- Email: [email protected]
Localization of soluble guanylyl cyclase α-subunit in identified insect neurons
Brain Research 800 Ž1998. 174–179
Short communication
Localization of soluble guanylyl cyclase a-subunit in identified insect neurons Maurice R. Elphick ) , Ian W. Jones School of Biological Sciences, Queen Mary and Westfield College, UniÕersity of London, Mile End Road, London E1 4NS, UK Accepted 5 May 1998
Abstract The distribution of soluble guanylyl cyclase in the brain of the locust Schistocerca gregaria was analysed using antisera to a highly conserved region ŽX-peptide. of the Drosophila soluble guanylyl cyclase a-subunit ŽSGCa .. Analysis of locust brain and locust eye homogenates in Western blots using X-peptide antisera revealed specific staining of a ; 65 kDa band, which is similar to the expected molecular mass for a SGCa-subunit. SGCa-immunoreactivity was localized in identified neuronal components of several sensory systems including photoreceptors of the compound eyes and ocelli, large ocellar interneurons, antennal mechanosensory neurons and olfactory interneurons. These neurons are putative targets for the gas nitric oxide which activates guanylyl cyclase activity in the locust brain. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Soluble guanylyl cyclase; Cyclic-GMP; Schistocerca gregaria; Locust; Nitric oxide
Soluble guanylyl cyclase ŽSGC. catalyses the conversion of guanosine triphosphate ŽGTP. to cyclic guanosine monophosphate ŽcGMP. which is an important intracellular signaling molecule. SGC is a heterodimeric protein comprising a ŽSGCa . and b ŽSGCb . subunits with molecular masses of 70–80 kDa w28x. In the nervous system, the main activator of SGC is thought to be the gaseous signaling molecule, nitric oxide ŽNO. w3,17x. The importance of SGC as a target protein for NO is evident in the occurrence of NO–cGMP signaling in the nervous systems of a wide variety of animal types including vertebrates w3,10,17x, insects w1,6,20x and molluscs w7,11x. Neurons containing nitric oxide synthase ŽNOS., the enzyme that produces NO from L-arginine, have been identified in the brain of the locust Schistocerca gregaria w8,9x, an insect species which is utilized as a model preparation in neurobiology w4x. NOS is present in a discrete population of interneurons in the locust brain which are most striking and abundant in the olfactory and visual processing centers, the antennal and optic lobes. This suggests that NO is likely to be involved in sensory
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processing in the insect brain and this hypothesis is supported by behavioral experiments w20,21x. However, if we are to gain further understanding of the role of NO in the insect brain, the neuronal targets of NO need to be identified. We have started to address this question here by identifying neurons in the locust brain which express SGCa using novel antisera to this protein. The cDNA sequences encoding SGCa- and SGCb-subunits have been obtained from several mammalian species w15,18,22x and recently a SGCa cDNA sequence was identified in an insect species, Drosophila melanogaster w27x. We have analysed the amino acid sequence of the SGCa-subunit from Drosophila Ž Dro.SGC a . to identify regions which are conserved in mammalian SGCa proteins and which could be used for the development of SGCa-specific antibodies. Several regions of Dro.SGC a share significant sequence identity Ž) 80%. with mammalian SGCa proteins but not all of these regions are uniquely conserved amongst SGCs. One region which is unique to SGCs is a 25 amino acid sequence between residues 386 and 410 in Dro.SGC a , which we have designated region-X ŽFig. 1A.. The region-X of Dro.SGC a shares 80–88% sequence identity with mammalian SGCa-subunits, 60% sequence identity with mammalian SGCb-subunits but less than 37% sequence identity with all other known protein sequences.
M.R. Elphick, I.W. Jonesr Brain Research 800 (1998) 174–179
Fig. 1. ŽA. Diagram showing the position of region-X in the Drosophila SGCa subunit Ž Dro.SGC a; Ref. w27x. and sequence identity with human ŽHum.SGCa 2; Ref. w15x., rat ŽRat.SGCa1; Ref. w22x. and bovine ŽBov.SGCa1; Ref. w18x. SGCa-subunits. ŽB. Characterization of Xpeptide antisera by competition radioimmunoassay. The graphs show displacement of 125 I-labelled X-peptide trace Ž10,000 cpm. from antibodies in XA-4, XB-3 and XC-3 sera Žfinal dilution 1r750. by unlabelled X-peptide.
