Bredt et al., 1992) or is an accessible invertebrate preparation to study NO signal cultured endothelial cells (Murphy et al., 1991). Before the measure- transduction. ment, dissociated cells of the various nervous tissues (al, br, tg) were washed with locust Ringer of the following composition (in mrvr):NaCl, Materials and Methods 180; MgCl,, 15; CaCl,, 2; KCl, 10; HEPES, 10; pH 7.0. They were Experiments were performed on Schistocerca gregaria reared in a crowd- subsequently resuspended in Ringer containing 10 PM oxyhemoglobin ed colony by H. J. Plhiger’s research group at our institute. All reagents, and seeded in microtiter plates at a density of 1400 cells in 100 ~1 per salts, and solvents were of analytical grade and were obtained from well. The conversion of oxyhemoglobin to methemoglobin was moni- Sigma, GIBCO, or Boehringer. tored continuously as described above in the microtiter plates with a NADPH diaphorase histochemistry. Locusts were chilled on ice and modified Reader (SLT Labinstruments. Austria). To block calcium parts of the nervous tissue including the cerebral ganglion and the ventral stimulation, incubations were performed is follows. Immediately before nerve cord were dissected. After careful removal of connective tissue measuring the dissociated cells were split in half. One half was washed surrounding the ganglia, the tissues were fixed in 4% paraformaldehyde and resuspended in normal locust Ringer containing oxyhemoglobin, in PBS for 2 hr at 4°C followed by washes in PBS. For permeabilization, whereas the other half was washed and resuspended with locust Ringer the tissues were incubated overnight in PBS containing 0.5% Saponin containing Co2+ instead of Ca 2+. For the inhibition of the NO synthase, at 4°C. After washing with PBS the visualization of fixation-insensitive cells were dissociated and preincubated for 1 hr in locust Ringer con- NADPH diaphorase activity was performed by incubation ofthe tissues taining 1 mM NC-monomethyl-L-arginine or NC-nitro-L-arginine. The in 50 mM Tris-HCl, pH 7.8,O. 1% Triton X- 100, and 0.1 mM nitro blue conversion of oxyhemoglobin after adding the various agents was re- tetrazolium in the presence or absence of 0.1 mM /3-NADPH at 25°C corded for 60 min and compared to the photometric signal before stim- for 60-90 min. After termination of staining the tissues were washed ulation. The difference in the signals was normalized with respect to in PBS and mounted in PBSglycerol 1:9. A similar procedure with the control wells measured in each cell preparation. exception of the permeabilization step was applied to cultured cells, To determine the possible interference of the agents and buffers used Cell culture. Details of the dissection of the locusts and the establish- on the oxyhemoglobin assay, all substances were tested in control ex- ment of primary cell cultures have been described in detail elsewhere periments with or without cell homogenates or viable cells, respectively. (Kirchhofand Bicker, 1992). Briefly, the cerebral ganglion and the three The locust Ringer as well as the used agents, like A23 187 (10 PM), Co2+ thoracic ganglia (tg) were dissected under a sterile Leibovitz L- 15 me- (2 mM), and acetylcholine (3 PM), have no effect on the loss of oxyhe- dium (GIBCO) containing 50 fig/ml gentamicin. After removal of the moglobin or the conversion of oxyhemoglobin to methemoglobin. optic lobes the central brain (br) and the antenna1 lobes (al) were sep- Omission of catalase or superoxide dismutase in the assay mixture did arated. The tissues were enzymatically treated with collagenase/dispase not affect the NO production of cell homogenates or viable cells, re- (Boehringer) according to the protocol of Kirchhof and Bicker (1992). spectively. After dissociation, cells were used for experiments on the same day or cultured for a week to allow for neurite outgrowth. NO synthase activity of cell homogenates. The dissociated cells of the Results central brain (br), the antenna1 lobe (al), and the thoracic ganglia (tg) of Histochemical detection of NADPHd four experimental animals of larval stage 5 were homogenized in 100 NADPHd histochemistry was performed on whole-mounts of ~1 of 50 mM Tris-HCl, pH 