Cell and Tissue Research

, Volume 337, Issue 2, pp 327–340 | Cite as

NO/cGMP signalling: L-citrulline and cGMP immunostaining in the central complex of the desert locust Schistocerca gregaria

  • Theresa Siegl
  • Joachim Schachtner
  • Gay R. Holstein
  • Uwe Homberg
Regular Article


Nitric oxide (NO) is a gaseous messenger molecule formed during conversion of L-arginine into L-citrulline by the enzyme NO synthase (NOS), which belongs to a group of NADPH diaphorases. Because of its gaseous diffusion properties, NO differs from classical neurotransmitters in that it is not restricted to synaptic terminals. In target cells, NO activates soluble guanylyl cyclase leading to an increase in cGMP levels. In insects, this NO/cGMP-signalling pathway is involved in development, memory formation and processing of visual, olfactory and mechanosensory information. We have analysed the distribution of putative NO donor and target cells in the central complex, a brain area involved in sky-compass orientation, of the locust Schistocerca gregaria by immunostaining for L-citrulline and cGMP. Six types of citrulline-immunostained neurons have been identified including a bilateral pair of hitherto undescribed neurons that connect the lateral accessory lobes with areas anterior to the medial lobes of the mushroom bodies. Three-dimensional reconstructions have revealed the connectivity pattern of a set of 18 immunostained pontine neurons of the central body. All these neurons appear to be a subset of previously mapped NADPH-diaphorase-positive neurons of the central complex. At least three types of central-complex neurons show cGMP immunostaining including a system of novel columnar neurons connecting the upper division of the central body and the lateral triangle of the lateral accessory lobe. Our results provide the morphological basis for further studies of the function of the labelled neurons and new insights into NO/cGMP signalling.


L-citrulline cGMP  Brain Central complex Schistocerca gregaria (Insecta) 

List of abbreviations


Bovine serum albumin


Lower division of the central body


Upper division of the central body


Cyclic adenosine monophosphate


Cyclic guanosine monophosphate


Columnar (neuron) of the CBU, type 1a


3,3'-Diaminobenzidine tetrahydrochloride


Dopamine-immunoreactive (neuron) of the pars intercerebralis, type 2


Guanosine trisphosphate


3-Isobutyl-1-methylxanthine (inhibitor of phosphodiesterases)


Lateral accessory lobe


Reduced form of nicotinamide adenine dinucleotide phosphate


NADPH diaphorase


Normal goat serum


Nitric oxide


Nitric oxide synthase


Protocerebral bridge


Phosphate-buffered saline




Posterior optic tubercle


Pontine (neuron) of the CBU, type 1


Sodium nitroprusside (nitric oxide donor)


Saline substituted TRIS buffer


Tangential (neuron) of the PB, type 2


TRIS-buffered saline

TL1, 2, 4

Tangential (neurons) of the CBL, types 1, 2, 4


Triton X-100


3-[5′-Hyrdroxymethyl-2′-furyl]-1-benzyl indazole (activator of soluble guanylate cyclase)



We thank Dr. Jan de Vente (Maastricht University, The Netherlands) for the generous gift of cGMP antiserum and Pfizer for the gift of sildenafil citrate.