A peptide comprising the region-X of Dro.SGC a was synthesized by the Peptide Synthesis and Protein Sequencing Service at the Sussex Center for Neuroscience, University of Sussex, England. N-terminal lysine and tyrosine residues were included to provide respective sites for coupling to a carrier protein Žthyroglobulin. and radioactive labelling with 125 I. A conjugate of X-peptide and thyroglobulin was used to immunize three New Zealand white rabbits Ždesignated A, B and C; 75 nmol X-peptide per rabbit.. Similar booster injections were administered at 2-month intervals. Blood was collected from the rabbits 7 days after each booster injection and the serum stored at y208C. Sera from the rabbits were designated XA, XB and XC, respectively, with a number indicating which bleed the serum was obtained from. Each serum was tested for the presence of antibodies to the X-peptide sequence by radioimmunoassay ŽFig. 1B. using methods described previously w5x. Analysis of sera by radioimmunoassay showed that antibodies to the X-peptide antigen had been produced in the three immunized rabbits. Three antisera ŽXA-4, XB-3 and XC-3. displayed the highest binding of 125 I-labelled X-peptide and were further analysed in competition radioimmunoassays using unlabelled X-peptide
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ŽFig. 1B.. With XC-3 and XA-4, X-peptide was detectable at subnanomolar concentrations. XB-3, in contrast with XA-4 and XC-3, displayed a 10-fold lower sensitivity for X-peptide in radioimmunoassays ŽFig. 1B.. Western blotting methods were used to establish whether the X-peptide antisera recognise a SGC-like protein in locust tissue. Homogenates of locust brains and locust eyes were separated by standard SDS-PAGE electrophoresis Žnon-reducing conditions. and transferred to a nitro-cellulose membrane. The membrane was blocked in 4% non-fat milk ŽMarvele; Premier Beverages, Stafford, England .r0.2% Tween-20rphosphate buffered saline ŽPBSTM. for 1 h on a shaker at room temperature, and strips incubated overnight at 48C in either X-peptide antisera, antisera pre-absorbed with 1 mM X-peptide or pre-immune sera Žeach diluted 1r10,000 in PBSTM.. The membrane strips were then washed in PBS and incubated in goat anti-rabbit IgG conjugated to alkaline phosphatase ŽVector Labs, Burlingham, CA., diluted 1r1000 in PBSTM, at room temperature for 1 h on a shaker. Following a rinse in PBS, the membrane strips were incubated in VectorBlacke alkaline phosphatase substrate ŽVector Labs, Burlingham, CA. for 30 min at room temperature in the dark on a shaker. The reaction was stopped by rinsing in distilled water. Western blot analysis of the locust eye using XA-4, XC-3, and XB-3 revealed a stained band with a molecular mass of about 65 kDa which was not immunoreactive with pre-immune sera ŽXA-0, XB-0, XC-0. and pre-absorbed antisera ŽFig. 2.. The ; 65 kDa band was also evident in locust brain homogenates but was fainter than that seen in
Fig. 2. Western blot showing a ;65 kDa protein in locust eye homogenate which is immunoreactive with the X-peptide antisera XA-4 Žlane 1., XC-3 Žlane 4., and XB-3 Žlane 7.. Staining of this band is abolished when XA-4 Žlane 2., XC-3 Žlane 5. and XB-3 Žlane 8. are pre-absorbed with X-peptide. Pre-immune sera ŽXA-0, XC-0, XB-0. do not stain any bands in eye homogenates Žlanes 3, 6, and 9, respectively.. XA-4 Žlane 1. also stains a band with a molecular mass of about 40 kDa but staining of this band is not blocked when XA-4 is pre-absorbed with X-peptide Žlane 2..
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M.R. Elphick, I.W. Jonesr Brain Research 800 (1998) 174–179
the eye Žnot shown.. The XA-4 antiserum also stained a band from locust brain and locust eye with a molecular mass of about 40 kDa but staining of this band was not pre-absorbable with the X-peptide antigen ŽFig. 2.. These results show that antibodies generated against the X-peptide specifically recognise a ; 65 kDa protein present in the locust brain and locust eye. This molecular mass is less than that predicted for Dro.SGC a , which is 74 kDa, although similar variation in the size of SGC subunits is also seen in mammals w28x. These data indicate that the antisera raised here recognise the X-peptide sequence in an intact SGCa-subunit from locust tissues and therefore could be used to localise this protein in the locust using immunocytochemistry. For immunocytochemical analysis, Bouin’s fixed adult locust brains were embedded in paraffin wax and sections Ž10 mm or 20 mm. prepared using a rotary microtome were collected on chrome–alum coated slides. The sections were dewaxed and rehydrated through an ethanol series into PBS. Non-specific binding sites were blocked by incubation in 1.5% normal goat serum ŽVector Labs, Burlingham, CA.rPBSq 0.2% Triton X-100 ŽPBST. for 1 h at room temperature. The sections were then rinsed in PBST and incubated in X-peptide antisera, diluted 1r1000 in PBST, overnight at 48C. Pre-immune sera or antisera pre-absorbed with 1 mM X-peptide were used as negative controls. Following the primary incubation, the sections were washed in PBST and then incubated for 4 h at room temperature in goat anti-rabbit IgG conjugated to alkaline phosphatase ŽVector Labs, Burlingham, CA. diluted 1r500 in PBST. After further washes in PBST, the sections were incubated in VectorBlacke alkaline phosphatase substrate ŽVector Labs, Burlingham, CA.. The reaction was stopped by rinsing the sections in distilled water. The sections were then dehydrated through an ethanol series, cleared in xylene and mounted. Immunocytochemical analysis of locust brain sections using XC-3 and XA-4 revealed selective staining of a specific population of neuronal elements ŽFig. 3.. No staining was