7.7, containing 0.1 mM EGTA. To normalize the protein concentration in the samples, the homogenate of brain and the cerebral, thoracic, and abdominal ganglia, from both adult thoracic ganglia was diluted. The NO synthase activity was determined and larval stages.NADPHd activity was detected in cell bodies by the conversion of oxyhemoglobin to methemoglobin by nitric oxide and their neuropilar processeswithin the ganglia. Due to the (Feelisch and Noak, 1987; Murphy et al., 1991; Mayer et al., 1992). easierpenetration ofthe staining solution through the perineural The oxyhemoglobin was always freshly prepared as described (Feelisch sheath in larvae as compared to adult tissue, the following de- and Noack, 1987). Immediately after homogenization, 30 ~1 of the equivalent homogenate was mixed with 70 ~1 of 50 mM Tris-HCl, pH scription is basedon the examination of larval stages3-5, which 7.7, containing 10 PM oxyhemoglobin, 0.1 mM fl-NADPH, 0.1 FM cal- gave the most satisfying histological results. Despite some dif- modulin, and 0.1 mM L-arginine, with either 1 mM EGTA or 0.2 mM ferencesin the staining intensity of neuropilar processes,there Ca2+. Reactions were started by the addition of NADPH and performed were no differencesin the pattern of the cell body staining be- at 27°C. Additional control experiments in which fl-NADPH and L-arginine were omitted from the reaction mixtures were also performed. tween the larval and adult stages.It should be stressed,however, The conversion of oxyhemoglobin to methemoglobin was monitored that certain identifiable groupsof somataconsistently expressed with a Pharmacia spectrophotometer or a modified Reader (SLT La- a different intensity of staining than others in both larvae and binstruments, Austria). The extinction difference between 40 1 and 4 11 adults. nm was recorded using a AC of 19.7 mM-lcm-’ (Feelisch and Noack, In the cerebral ganglion, the most striking NADPHd activity 1987) or in the presence of catalase and superoxide dismutase, the change of 577 versus 59 1 nm was recorded using a ACof 11.2 mMmlcm-l was concentrated in a cluster of approximately 45 cell bodies (Murphy et al., 199 1). Standards of nitric oxide release were prepared in the dorsal region of the antenna1lobe. As shown in Figure 1, as described by Feelisch and Noack (1987). the majority of stained cell bodies sendtheir neurites into the The Journal of Neuroscience, December 1994, f4(12) 7523

Figure I. NADPHd histochemistry of the cerebral ganglion. a, Frontal view of a cerebral ganglion from larval stage 5 showing staining in cell bodies in the dorsal part of the antenna1 lobe (al) and in the glomeruli (g). The trimgZe indicates a soma (s) belonging to a group of neurosecretoty cells. Light-refracting structures are tracheae (tr). Tritocerebrum (tc) is positioned out of the focal plane. b, Frontal view of the deuto- and tritocerebntm from larval stage 3 showing primary neurites @n) of antenna1 lobe neurons entering the stained neuropil of the antennal lobe. Two stained somata (s) are indicated in the tritocerebrum. Scale bars: A, 200 pm; B, 100 pm.

Figure 2. NADPHd histochemistry in the ventral nerve cord. a, Dorsal view of metathoracic ganglion of adult animal showing two stained somata (s) at the entrance of the connectives and cell bodies in various other locations. Arrow points toward anterior. b, Anterior region of abdominal ganglion 7 of adult animal showing stained pair of neurons with ascending axon (a) and varicose arborizations in portions of neuropil. Arrow points toward anterior. c, Connective between abdominal ganglion 7 and 6 showing two stained axons (a) in the plane of focus. Scale bars, 200 pm. 7524 Mijller and Bicker l Nitric Oxide Release by Locust Neurons

Cl NADPH diaphorase q NO synthase

Figure 4. Ca*+/calmodulin-dependent NO synthase activity and fixation-insensitive NADPH dianhorase activities in homoeenates of cells from antenna1 lobes (al), brain (br), and thoracic ganglya (tg), respectively. Equal amounts of protein from homogenates of the dissociated cells were compared. Each column shows the means + SEM of at least four separate experiments. The data were normalized with respect to the highest activities.