  1. Bellamy TC, Garthwaite J (2002) The receptor-like properties of nitric oxide-activated soluble guanylyl cyclase in intact cells. Mol Cell Biochem 230:165–176PubMedCrossRefGoogle Scholar
  2. Bicker G (2001a) Nitric oxide: an unconventional messenger in the nervous system of an orthopteroid insect. Arch Insect Biochem Physiol 48:100–110PubMedCrossRefGoogle Scholar
  3. Bicker G (2001b) Sources and targets of nitric oxide signalling in insect nervous systems. Cell Tissue Res 303:137–146PubMedCrossRefGoogle Scholar
  4. Bicker G, Schmachtenberg O (1997) Cytochemical evidence for nitric oxide/cyclic GMP signal transmission in the visual system of the locust. Eur J Neurosci 9:189–193PubMedCrossRefGoogle Scholar
  5. Blottner D, Grozdanovic Z, Gossrau R (1995) Histochemistry of nitric oxide synthase in the nervous system. Histochem J 27:785–811PubMedGoogle Scholar
  6. Boehning D, Snyder SH (2003) Novel neural modulators. Annu Rev Neurosci 26:105–131PubMedCrossRefGoogle Scholar
  7. Bredt DS (2003) Nitric oxide signaling in brain: potentiating the gain with YC-1. Mol Pharmacol 63:1206–1208PubMedCrossRefGoogle Scholar
  8. Clements AN, May TE (1974) Studies on locust neuromuscular physiology in relation to glutamic acid. J Exp Biol 60:673–705PubMedGoogle Scholar
  9. Collmann C, Carlsson MA, Hansson BS, Nighorn A (2004) Odorant-evoked nitric oxide signals in the antennal lobe of Manduca sexta. J Neurosci 24:6070–6077PubMedCrossRefGoogle Scholar
  10. Corbin JD, Francis SH (1999) Cyclic GMP phosphodiesterase-5: target of sildenafil. J Biol Chem 274:13729–13732PubMedCrossRefGoogle Scholar
  11. Davies S-A (2000) Nitric oxide signalling in insects. Insect Biochem Mol Biol 30:1123–1138PubMedCrossRefGoogle Scholar
  12. Davies S-A (2005) Signalling via cGMP: lessons from Drosophila. Cell Signal 18:409–421PubMedCrossRefGoogle Scholar
  13. Dawson TM, Snyder SH (1994) Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci 14:5147–5159PubMedGoogle Scholar
  14. Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 88:7797–7801PubMedCrossRefGoogle Scholar
  15. Day JP, Dow JA, Houslay MD, Davies S-A (2005) Cyclic nucleotide phosphodiesterases in Drosophila melanogaster. Biochem J 388:333–342PubMedCrossRefGoogle Scholar
  16. De Vente J, Steinbusch HWM, Schipper J (1987) A new approach to immunocytochemistry of 3’,5’-cyclic guanosine monophosphate: preparation, specificity, and initial application of a new antiserum against formaldehyde-fixed 3’,5’-cyclic guanosine monophosphate. Neuroscience 22:361–373PubMedCrossRefGoogle Scholar
  17. Dircksen H, Homberg U (1995) Crustacean cardioactive peptide-immunoreactive neurons innervating brain neuropils, retrocerebral complex and stomatogastric nervous system of the locust, Locusta migratoria. Cell Tissue Res 279:495–515CrossRefGoogle Scholar
  18. Elphick MR, Rayne RC, Riveros-Moreno V, Moncada S, O’Shea M (1995) Nitric oxide synthesis in locust olfactory interneurones. J Exp Biol 198:821–829PubMedGoogle Scholar
  19. Elphick MR, Williams L, O’Shea M (1996) New features of the locust optic lobe: evidence of a role for nitric oxide in insect vision. J Exp Biol 199:2395–2407PubMedGoogle Scholar
  20. Evers JF, Schmitt S, Sibila M, Duch C (2005) Progress in functional neuroanatomy: precise automatic geometric reconstruction of neuronal morphology from confocal image stacks. J Neurophysiol 93:2331–2342PubMedCrossRefGoogle Scholar