observed with XB-3. The pattern of staining observed with XC-3 and XA-4 ŽFig. 3A and B. was identical but was not seen with pre-absorbed antisera or pre-immune sera Žsee Fig. 3C for an example.. A complete description of all of the neuronal elements in the locust brain which are stained by the X-peptide antisera is beyond the scope of this paper. The most obvious features can be described, however, and interestingly these are associated with several sensory systems of the brain. Perhaps the most striking feature stained by the X-peptide antisera is seen in the retina of the compound eyes ŽFig. 3A and B.. The widespread and intense staining in the compound eyes correlates with the detection of the ; 65 kDa SGCa-like protein in Western blots of locust eye homogenates ŽFig. 2.. The locust compound eyes are comprised of functional units known as ommatidia, each of which contain eight
photoreceptor cells organised in a ring around a central rhabdom where phototransduction occurs w26x. The rhabdom is formed by microvillar projections from each of the eight photoreceptor cells. Very strong immunostaining is observed in the rhabdomeric compartment of the photoreceptor cells, extending along the length of the rhabdom from the basement membrane, which separates the eye from the brain, to the base of the cone cells which focus light onto the rhabdom ŽFig. 3A and B.. Previous studies have shown that NO elevates cGMP levels in locust photoreceptors w2,25x and that the SGCa-subunit is expressed in the Drosophila retina w23x. Our data show that SGCa-immunoreactivity is located specifically within the phototransducing rhabdomeric compartment of locust photoreceptors. This is of particular interest because it suggests that SGC and cGMP may be involved in the regulation of phototransduction. Moreover, experiments performed on the compound eye of the fly Musca domestica have shown that NO donors and cGMP can stimulate migration of pigment granules out of the rhabdom, a process that normally occurs under dark conditions w14x. Adaptive movement of organelles in response to changes in illumination also occurs in locust photoreceptors w24x and it will be interesting to investigate the involvement of SGC and cGMP in regulating this process. In addition to the two large compound eyes, the locust head has three simple eyes Žocelli., two located laterally and one medially w13x. These ocelli consist of large numbers of photoreceptor cells located under a common cuticular lens. The ocellar photoreceptors, like their counterparts in the compound eyes, are also stained by the X-peptide antisera ŽFig. 3D.. Photosensory information gathered by the photoreceptor cells of the locust ocelli is transmitted to the brain via the ocellar nerves by a population of ocellar interneurons whose cell bodies are located within the brain w13x. Immunostaining is observed in a subset of six or seven axons within the ocellar nerves with a mean diameter of 13 mm ŽFig. 3E and F.. These correspond exactly with the axons of large ocellar interneurons described by Goodman w13x. Moreover, the position of several immunostained cell bodies located in the dorsal compartment of the pars intercerebralis of the brain ŽFig. 3E and F. is consistent with that of the seventeen large ocellar interneurons described by Goodman w13x. The detection of SGCaimmunoreactivity in these identified interneurons is an important finding because it opens up an opportunity for the analysis of SGC function in neurons which can be repeatably recognised from animal to animal. The main olfactory processing centres of the insect brain are the antennal lobes where the axons of antennal olfactory receptor neurons synapse with interneurons. A subset Žabout 20. of these olfactory interneurons whose cell bodies are located within the anterior-medial quadrant of the antennal lobe are immunoreactive with the X-peptide antisera ŽFig. 3G.. These neurons are putative targets for NO derived from a population of about 50 NADPH-di-
M.R. Elphick, I.W. Jonesr Brain Research 800 (1998) 174–179
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Fig. 3. Immunocytochemical localization of SGCa expression in the locust brain and eyes as revealed using X-peptide antisera ŽXC-3 or XA-4.. ŽA. Photoreceptors of the locust compound eye seen in transverse section; note the intense staining in the central rhabdomeric compartment of each ommatidium Ž=1150.. ŽB. Photoreceptors of the compound eye seen in longitudinal section; note the stained rhabdom extends from the basement membrane Žblack arrow. of the retina to the cone cell layer Žwhite arrow. Ž=160.. ŽC. Photoreceptors as seen in ŽA. but showing that staining is blocked when the antiserum ŽXC-3. is pre-absorbed with X-peptide Ž=1150.. ŽD. The right lateral ocellus seen in transverse section showing stained clusters of photoreceptor cells Ž=320.. ŽE. The main picture shows the locust brain seen in horizontal section with stained neuronal cell bodies Žwhite arrow. in the pars intercerebralis and stained axons in the left lateral ocellar nerve tract Žblack arrows. Ž=80.; the inset shows the median ocellar nerve in transverse section with stained axons of the large ocellar interneurons Ž=280.. ŽF. Parasaggital section of the locust brain showing stained cell bodies of large ocellar interneurons in the dorsal region of the pars intercerebralis Ž=200.. ŽG. Parasaggital section of the locust brain showing stained neuronal cell bodies in the anterior cortex the antennal lobe Ž=260.. ŽH. Stained axons seen in a transverse section of the antennal nerve Žopen arrows. and its branches Žblack arrow. Ž=300.. ŽI. Stained axons projecting from the antennal nerve Žblack arrow. around the lateral margin of the antennal lobe Žopen arrow. Ž=270..