somatain a more lateral position. Similarly, an intensely stained soma was found close to the entrance of the connective in the meso- and metathoracic ganglia(Fig. 2a), suggestinga segmen- tally repeated organization. Approximately 20 stainedcell bodies were found in other ganglionic locations in the second and third thoracic ganglion. According to Goodman and Bate (198 l), each thoracic ganglion contains about 3000 somata. Thus, the Figure 3. NADPHd histochemistry in vitro. a, Dissociated cells from NADPHd-positive cells constitute lessthan 1% of the popula- the cerebral ganglionwere stained after 1 weekof primaryculture. The tion of neurons in the ventral nerve cord. transilluminationreveals NADPHd exnressionin the cell bodv and The whole-mount histochemistry revealed clearly the differ- regeneratedneurites of a singlecell. LJ, Hi‘stochemistry performed &thin hoursafter dissociationshowing different staining intensities in the cell ential staining of thoracic and abdominal neurons (Fig. 2a,b), bodies. Scale bar, 50 pm. their varicose innervation of large portions of the neuropil (Fig. 2b), and intersegmentally projecting axons in the connectives (Fig. 2~). Despite the fact that viscera, neurohemal, and genital glomeruli of the antenna1lobe. Thus, the glomeruli contain a organswere ensheathedby NADPHd-positive non-neural cells network of fine-grained arborizations originating from inter- (data not shown), we could not detect staining of motor or neurons. Figure 1b reveals the dense innervation from the sensoryaxons in the thoracic and abdominal nerve roots. NADPHd-positive soma group in the deutocerebral antenna1 Due to the smallernumber of cells, identifiable neuronscould lobe in contrast to a few scatteredstained cell bodies and rather be most easily detected in the unfused abdominal ganglia and sparselystained fibers in the tritocerebrum. No specificstaining the terminal ganglion. For example, in both femalesand males was detected in the antenna1 nerve, as has been reported in the abdominal ganglion AG 7 contains a symmetrical pair of Drosophila and Apis (Miiller and Buchner, 1993; Miiller, 1994). neurons (Fig. 2b) whose ascendingaxons cross to the contra- In the protocerebrum slight staining was also found in the lateral side of the ganglion before projecting anteriorly (Fig 2~). lobesof the mushroom bodies.Presumably, the neuropilar label originatesfrom neuronsextrinsic to the mushroombody as the somata of the intrinsic globuli cells in the calycal region were Histochemical detection of NADPHd in cell cultures not stained. Neurosecretory cells of the pars intercebralis were Dissociatedcell cultures provide a powerful system for studying stainedwith an intermediate intensity (Fig. 1a) and labeledcell the chemistry of neuronsin a controlled environment, and locust bodies were also found in the remaining parts of the cerebral neuronsgrown in primary cell culture have been shown to retain ganglion including the optic ganglia. The flat, spindle-shaped markers related to their transmitter metabolism (Kirchhof and morphology of single stainedcell bodies in several locations of Bicker, 1992). We cultured dissociatedcells of the cerebral gan- the cerebral ganglion suggeststhat they are not neurons,but of glion for 1 week to allow for neurite outgrowth and applied the glial origin. NADPHd staining protocol after fixation. The culture dishes Each of the thoracic and abdominal ganglia showsa distinct contained 3.5% NADPHd-positive cells that expressedthe label pattern of stained cells. The prothoracic ganglion contains a both in the cell body and in the regeneratedneurites (Fig. 3a). single bilateral, intensely labeled soma, ventral to the anterior Furthermore, NADPHd-positive somatacould also be detected entrance of the connective, and approximately 10 lightly stained immediately after the dissociation process(Fig. 3b). The Journal of Neuroscience, December 1994, 14(12) 7525

H + Cakalmodulin 600 E4 A23187 800 1. 0 +EGTA 0 Ringer control '3.F - zz 600 $0 coo gz 400 $8 O-z 200

0 al br al-co tg al br tg

