Regulatory Peptides, 42 (1992) 123-134
123
© 1992Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00 REGPEP 01239
Autoradiographic localization of 125I-galanin binding sites in the blowfly brain Helena A. D. Johard
a, C. Tomas Lundquist a, Ake ROkaeus b and Dick R. N~ssel a
aDepartment of Zoology, Stockholm University, Stockholm (Sweden) and b Department of Biochemistry I. Karolinska Institute, Stockholm (Sweden)
(Received 4 May 1992; revisedversion receivedand accepted 13 August 1992) K e y words." Galanin; Neuropeptide; Insect nervous system; Peptide receptor;
Autoradiography
Summary The localization of porcine galanin (pGAL) binding sites in the brain of the blowfly Phormia terraenovae was investigated by autoradiography using the following radioiodinated ligands: pGAL 1-29 (two isoforms), pGAL 15-29 and rat (r) GAL 129. The different porcine radioligands bound specifically with the following intensity: 125I-[Tyr26]-pGAL 15-29 > > 125I-[Tyr26]-pGAL 1-29 > > 125I-[Tyr9]-pGAL 1-29. With rat galanin 125I-[Tyr9]-rGAL1-29 no specific binding could be shown. In addition, displacement of 125I-[TyrZ6]-pGAL1-29 was tested with pGAL 1-29, pGAL 1-22 and pGAL 15-29 (at 0.1 n M - 1 laM). A gradual displacement was achieved with increasing concentrations of pGAL 1-29 and pGAL15-29, whereas no displacement with p G A L 1-22 was detected. The results indicate that the C-terminal portion of pGAL is important for binding in the blowfly. The pGAL binding sites were localized in synaptic neuropils of the central body, the antennal lobes, the optic lobes, the pars intercerebralis and the subesophageal ganglion, all of which contain GAL-like immunoreactive neural processes.
Correspondence to: D.R. Nassel, Department of Zoology, Stockholm University, S-106 91 Stockholm,
Sweden.
124 Introduction
Galanin (GAL) is an amidated 29 amino acid residue peptide, originally isolated from pig intestine [ 1], which is widely distributed in the central and peripheral nervous system of several mammalian [2-4] and submammalian species [5-7]. In close association with GAL-immunoreactive (GAL-IR) neurons, high affinity GAL binding sites have been visualized by autoradiography and characterized by binding assays, using radioiodinated GAL [8-16]. As functional correlate to the localization of GAL-IR neurons and receptors, a number of biological actions of GAL have been described, e.g., modulation of neurotransmitter release [ 17,18 ], regulation of hormone secretion [ 19-21 ], regulation of smooth muscle activity [ 3,17,22 ] and control of feeding behavior [23]. GAL-IR neurons have also been demonstrated in two invertebrate species. In the mollusc, Bulla gouldiana, a single pair of GAL-IR neurons was detected in the cerebral ganglion [24]. A more extensive distribution of GAL-IR neurons was demonstrated in the central nervous system of the blowfly, Phorrnia terraenovae [25]. Chromatographical analysis of the GAL-IR indicated the presence of several forms of GAL-like peptide in the blowfly [25]. Quite a number of other neuropeptides have been demonstrated in neurons and neurosecretory cells in the brain of insects [26-28]. Most of these insect peptides were isolated as neurohormones and their action has mainly been studied at peripheral targets [27-29]. Considering the large number of peptidecontaining interneurons in the insect brain it is to be expected that different neuropeptides have a variety of central modulatory actions. One strategy to search for central sites of action of neuropeptides is to analyze neuropeptide binding. In the present study we have thus investigated the presence of 125I-GAL binding sites in sections of the blowfly brain by autoradiography.
Materials and Methods
Tissue preparation Brains of the blowfly, Phormia terraenovae, were rapidly dissected in cold 0.1 M sodium phosphate buffer solution (pH 7.4) and immediately frozen in Tissue-Tek ® (Miles, Elkhart, IN). Frontal 20 lam-thick sections of the brain were cut on a cryostat ( - 14 ° C) and thaw-mounted on chrome-alun coated slides. Subsequently, the sections were dried at 35 °C for 30 min and stored dessicated at -80°C until used for autoradiography (within 2 weeks). Preparation of radioligands Synthetic porcine (p) G A L l - 2 9 (Peninsula, UK), rat (r) G A L l - 2 9 and pGAL15-29 (gift from P. Padgett, Laboratory of Cell Biology, NIMH-NIH, Bethesda, USA) were labeled by the chloramine T method using 1251 (Amersham, UK). Following radioiodination, peptides were rapidly purified from unincorporated iodine and other salts on a Sep Pak C18 cartridge (Waters, Millipore). A further purification of the labeled peptides was performed by reverse phase HPLC, using a linear gradient of 20-38~o
125 acetonitrile with 0.1 ~o trifluoroacetic acid over 70 rain at a flow rate of 0.5 ml/min. These conditions allowed separation of unlabeled peptides from labeled peptides as well as separation of two iodinated pGAL isoforms (125I-[TyrZ6]-pGAL1-29 and 125I-[Tyr9]-pGAL1-29; ROkaeus, unpublished data). The radioactivity of the separated fractions was measured in a gamma counter. The specific activity of the iodinated peptides was estimated to be higher than 2000 Ci/mmol.