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M.R. Elphick, I.W. Jonesr Brain Research 800 (1998) 174–179
aphorase-positive neurons previously described in the locust antennal lobe w8x. Moreover, the detection of SGCaimmunoreactivity in a population of olfactory interneurons in the locust provides further evidence that the NO–cGMP pathway is likely to participate in mechanisms of olfactory processing in insects. In the mollusc Limax, inhibition of NO production or release slows or stops oscillatory electrical activity in the procerebral lobe where olfactory processing occurs w11x. Similar oscillatory electrical activity has been observed in the locust antennal lobes and linked with encoding of chemosensory information w19x. Pharmacological and electrophysiological analysis of the locust antennal lobe using NO donors, cGMP, NOS inhibitors and SGC inhibitors may reveal how the NO–cGMP pathway participates in olfactory processing mechanisms. Another striking feature of the locust antennal system is a discrete population of stained axons in the antennal nerves ŽFig. 3H.. These are not part of the olfactory system, however, because they do not project into the olfactory neuropiles of the antennal lobes. Instead they bypass the antennal lobe neuropile ŽFig. 3I., arborise in a more dorsal neuropile region known as the dorsal lobe or antennal mechanosensory and motor center w16x and then project via the circumesophageal connectives to the subesophageal ganglion Žnot shown.. The anatomy of these stained antennal nerve fibers matches exactly with previously described antennal mechanosensory neurons thought to be involved in monitoring wind movement during flight in the locust w12x. In summary, our observations indicate that SGC is expressed in both primary sensory receptor cells Žantennal mechanoreceptors and photoreceptors of the eyes. and interneurons Žantennal lobe olfactory interneurons and ocellar interneurons. of the locust brain. It remains to be shown that the SGCa-immunoreactivity described here reflects functional SGC activity at the cellular level, but the widespread immunoreactivity is consistent with the detection of measurable SGC activity in isolated locust brains exposed to NO donors w6x. SGC activity appears to be dependent on the co-expression of both a and b SGC-subunits w15x, so it will be important to analyse both SGCa and SGCb expression in the locust brain. While this work was in progress, the sequence of a Drosophila SGCb-subunit was reported w23x and so in the future it should be possible to analyse SGCb expression in the locust brain using a similar approach to that adopted here for SGCa.
Acknowledgements We are grateful to Richard Melarange for technical assistance and to Prof. Bob Sinden’s lab ŽImperial College. for use of their Western blot apparatus. This work was supported by Grant S03858 from the BBSRC to M.R.E.
References w1x G. Bicker, O. Schmachtenberg, J. de Vente, The nitric oxidercyclic GMP messenger system in olfactory pathways of the locust brain, Eur. J. Neurosci. 8 Ž1996. 2635–2643. w2x G. Bicker, O. Schmachtenberg, Cytochemical evidence for nitric oxidercyclic GMP signal transmission in the visual system of the locust, Eur. J. Neurosci. 9 Ž1997. 189–193. w3x D.S. Bredt, S.H. Snyder, Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum, Proc. Natl. Acad. Sci. USA 86 Ž1989. 9030–9033. w4x M. Burrows, The Neurobiology of an Insect Brain, Oxford University Press, Oxford, 1996. w5x M.R. Elphick, J.R. Reeve, R.D. Burke, M.C. Thorndyke, Isolation of the neuropeptide SALMFamide-1 from starfish using a new antiserum, Peptides 12 Ž1991. 455–459. w6x M.R. Elphick, I.C. Green, M. O’Shea, Nitric oxide synthesis and action in an invertebrate brain, Brain Res. 619 Ž1993. 344–346. w7x M.R. Elphick, G. Kemenes, K. Staras, M. O’Shea, Behavioral role for nitric oxide in chemosensory activation of feeding in a mollusc, J. Neurosci. 15 Ž1995. 7653–7664. w8x M.R. Elphick, R.C. Rayne, V. Riveros-Moreno, S. Moncada, M. O’Shea, Nitric oxide synthesis in locust olfactory interneurons, J. Exp. Biol. 198 Ž1995. 821–829. w9x M.R. Elphick, L. Williams, M. O’Shea, New features of the locust optic lobe: evidence of a role for nitric oxide in insect vision, J. Exp. Biol. 199 Ž1996. 2395–2407. w10x J. Garthwaite, S.L. Charles, R. Chess-Williams, Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intracellular messengers in the brain, Nature 336 Ž1988. 385–388. w11x A. Gelperin, Nitric oxide mediates network oscillations of olfactory interneurons in a terrestrial mollusc, Nature 369 Ž1994. 61–63. w12x M. Gewecke, Central projection of antennal afferents for the flight motor in Locusta migratoria ŽOrthoptera: Acrididae., Entomologia Generalis 5 Ž1979. 317–320. w13x C.S. Goodman, Anatomy of the ocellar interneurons of acridid grasshoppers, Cell Tiss. Res. 175 Ž1976. 183–202. w14x Y. Hanyu, N. Franceschini, Pigment granule migration and phototransduction are triggered by separate pathways in fly photoreceptor cells, NeuroReport 4 Ž1993. 215–218. w15x C. Harteneck, B. Wedel, D. Koesling, J. Malkewitz, E. Bohme, G. Schultz, Expression of soluble guanylate cyclase: catalytic activity requires two enzyme subunits, FEBS Lett. 292 Ž1991. 217–222. w16x U. Homberg, Distribution of neurotransmitters in the insect brain, Gustav Fischer Verlag, Stuttgart, 1994. w17x R.G. Knowles, M. Palacios, R.M.G. Palmer, S. Moncada, Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. USA 86 Ž1989. 5159–5162. w18x D. Koesling, C. Harteneck, P. Humbert, A. Bosserhoff, R. Frank, G. Schultz, E. Bohme, The primary structure of the larger subunit of soluble guanylate cyclase from bovine lung; homology between the two subunits of the enzyme, FEBS Lett. 266 Ž1990. 128–132. w19x G. Laurent, M. Wehr, K. Macleod, M. Stopfer, B. Leitch, H. Davidowitz, Dynamic encoding of odors with oscillating neuronal assemblies in the locust brain, Biol. Bull. 191 Ž1996. 70–75. w20x U. Muller, The nitric oxide system in insects, Prog. Neurobiol. 51 ¨ Ž1997. 363–381. w21x U. Muller, H. Hildebrandt, The nitric oxidercGMP system in the ¨ antennal lobe of Apis mellifera is implicated in integrative processing of chemosensory stimuli, Eur. J. Neurosci. 7 Ž1995. 2240–2248. w22x M. Nakane, K. Arai, S. Saheki, T. Kuno, W. Buechler, F. Murad, Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung, J. Biol. Chem. 265 Ž1990. 16841– 16845.