Figure 5. NO synthaseactivity in homogenatesobtained from the Figure 6. Stimulationof NO synthaseactivity in dissociatedintact antenna1lobes (al), brain (br), and thoracicganglia (tg). The antenna1 cellsobtained from the antenna1lobes (al), brain (br), and thoracic lobe-control(al-co) column showsresults from antenna1lobe cell ho- ganglia(tg) by treatmentwith the Ca2+ionophore A23 187(5 PM).Since mogenatesthat wereincubated in reactionmixtures without P-NADPH the basalenzyme activity in the untreatedcells is similar,the datawere andL-arginine. The Ca2+/calmodulin-stimulatedNO synthaseactivities normalizedwith respectto the averageof all Ringercontrols. Each weredetermined in equalamounts of proteinfrom cell homogenates. column shows the means k SEM of at least four separate experiments. Sincethe NO synthaseactivities in nonstimulated(EGTA) cell ho- The data from stimulated and unstimulated cells are significantly dif- mogenatesfrom al, br, and tg showsimilar levels, the data werenor- ferent (p < 0.0 1, Student’s t test, two tailed), with the exception of the malizedwith respectto the averageof all EGTA controls.Each column thoracic ganglia (tg). showsthe means? SEM of at leastfive separateexperiments. All the data from stimulatedand unstimulatedhomogenates are significantly different(p < 0.01,Student’s t test,two tailed). nificantly different from controls in the absenceof calcium (Fig. 5). Neurochemicaldetection of NADPHd and NO synthase Releaseof NO by dissociatedneurons The histochemical staining for NADPHd in whole-mounts and The histochemical demonstration of NADPHd expressionin isolatedcells supports the existenceof an NO messengersystem dissociated neurons prompted us to explore a direct on-line in the locust. To strengthen the evidence that Ca*+/calmodulin- measurementof NO releasefrom viable cells with the oxyhe- dependent NO synthase is responsible for the histochemical moglobin assay(Feelisch and Noak, 1987; Murphy et al., 1991; staining in fixed nervous tissue of insects (Miiller, 1994) we Mayer et al., 1992). After dissociation cells were seededin the subjecteddissociated cells from specific parts of the locust CNS presenceof oxyhemoglobin into the wells of an ELISA plate to a biochemical analysis. These parts included the antenna1 and challengedwith the Ca*+ ionophore A23 187. Elevating cy- lobes(al), the central brain comprising cerebral ganglionwithout toplasmic Ca2+ levels by the ionophore caused a fivefold in- optic and antenna1lobes (br), and the three thoracic ganglia(tg). crease of NO synthase activity in antenna1 lobe cells and a The cells were homogenizedand equal amounts of protein were twofold increaseover basallevels in cells from the central brain compared for NO synthaseand NADPHd activity. NO synthase (Fig. 6). The Caz+-induced NO production by thoracic cells was activity was determined by the conversion of oxyhemoglobin not significantly different from the unstimulated control and, to methemoglobin (Feelisch and Noak, 1987; Murphy et al., given the limited cell density, presumably was at the detection 1991; Mayer et al., 1992). Becausethe histochemical staining threshold of the assay.However, a comparison of the NO syn- was performed on fixed tissue,NADPH diaphoraseactivity was thase activity in homogenates(Fig. 5) and the corresponding quantified after blotting the cell homogenatesto nitrocellulose viable cells (Fig. 6) reveals both the tissue-specificdistribution in the presenceof fixative. After normalizing to the maximal and the CaZ+dependence of the enzyme. values, both enzyme activities showed an almost perfect cor- A basic requirement for identifying NO as a messengermol- relation (Fig. 4), with the highestactivities in the antenna1lobe, ecule is the calcium-dependent releaseafter depolarization of intermediate in the central brain, and lowest in the thoracic viable cells. Therefore, we challenged the dissociatedcells with ganglia. To this end, the biochemical measurementsof enzyme depolarizing agentsin the presenceand absenceof extracellular activities (Fig. 4) demonstratedthe tissue-specificexpression of Ca2+. As with treatment with the Ca*+ ionophore, depolarizing NO synthase and fixation-insensitive NADPHd that corre- the cells by the application of high K+ (30 mM KCl) causedan spondswell to the histochemical staining (Figs. 1, 2). increasein enzyme activity (Fig. 7). Replacingextracellular Ca2+ To determine the Ca*+ dependenceof enzyme activity, we with 2 mM CO*+reduced the activity below Ringer control levels. measuredNO formation in the cell homogenatesin the presence Dissociation and preincubation of cells in the presenceof the and absenceof Ca*+/calmodulin stimulation. Again, cell ho- NO synthaseblocker nitroarginine prevented the K+ -induced mogenatesprepared from the antenna1lobes showed the highest increase,demonstrating a specificinhibitory action on NO syn- activity, with an eightfold stimulation over basallevels (Fig. 5). thase.To mimic more physiological conditions, we applied ace- Enzyme activities from the central brain were in the interme- tylcholine (ACh), which is thought to act predominantly as an diate range, whereas the thoracic ganglia showed the lowest excitatory neurotransmitter in the insectnervous system(Breer, levels of inducible activity, which were nevertheless still sig- 1987). ACh causeda stimulation of enzyme activity, suggesting 7526 Mijller and Bicker * Nitric Oxide Release by Locust Neurons