  21. Flam BR, Eichler DC, Solomonson LP (2007) Endothelial nitric oxide production is tightly coupled to the citrulline-NO cycle. Nitric Oxide 17:115–121PubMedCrossRefGoogle Scholar
  22. Friebe A, Koesling D (2003) Regulation of nitric oxide-sensitive guanylyl cyclase. Circ Res 93:96–105PubMedCrossRefGoogle Scholar
  23. Garthwaite J (2008) Concepts of neural nitric oxide-mediated transmission. Eur J Neurosci 27:2783–2802PubMedCrossRefGoogle Scholar
  24. Garthwaite J, Boulton CL (1995) Nitric oxide signaling in the central nervous system. Annu Rev Physiol 57:683–706PubMedCrossRefGoogle Scholar
  25. Gibson NJ, Nighorn A (2000) Expression of nitric oxide synthase and soluble guanylyl cyclase in the developing olfactory system of Manduca sexta. J Comp Neurol 422:191–205PubMedCrossRefGoogle Scholar
  26. Hanesch U, Fischbach K-F, Heisenberg M (1989) Neuronal architecture of the central complex in Drosophila melanogaster. Cell Tissue Res 257:343–366CrossRefGoogle Scholar
  27. Heinze S, Homberg U (2007) Maplike representation of celestial E-vector orientations in the brain of an insect. Science 315:995–997PubMedCrossRefGoogle Scholar
  28. Heinze S, Homberg U (2008) Neuroarchitecture of the central complex of the desert locust: intrinsic and columnar neurons. J Comp Neurol 511:454–478PubMedCrossRefGoogle Scholar
  29. Heinze S, Homberg U (2009) Linking the input to the output—new sets of neurons complement the polarization vision network in the locust central complex. J Neurosci 29:4911–4921PubMedCrossRefGoogle Scholar
  30. Homberg U (1985) Interneurons of the central complex in the bee brain (Apis mellifera, L.). J Insect Physiol 31:251–264CrossRefGoogle Scholar
  31. Homberg U (1991) Neuroarchitecture of the central complex in the brain of the locust Schistocerca gregaria and S. americana as revealed by serotonin immunocytochemistry. J Comp Neurol 303:245–254PubMedCrossRefGoogle Scholar
  32. Homberg U (2004) In search of the sky compass in the insect brain. Naturwissenschaften 91:199–208PubMedCrossRefGoogle Scholar
  33. Hope BT, Michael GJ, Knigge KM, Vincent SR (1991) Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 88:2811–2814PubMedCrossRefGoogle Scholar
  34. Jones IW, Elphick MR (1999) Dark-dependent soluble guanylyl cyclase activity in locust photoreceptor cells. Proc R Soc Lond [Biol] 266:413–419CrossRefGoogle Scholar
  35. Kasai H, Petersen OH (1994) Spatial dynamics of second messengers: IP3 and cAMP as long-range and associative messengers. Trends Neurosci 17:95–101PubMedCrossRefGoogle Scholar
  36. Ko FN, Wu CC, Kuo SC, Lee FY, Teng CM (1994) YC-1, a novel activator of platelet guanylate cyclase. Blood 84:4226–4233PubMedGoogle Scholar
  37. Koesling D, Russwurm M, Mergia E, Müllershausen F, Friebe A (2004) Nitric oxide-sensitive guanylyl cyclase: structure and regulation. Neurochem Int 45:813–819PubMedCrossRefGoogle Scholar
  38. Krumenacker JS, Hanafy KA, Murad F (2004) Regulation of nitric oxide and soluble guanylyl cyclase. Brain Res Bull 62:505–515PubMedCrossRefGoogle Scholar
  39. Kurylas AE, Ott SR, Schachtner J, Elphick MR, Williams L, Homberg U (2005) Localization of nitric oxide synthase in the central complex and surrounding midbrain neuropils of the locust Schistocerca gregaria. J Comp Neurol 484:206–223PubMedCrossRefGoogle Scholar
  40. Kurylas AE, Rohlfing T, Krofczik S, Jenett A, Homberg U (2008) Standardized atlas of the brain of the desert locust, Schistocerca gregaria. Cell Tissue Res 333:125–145PubMedCrossRefGoogle Scholar