M.R. Elphick, I.W. Jonesr Brain Research 800 (1998) 174–179 w23x S. Shah, D.R. Hyde, Two Drosophila genes that encode the a- and b-subunits of the brain soluble guanylyl cyclase, J. Biol. Chem. 270 Ž1995. 15368–15376. w24x K. Sturmer, O. Baumann, B. Walz, Actin-dependent light-induced ¨ translocation of mitochondria and ER cisternae in the photoreceptor cells of the locust Schistocerca gregaria, J. Cell Sci. 108 Ž1995. 2273–2283. w25x J.W. Truman, J. de Vente, E.E. Ball, Nitric oxide-sensitive guanylate cyclase activity is associated with the maturational phase of
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neuronal development in insects, Development 122 Ž1996. 3949– 3958. w26x M. Wilson, P. Garrard, S. McGinness, The unit structure of the locust compound eye, Cell Tiss. Res. 195 Ž1978. 205–226. w27x S. Yoshikawa, I. Miyamoto, J. Aruga, T. Furuichi, H. Okano, K. Mikoshiba, Isolation of a Drosophila gene encoding a head-specific guanylyl cyclase, J. Neurochem. 60 Ž1993. 1570–1573. w28x P.S.T. Yuen, D.L. Garbers, Guanylyl cyclase-linked receptors, Ann. Rev. Neurosci. 15 Ž1992. 193–225.
Short communication
Localization of soluble guanylyl cyclase a-subunit in identified insect neurons Maurice R. Elphick ) , Ian W. Jones School of Biological Sciences, Queen Mary and Westfield College, UniÕersity of London, Mile End Road, London E1 4NS, UK Accepted 5 May 1998
Abstract The distribution of soluble guanylyl cyclase in the brain of the locust Schistocerca gregaria was analysed using antisera to a highly conserved region ŽX-peptide. of the Drosophila soluble guanylyl cyclase a-subunit ŽSGCa .. Analysis of locust brain and locust eye homogenates in Western blots using X-peptide antisera revealed specific staining of a ; 65 kDa band, which is similar to the expected molecular mass for a SGCa-subunit. SGCa-immunoreactivity was localized in identified neuronal components of several sensory systems including photoreceptors of the compound eyes and ocelli, large ocellar interneurons, antennal mechanosensory neurons and olfactory interneurons. These neurons are putative targets for the gas nitric oxide which activates guanylyl cyclase activity in the locust brain. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Soluble guanylyl cyclase; Cyclic-GMP; Schistocerca gregaria; Locust; Nitric oxide
Soluble guanylyl cyclase ŽSGC. catalyses the conversion of guanosine triphosphate ŽGTP. to cyclic guanosine monophosphate ŽcGMP. which is an important intracellular signaling molecule. SGC is a heterodimeric protein comprising a ŽSGCa . and b ŽSGCb . subunits with molecular masses of 70–80 kDa w28x. In the nervous system, the main activator of SGC is thought to be the gaseous signaling molecule, nitric oxide ŽNO. w3,17x. The importance of SGC as a target protein for NO is evident in the occurrence of NO–cGMP signaling in the nervous systems of a wide variety of animal types including vertebrates w3,10,17x, insects w1,6,20x and molluscs w7,11x. Neurons containing nitric oxide synthase ŽNOS., the enzyme that produces NO from L-arginine, have been identified in the brain of the locust Schistocerca gregaria w8,9x, an insect species which is utilized as a model preparation in neurobiology w4x. NOS is present in a discrete population of interneurons in the locust brain which are most striking and abundant in the olfactory and visual processing centers, the antennal and optic lobes. This suggests that NO is likely to be involved in sensory
) Corresponding author. [email protected]
Fax:
q44-181-983-0973;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 5 2 2 - 8
processing in the insect brain and this hypothesis is supported by behavioral experiments w20,21x. However, if we are to gain further understanding of the role of NO in the insect brain, the neuronal targets of NO need to be identified. We have started to address this question here by identifying neurons in the locust brain which express SGCa using novel antisera to this protein. The cDNA sequences encoding SGCa- and SGCb-subunits have been obtained from several mammalian species w15,18,22x and recently a SGCa cDNA sequence was identified in an insect species, Drosophila melanogaster w27x. We have analysed the amino acid sequence of the SGCa-subunit from Drosophila Ž Dro.SGC a . to identify regions which are conserved in mammalian SGCa proteins and which could be used for the development of SGCa-specific antibodies. Several regions of Dro.SGC a share significant sequence identity Ž) 80%. with mammalian SGCa proteins but not all of these regions are uniquely conserved amongst SGCs. One region which is unique to SGCs is a 25 amino acid sequence between residues 386 and 410 in Dro.SGC a , which we have designated region-X ŽFig. 1A.. The region-X of Dro.SGC a shares 80–88% sequence identity with mammalian SGCa-subunits, 60% sequence identity with mammalian SGCb-subunits but less than 37% sequence identity with all other known protein sequences.