A231 87 KCI KCI KCI Ringer Pa-l Cell (5pM) (30 mM) Co NArg (3 FM) homogenate

Figure 7. Regulation of NO synthase activity in dissociated cells from the cerebral ganglion by elevation of cytoplasmic Ca2+ levels. The cells were stimulated with the Ca2+ ionophore A23 187 (5 PM), KC1 (30 mM), and ACh (3 PM). Increase of activity was blocked by replacing extracellular Ca*+ with Co2+ (KC1 Co), or by pretreatment with 1 mM of the NO synthase inhibitor nitroarginine (KCINArg). Parallel treatment ofcell homogenates with Ca2+ ionophore, KCl, and ACh does not activate NO synthase since cofactors and cosubstrates are missing. The data were normalized with respect to the average of Ringer controls. Each column shows the means + SEM of at least eight separate experiments. that neurotransmitter stimulation is sufficient to releaseNO in the ventral nerve cord (Fig. 2) but a group of 45 intensely stained viva. Finally, to rule out unspecific artifacts of cell damagethat cells in the antenna1lobe (Fig. 1) of the cerebral ganglion. The might not require Ca2+ influx into intact cells, we exposed cell cerebral ganglion of the locust proved ideal to analyze NO re- homogenatesto the agents, but failed to induce any enzyme lease because of a rather high portion of candidate NO-synthe- activity. Since the assaydid not contain any cofactors or co- sizing neurons and the easeof establishingviable dissociated substratesof NO synthase, NO production depends on intact cells that preserved the NADPHd phenotype (Fig. 3). cells. This finding allowed the chemical detection of NO release from dissociatedneurons by the conversion of oxyhemoglobin Discussion to methemoglobin. The spectrophotometric assayhas already The mechanismsby which nerve cells releasetransmitters and been usedto measureNO production by cultured endothelial other signaling molecules are as fundamental to neurobiology cells after stimulation with the Ca*+ ionophore A23 187 (Mur- as are the mechanismsby which thesesame messengers interact phy et al., 1991). NO is synthesized by a Ca2+/calmodulin- with their target receptors. Based on NADPHd histochemistry stimulated enzyme (Garthwaite, 1991; Bredt and Snyder, 1992) (Mtiller and Buchner, 1993; Miiller, 1994) and biochemical which proved to be Ca*+ dependent in homogenates(Fig. 5). measurementof NO synthaseactivity in homogenates(Elphick Therefore, the following experiments provided strong argu- et al., 1993) nitric oxide is a candidate messengerin the nervous ments for interpreting the changesof the photometric signal in system of fruit flies, bees, and locusts. NADPH diaphorase- the oxyhemoglobin assay on intact cells as releasedNO that positive neurons have recently also been described in several binds to the hemegroup. Significant changeswere detected only nonarthropod invertebrate phyla (Elofsson et al., 1993). How- after adding agents to the incubation medium that are known ever, the actual demonstration of NO releasefrom invertebrate to elevate cytoplasmic Ca2+ levels (Figs. 6, 7). A signal was nerve cells has so far not been obtained. One of the principal generatedby membrane depolarization with KCl, which causes identification criteria for a neurotransmitter is its Caz+-depen- Ca2+influx via voltage-operated calcium channelsand by direct dent releaseduring nerve cell depolarization (Werman, 1966). calcium influx via an ionophore. Replacing extracellular Ca2+ Analogous criteria should be met for the activity-dependent by cobalt reduced the signal below Ringer control levels. Ace- releaseof any type of signalingmolecule in the nervous system. tylcholine is one of the main excitatory neurotransmittersof the In practice, however, the accurate chemical detection of a CaZ+- insect nervous system (Breer, 1987) and recent Ca2+ imaging dependent releasefrom intact nerve cells is difficult to prove experiments have indeed shown that ACh induces elevation of for transmitter identification. In most casesit is experimentally