  41. Liu G, Seiler H, Wen A, Zars T, Ito K, Wolf R, Heisenberg M, Liu L (2006) Distinct memory traces for two visual features in the Drosophila brain. Nature 439:551–556PubMedCrossRefGoogle Scholar
  42. Martinelli GPT, Friedrich VL Jr, Hostein GR (2002) L-citrulline immunostaining identifies nitric oxide production sites within neurons. Neuroscience 114:111–122PubMedCrossRefGoogle Scholar
  43. Mehats C, Andersen CB, Filopanti M, Jin SLC, Conti M (2002) Cyclic nucleotide phosphodiesterases and their role in endocrine cell signaling. Trends Endocrinol Metab 13:29–35PubMedCrossRefGoogle Scholar
  44. Müller U (1996) Inhibition of nitric oxide synthase impairs a distinct form of long-term memory in the honeybee, Apis mellifera. Neuron 16:541–549PubMedCrossRefGoogle Scholar
  45. Müller U (1997) The nitric oxide system in insects. Prog Neurobiol 51:363–381PubMedCrossRefGoogle Scholar
  46. Müller U, Bicker G (1994) Calcium-activated release of nitric oxide and cellular distribution of nitric oxide-synthesizing neurons in the nervous system of the locust. J Neurosci 14:7521–7528PubMedGoogle Scholar
  47. Müller U, Hildebrandt H (1995) The nitric oxide/cGMP system in the antennal lobe of Apis mellifera is implicated in integrative processing of chemosensory stimuli. Eur J Neurosci 7:2240–2248PubMedCrossRefGoogle Scholar
  48. Neuser K, Triphan T, Mronz M, Poeck B, Strauss R (2008) Analysis of a spatial orientation memory in Drosophila. Nature 453:1244–1247PubMedCrossRefGoogle Scholar
  49. O’Shea M, Colbert R, Williams L, Dunn S (1998) Nitric oxide compartments in the mushroom bodies of the locust brain. Neuroreport 9:333–336PubMedCrossRefGoogle Scholar
  50. Ott SR, Burrows M (1998) Nitric oxide synthase in the thoracic ganglia of the locust: distribution in the neuropiles and morphology of neurones. J Comp Neurol 396:217–230CrossRefGoogle Scholar
  51. Ott SR, Elphick MR (2002) Nitric oxide synthase histochemistry in insect nervous systems: methanol/formalin fixation reveals the neuroarchitecture of formaldehyde-sensitive NADPH diaphorase in the cockroach Periplaneta americana. J Comp Neurol 448:165–185PubMedCrossRefGoogle Scholar
  52. Ott SR, Jones IW, Burrows M, Elphick MR (2000) Sensory afferents and motor neurons as targets for nitric oxide in the locust. J Comp Neurol 422:521–532PubMedCrossRefGoogle Scholar
  53. Ott SR, Burrows M, Elphick MR (2001) The neuroanatomy of nitric oxide-cyclic GMP signaling in the locust: functional implications for sensory systems. Am Zool 41:321–331CrossRefGoogle Scholar
  54. Ott SR, Delago A, Elphick MR (2004) An evolutionarily conserved mechanism for sensitization of soluble guanylyl cyclase reveals extensive nitric oxide-mediated upregulation of cyclic GMP in insect brain. Eur J Neurosci 20:1231–1244PubMedCrossRefGoogle Scholar
  55. Ott SR, Philippides A, Elphick MR, O’Shea M (2007) Enhanced fidelity of diffuse nitric oxide signalling by the spatial segregation of source and target neurones in the memory centre of an insect brain. Eur J Neurosci 25:181–190PubMedCrossRefGoogle Scholar
  56. Schachtner J, Klaassen L, Truman JW (1998) Metamorphic control of cyclic guanosine monophosphate expression in the nervous system of the tobacco hornworm, Manduca sexta. J Comp Neurol 396:238–252PubMedCrossRefGoogle Scholar
  57. Schachtner J, Homberg U, Truman JW (1999) Regulation of cyclic GMP elevation in the developing antennal lobe of the sphinx moth, Manduca sexta. J Neurobiol 41:359–375PubMedCrossRefGoogle Scholar