M.R. Elphick, I.W. Jonesr Brain Research 800 (1998) 174–179
Fig. 1. ŽA. Diagram showing the position of region-X in the Drosophila SGCa subunit Ž Dro.SGC a; Ref. w27x. and sequence identity with human ŽHum.SGCa 2; Ref. w15x., rat ŽRat.SGCa1; Ref. w22x. and bovine ŽBov.SGCa1; Ref. w18x. SGCa-subunits. ŽB. Characterization of Xpeptide antisera by competition radioimmunoassay. The graphs show displacement of 125 I-labelled X-peptide trace Ž10,000 cpm. from antibodies in XA-4, XB-3 and XC-3 sera Žfinal dilution 1r750. by unlabelled X-peptide.
A peptide comprising the region-X of Dro.SGC a was synthesized by the Peptide Synthesis and Protein Sequencing Service at the Sussex Center for Neuroscience, University of Sussex, England. N-terminal lysine and tyrosine residues were included to provide respective sites for coupling to a carrier protein Žthyroglobulin. and radioactive labelling with 125 I. A conjugate of X-peptide and thyroglobulin was used to immunize three New Zealand white rabbits Ždesignated A, B and C; 75 nmol X-peptide per rabbit.. Similar booster injections were administered at 2-month intervals. Blood was collected from the rabbits 7 days after each booster injection and the serum stored at y208C. Sera from the rabbits were designated XA, XB and XC, respectively, with a number indicating which bleed the serum was obtained from. Each serum was tested for the presence of antibodies to the X-peptide sequence by radioimmunoassay ŽFig. 1B. using methods described previously w5x. Analysis of sera by radioimmunoassay showed that antibodies to the X-peptide antigen had been produced in the three immunized rabbits. Three antisera ŽXA-4, XB-3 and XC-3. displayed the highest binding of 125 I-labelled X-peptide and were further analysed in competition radioimmunoassays using unlabelled X-peptide
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ŽFig. 1B.. With XC-3 and XA-4, X-peptide was detectable at subnanomolar concentrations. XB-3, in contrast with XA-4 and XC-3, displayed a 10-fold lower sensitivity for X-peptide in radioimmunoassays ŽFig. 1B.. Western blotting methods were used to establish whether the X-peptide antisera recognise a SGC-like protein in locust tissue. Homogenates of locust brains and locust eyes were separated by standard SDS-PAGE electrophoresis Žnon-reducing conditions. and transferred to a nitro-cellulose membrane. The membrane was blocked in 4% non-fat milk ŽMarvele; Premier Beverages, Stafford, England .r0.2% Tween-20rphosphate buffered saline ŽPBSTM. for 1 h on a shaker at room temperature, and strips incubated overnight at 48C in either X-peptide antisera, antisera pre-absorbed with 1 mM X-peptide or pre-immune sera Žeach diluted 1r10,000 in PBSTM.. The membrane strips were then washed in PBS and incubated in goat anti-rabbit IgG conjugated to alkaline phosphatase ŽVector Labs, Burlingham, CA., diluted 1r1000 in PBSTM, at room temperature for 1 h on a shaker. Following a rinse in PBS, the membrane strips were incubated in VectorBlacke alkaline phosphatase substrate ŽVector Labs, Burlingham, CA. for 30 min at room temperature in the dark on a shaker. The reaction was stopped by rinsing in distilled water. Western blot analysis of the locust eye using XA-4, XC-3, and XB-3 revealed a stained band with a molecular mass of about 65 kDa which was not immunoreactive with pre-immune sera ŽXA-0, XB-0, XC-0. and pre-absorbed antisera ŽFig. 2.. The ; 65 kDa band was also evident in locust brain homogenates but was fainter than that seen in
Fig. 2. Western blot showing a ;65 kDa protein in locust eye homogenate which is immunoreactive with the X-peptide antisera XA-4 Žlane 1., XC-3 Žlane 4., and XB-3 Žlane 7.. Staining of this band is abolished when XA-4 Žlane 2., XC-3 Žlane 5. and XB-3 Žlane 8. are pre-absorbed with X-peptide. Pre-immune sera ŽXA-0, XC-0, XB-0. do not stain any bands in eye homogenates Žlanes 3, 6, and 9, respectively.. XA-4 Žlane 1. also stains a band with a molecular mass of about 40 kDa but staining of this band is not blocked when XA-4 is pre-absorbed with X-peptide Žlane 2..