cytoplasmic calcium levels in neuronsof an insect brain (Bicker performed on synaptosomes,perfused brain slices,or peripheral and Kreissl, 1994). Again, treatment of dissociatedlocust neu- glandular and neuromuscular preparations. This experimental rons with ACh in the micromolar range causeda photometric obstacle applies especially to the detection of an extremely re- signal, presumably by activating NO synthasevia an influx of active compound such as nitric oxide (Stamler et al., 1993). Ca2+,mainly through voltage-operated calcium channels.High- Initially we set out to identify candidate NO-releasing cells voltage-activated Ca*+ currents have also been demonstrated by simpleNADPHd whole-mount histochemistry. The anatom- in isolatedlocust neurons(Pearson et al., 1993). All our reported ical screeningrevealed lessthan 1% NADPHd-positive cells in experiments argue strongly for a CaZ+-dependent process in the The Journal of Neuroscience, December 1994, 14(12) 7527 generation of the photometric signal. We interpret the remaining the conventional transmitters but constitute a new separate sig- signal in the Ringer control as NO that is continuously released naling pathway. during the assay. Application of the NO synthase blocker n-ar- Since NADPHd activity could be detected in the neuropils ginine caused a reduction of the KCl-induced signal to levels (Fig. 2b) and connectives (Fig. 2~) of the ventral ganglia, but comparable to the cobalt block. This is in agreement with ex- not in the peripheral nerves, NO is produced only by interneu- periments demonstrating the effectiveness of n-arginine as in- rons but not by moto- and sensory neurons. With the caveat hibitor of NO synthase in homogenates of locust (Elphick et al., that some neurons, for example, dorsal unpaired median (DUM) 1993) and other insect nervous tissue (Miiller, 1994). The com- cells (Hoyle et al., 1974; Evans and O’Shea, 1978) have very bined results are consistent with the hypothesis that at least the thin peripherally projecting neurites, which may be difficult to proportion of the n-arginine-blocked signal represents NO that resolve in simple histological staining procedures, we propose is released by cells of the locust nervous system in a Ca*+- that NO acts mainly as a signaling molecule in the central parts dependent process. of the locust ventral nerve cord. The photometric detection of NO synthase activity of dis- This study has been concerned only with the Ca2+-activated sociated neurons taken from the brain, the antenna1 lobe, and release of NO, but it is obviously desirable to address the re- the thoracic ganglia (Fig. 6) correlated well to the histochemi- sponses of the cellular targets in the CNS to the endogenously tally mapped distribution of NADPHd-positive cell bodies in produced messenger molecule. Since locust neurons can be readily situ (Figs. 1, 2). Furthermore, the ratio of NADPd and NO grown in primary culture, the various signal transduction mech- synthase activity in cell homogenates (Fig. 4) indicated the iden- anisms of NO in elevating cGMP levels in target cells (Garth- tity of the enzyme in the CNS of the locust, as has been shown Waite, 1991; Bredt and Snyder, 1992) as modifier of synaptic for vertebrates (Dawson et al., 1991; Hope et al., 1991; Mat- efficacy (Zhuo et al., 1993) as regulator of neurite growth (Hess sumoto et al., 1993) and for Drosophila and Apis (Mtiller, 1994). et al., 1993), and as neurotoxic or neuroprotective agent (Lipton We suggest that in the locust nervous system the expression of et al., 1993), will also be amenable to a cellular analysis. Ulti- NADPHd activity after fixation is equivalent to NO synthase. mately, locusts could provide useful preparations in which re- Therefore, we feel confident that the organization of the release lease and effect of NO can be studied in an accessible inverte- sites of the NO signal transduction system can be derived from brate nervous system at the level of single identified neurons. the NADPHd staining pattern. The hallmark of the NADPHd histochemistry in the brain is References the concentration of NADPHd-positive neurons in the olfactory Bicker G, Kreissl S (1994) Calcium imaging reveals nicotinic acetyl- neuropil of the antenna1 lobe. Despite some differences in the choline receptors on cultured mushroom body neurons. J Neurophy- anatomical distribution of NADPHd activity in the brains of siol 71:808-810. 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