  58. Schipper J, Tilders FJH (1983) A new technique for studying specificity of immunocytochemical procedures: specificity of serotonin immunostaining. J Histochem Cytochem 31:12–18PubMedGoogle Scholar
  59. Strausfeld NJ (1976) Atlas of an insect brain. Springer, HeidelbergGoogle Scholar
  60. Strauss R (2002) The central complex and the genetic dissection of locomotor behaviour. Curr Opin Neurobiol 12:633–638PubMedCrossRefGoogle Scholar
  61. Tanaka J, Markerink-van Ittersum M, Steinbusch HWM, De Vente J (1997) Nitric oxide-mediated cGMP synthesis in oligodendrocytes in the developing rat brain. Glia 19:286–297PubMedCrossRefGoogle Scholar
  62. Vitzthum H, Homberg U (1998) Immunocytochemical demonstration of locustatachykinin-related peptides in the central complex of the locust brain. J Comp Neurol 390:455–469PubMedCrossRefGoogle Scholar
  63. Vitzthum H, Müller M, Homberg U (2002) Neurons of the central complex of the locust Schistocerca gregaria are sensitive to polarized light. J Neurosci 22:1114–1125PubMedGoogle Scholar
  64. Wang Z, Pan Y, Li W, Jiang H, Chatzimanolis L, Chang J, Gong Z, Liu L (2008) Visual pattern memory requires foraging function in the central complex of Drosophila. Learn Mem 15:133–142PubMedCrossRefGoogle Scholar
  65. Weinrich A, Kunst M, Wirmer A, Holstein GR, Heinrich R (2008) Suppression of grasshopper sound production by nitric oxide-releasing neurons of the central complex. J Comp Physiol [A] 194:763–776CrossRefGoogle Scholar
  66. Wendt B, Homberg U (1992) Immunocytochemistry of dopamine in the brain of the locust Schistocerca gregaria. J Comp Neurol 321:387–403PubMedCrossRefGoogle Scholar
  67. Wenzel B, Kunst M, Günther C, Ganter GK, Lakes-Harlan R, Elsner N, Heinrich R (2005) Nitric oxide/cyclic guanosine monophosphate signaling in the central complex of the grasshopper brain inhibits singing behavior. J Comp Neurol 488:129–139PubMedCrossRefGoogle Scholar
  68. Williams JLD (1975) Anatomical studies of the insect central nervous system: a ground-plan of the midbrain and an introduction to the central complex in the locust, Schistocerca gregaria (Orthoptera). J Zool (Lond) 176:67–86CrossRefGoogle Scholar
  69. Wilson CH, Christensen TA, Nighorn AJ (2007) Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, Manduca sexta. J Comp Physiol [A] 193:715–728CrossRefGoogle Scholar
  70. Wood J, Garthwaite J (1994) Models of the diffusional spread of nitric oxide: implications for neural nitric oxide signalling and its pharmacological properties. Neuropharmacology 33:1235–1244PubMedCrossRefGoogle Scholar
  71. Wu C-L, Xia S, Fu T-F, Wang H, Chen Y-H, Leong D, Chiang A-S, Tully T (2007) Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body. Nat Neurosci 10:1578–1586PubMedCrossRefGoogle Scholar
  72. Zayas RM, Qazi S, Morton DB, Trimmer BA (2002) Nicotinic-acetylcholine receptors are functionally coupled to the nitric oxide/cGMP-pathway in insect neurons. J Neurochem 83:421–431PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Theresa Siegl
    • 1
  • Joachim Schachtner
    • 1
  • Gay R. Holstein
    • 2
  • Uwe Homberg
    • 1
  1. 1.Fachbereich Biologie, TierphysiologiePhilipps Universität MarburgMarburgGermany
  2. 2.Departments of Neurology and NeuroscienceMount Sinai School of MedicineNew YorkUSA

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