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the eye Žnot shown.. The XA-4 antiserum also stained a band from locust brain and locust eye with a molecular mass of about 40 kDa but staining of this band was not pre-absorbable with the X-peptide antigen ŽFig. 2.. These results show that antibodies generated against the X-peptide specifically recognise a ; 65 kDa protein present in the locust brain and locust eye. This molecular mass is less than that predicted for Dro.SGC a , which is 74 kDa, although similar variation in the size of SGC subunits is also seen in mammals w28x. These data indicate that the antisera raised here recognise the X-peptide sequence in an intact SGCa-subunit from locust tissues and therefore could be used to localise this protein in the locust using immunocytochemistry. For immunocytochemical analysis, Bouin’s fixed adult locust brains were embedded in paraffin wax and sections Ž10 mm or 20 mm. prepared using a rotary microtome were collected on chrome–alum coated slides. The sections were dewaxed and rehydrated through an ethanol series into PBS. Non-specific binding sites were blocked by incubation in 1.5% normal goat serum ŽVector Labs, Burlingham, CA.rPBSq 0.2% Triton X-100 ŽPBST. for 1 h at room temperature. The sections were then rinsed in PBST and incubated in X-peptide antisera, diluted 1r1000 in PBST, overnight at 48C. Pre-immune sera or antisera pre-absorbed with 1 mM X-peptide were used as negative controls. Following the primary incubation, the sections were washed in PBST and then incubated for 4 h at room temperature in goat anti-rabbit IgG conjugated to alkaline phosphatase ŽVector Labs, Burlingham, CA. diluted 1r500 in PBST. After further washes in PBST, the sections were incubated in VectorBlacke alkaline phosphatase substrate ŽVector Labs, Burlingham, CA.. The reaction was stopped by rinsing the sections in distilled water. The sections were then dehydrated through an ethanol series, cleared in xylene and mounted. Immunocytochemical analysis of locust brain sections using XC-3 and XA-4 revealed selective staining of a specific population of neuronal elements ŽFig. 3.. No staining was observed with XB-3. The pattern of staining observed with XC-3 and XA-4 ŽFig. 3A and B. was identical but was not seen with pre-absorbed antisera or pre-immune sera Žsee Fig. 3C for an example.. A complete description of all of the neuronal elements in the locust brain which are stained by the X-peptide antisera is beyond the scope of this paper. The most obvious features can be described, however, and interestingly these are associated with several sensory systems of the brain. Perhaps the most striking feature stained by the X-peptide antisera is seen in the retina of the compound eyes ŽFig. 3A and B.. The widespread and intense staining in the compound eyes correlates with the detection of the ; 65 kDa SGCa-like protein in Western blots of locust eye homogenates ŽFig. 2.. The locust compound eyes are comprised of functional units known as ommatidia, each of which contain eight
photoreceptor cells organised in a ring around a central rhabdom where phototransduction occurs w26x. The rhabdom is formed by microvillar projections from each of the eight photoreceptor cells. Very strong immunostaining is observed in the rhabdomeric compartment of the photoreceptor cells, extending along the length of the rhabdom from the basement membrane, which separates the eye from the brain, to the base of the cone cells which focus light onto the rhabdom ŽFig. 3A and B.. Previous studies have shown that NO elevates cGMP levels in locust photoreceptors w2,25x and that the SGCa-subunit is expressed in the Drosophila retina w23x. Our data show that SGCa-immunoreactivity is located specifically within the phototransducing rhabdomeric compartment of locust photoreceptors. This is of particular interest because it suggests that SGC and cGMP may be involved in the regulation of phototransduction. Moreover, experiments performed on the compound eye of the fly Musca domestica have shown that NO donors and cGMP can stimulate migration of pigment granules out of the rhabdom, a process that normally occurs under dark conditions w14x. Adaptive movement of organelles in response to changes in illumination also occurs in locust photoreceptors w24x and it will be interesting to investigate the involvement of SGC and cGMP in regulating this process. In addition to the two large compound eyes, the locust head has three simple eyes Žocelli., two located laterally and one medially w13x. These ocelli consist of large numbers of photoreceptor cells located under a common cuticular lens. The ocellar photoreceptors, like their counterparts in the compound eyes, are also stained by the X-peptide antisera ŽFig. 3D.. Photosensory information gathered by the photoreceptor cells of the locust ocelli is transmitted to the brain via the ocellar nerves by a population of ocellar interneurons whose cell bodies are located within the brain w13x. Immunostaining is observed in a subset of six or seven axons within the ocellar nerves with a mean diameter of 13 mm ŽFig. 3E and F.. These correspond exactly with the axons of large ocellar interneurons described by Goodman w13x. Moreover, the position of several immunostained cell bodies located in the dorsal compartment of the pars intercerebralis of the brain ŽFig. 3E and F. is consistent with that of the seventeen large ocellar interneurons described by Goodman w13x. The detection of SGCaimmunoreactivity in these identified interneurons is an important finding because it opens up an opportunity for the analysis of SGC function in neurons which can be repeatably recognised from animal to animal. The main olfactory processing centres of the insect brain are the antennal lobes where the axons of antennal olfactory receptor neurons synapse with interneurons. A subset Žabout 20. of these olfactory interneurons whose cell bodies are located within the anterior-medial quadrant of the antennal lobe are immunoreactive with the X-peptide antisera ŽFig. 3G.. These neurons are putative targets for NO derived from a population of about 50 NADPH-di-
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Fig. 3. Immunocytochemical localization of SGCa expression in the locust brain and eyes as revealed using X-peptide antisera ŽXC-3 or XA-4.. ŽA. Photoreceptors of the locust compound eye seen in transverse section; note the intense staining in the central rhabdomeric compartment of each ommatidium Ž=1150.. ŽB. Photoreceptors of the compound eye seen in longitudinal section; note the stained rhabdom extends from the basement membrane Žblack arrow. of the retina to the cone cell layer Žwhite arrow. Ž=160.. ŽC. Photoreceptors as seen in ŽA. but showing that staining is blocked when the antiserum ŽXC-3. is pre-absorbed with X-peptide Ž=1150.. ŽD. The right lateral ocellus seen in transverse section showing stained clusters of photoreceptor cells Ž=320.. ŽE. The main picture shows the locust brain seen in horizontal section with stained neuronal cell bodies Žwhite arrow. in the pars intercerebralis and stained axons in the left lateral ocellar nerve tract Žblack arrows. Ž=80.; the inset shows the median ocellar nerve in transverse section with stained axons of the large ocellar interneurons Ž=280.. ŽF. Parasaggital section of the locust brain showing stained cell bodies of large ocellar interneurons in the dorsal region of the pars intercerebralis Ž=200.. ŽG. Parasaggital section of the locust brain showing stained neuronal cell bodies in the anterior cortex the antennal lobe Ž=260.. ŽH. Stained axons seen in a transverse section of the antennal nerve Žopen arrows. and its branches Žblack arrow. Ž=300.. ŽI. Stained axons projecting from the antennal nerve Žblack arrow. around the lateral margin of the antennal lobe Žopen arrow. Ž=270..
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aphorase-positive neurons previously described in the locust antennal lobe w8x. Moreover, the detection of SGCaimmunoreactivity in a population of olfactory interneurons in the locust provides further evidence that the NO–cGMP pathway is likely to participate in mechanisms of olfactory processing in insects. In the mollusc Limax, inhibition of NO production or release slows or stops oscillatory electrical activity in the procerebral lobe where olfactory processing occurs w11x. Similar oscillatory electrical activity has been observed in the locust antennal lobes and linked with encoding of chemosensory information w19x. Pharmacological and electrophysiological analysis of the locust antennal lobe using NO donors, cGMP, NOS inhibitors and SGC inhibitors may reveal how the NO–cGMP pathway participates in olfactory processing mechanisms. Another striking feature of the locust antennal system is a discrete population of stained axons in the antennal nerves ŽFig. 3H.. These are not part of the olfactory system, however, because they do not project into the olfactory neuropiles of the antennal lobes. Instead they bypass the antennal lobe neuropile ŽFig. 3I., arborise in a more dorsal neuropile region known as the dorsal lobe or antennal mechanosensory and motor center w16x and then project via the circumesophageal connectives to the subesophageal ganglion Žnot shown.. The anatomy of these stained antennal nerve fibers matches exactly with previously described antennal mechanosensory neurons thought to be involved in monitoring wind movement during flight in the locust w12x. In summary, our observations indicate that SGC is expressed in both primary sensory receptor cells Žantennal mechanoreceptors and photoreceptors of the eyes. and interneurons Žantennal lobe olfactory interneurons and ocellar interneurons. of the locust brain. It remains to be shown that the SGCa-immunoreactivity described here reflects functional SGC activity at the cellular level, but the widespread immunoreactivity is consistent with the detection of measurable SGC activity in isolated locust brains exposed to NO donors w6x. SGC activity appears to be dependent on the co-expression of both a and b SGC-subunits w15x, so it will be important to analyse both SGCa and SGCb expression in the locust brain. While this work was in progress, the sequence of a Drosophila SGCb-subunit was reported w23x and so in the future it should be possible to analyse SGCb expression in the locust brain using a similar approach to that adopted here for SGCa.
Acknowledgements We are grateful to Richard Melarange for technical assistance and to Prof. Bob Sinden’s lab ŽImperial College. for use of their Western blot apparatus. This work was supported by Grant S03858 from the BBSRC to M.R.E.
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