Autoradiographic analysis of t25I-GAL binding Frozen sections of the fly brains were allowed to thaw for 15 min at room temperature before preincubation in 50 mM Tris buffer (pH 7.4) containing 5 mM MgC12, 2 mM EGTA, 0.1 ~o bovine serum albumin (B SA; Sigma) and 0.01 ~o bacitracin (Sigma) for 15 min at room temperature. Thereafter slides were incubated for 60 min in the same buffer as above, including 125I-[Tyr26]-pGAL1-29 at a concentration of 0.2-0.4 nM (500 cpm/~tl). Preliminary binding experiments were performed for 30, 60 and 120 min and 60 min was found to yield maximal specific binding. Incubations were terminated by rinsing four times 30 s in 50 mM Tris buffer containing 5 mM MgC1z (at 4°C) and 30 s in double distilled water (at 4°C). The sections were subsequently dried under a cold stream of air and fixed for 30 rain in paraformaldehyde vapor at 80°C. The slides were coated with Kodak NTB-2 emulsion, exposed for 14-21 days at 4°C, and developed in Kodak D-19 developer. Thereafter the sections were dehydrated in a series of alcohol/xylene steps, mounted with Permount and finally analyzed under light- and darkfield illumination in a Zeiss Axiophot microscope. A set of autoradiography slides were counterstained with Giemsa stain for more accurate localization of silver grains. In order to investigate the specificity of the GAL binding and to reveal which portion of the GAL molecule that was of importance for displacement of 125I-[Tyr26]pGAL 1-29 binding, unlabeled pGAL 1-29, pGAL 1-22 and pGAL 15-29, respectively, were added to the incubation buffer at six concentrations ranging from 0.1 nM to 10 I~M. To further analyze the structural requirements of the GAL binding sites, we investigated the ability of different iodinated GAL ligands to specifically visualize such sites at the incubation conditions described above. The following four radioligands were tested at a concentration of 0.2-0.4 nM: 125I-[TyrZ6]-pGAL1-29, ~25I-[Tyr9]pGAL1-29, 125I-[TyrZ6]-pGAL15-29 and rat galanin, 125I-[Tyr9]-rGAL1-29. Nonspecific binding was estimated by including 1 ~tM of pGAL1-29 in the incubation buffer. However, since 12sI-[Tyr26]-pGAL15-29 gave the best visualization of binding sites, the ability of unlabeled pGAL15-29 to displace this binding was tested in one experiment at five concentrations ranging from 0.1 nM to 1 ~tM. Triplets of brains were used for each condition described above and all experiments were repeated at least twice (except the displacement with pGAL15-29). The grain density on the brain sections was analyzed microscopically by several investigators, using six serially sectioned brains for each experimental condition. While the well delineated synaptic neuropil termed the central body is easy to distinguish and had the highest density of specific 125I-GAL-binding, it was used in all analysis of ligand displacement (see Fig. 4). To estimate the radioligand degradation during the incubation, the ~25I-[TyrZ6]pGAL1-29 in incubation buffer was retrieved from the sections for a comparison with
126
fresh ligand. This analysis was performed with reversed phase HPLC at the same conditions as used for the radioligand purification.
Results
Binding of 12sI-[ Tyr26]-pGAL1-29 In Fig. 1 a schematic diagram of the blowfly brain is shown for reference to the distribution of binding sites. It is important to note that the insect brain is organized with all the neuronal cell bodies in a superficial rind and with axonal tracts and synaptic neuropil regions in the core. The radioligand binding described below is in synaptic neuropil regions, as determined from counterstained sections (Fig. 2a). Specific binding of 1251-[TyrZ6]-pGAL1-29 was most apparent in the central body, a distinct synaptic neuropil region in the protocerebrum of the blowflybrain (Fig. 2b). Specific binding of radiolabeled GAL, albeit at lower density, could also be observed in the neuropil of the antennal lobes in the deutocerebrum of the brain, and at a barely detectable level in the medulla of the optic lobe (Fig. 2b,c). The integrity of the radioiodinated ligand ~25I-[Tyr26]-pGAL 1-29, in the incubation buffer retrieved from sections, incubated during standard conditions is demonstrated in Fig. 3. It was found that a major portion of the radioligand eluted as a single peak, although approx. 10~o of the total radioactivity eluted at other positions (Fig. 3). However, as apparent in the chromatogram, some degradation of the radioligand had
Fig. 1. Schematic diagram of the blowfly brain with some of the neuropil regions of interest in this account indicated (frontal compressed view). The galanin-like immunoreactive (GAL-IR) cell bodies are indicated in black. Note that all these cell bodies actually are located superficially in a rind and that all neuropils are located in the core. The central body neuropil (stippled) is supplied by a dense innervation of GAL-IR fibers. Also in the antennal lobes (AL), medulla (Me) of the optic lobe and subesophageal ganglion (SEG), GAL-IR fibers can be found. The pars intercerebralis is the region above the central body, between the clusters of small and the large GAL-IR cell bodies. Lo, lobula; M, mushroom bodies.
127
a
Fig. 2. (a) Bright field micrograph of autoradiograph of frontal section of fly brain that has been Giemsa counterstained. In bright field illumination labeling can barely be seen in central body neuropil (CB). All other dark-staining structures are neuronal and glial cell bodies surrounding brain neuropils. The synaptic neuropils of the lobula (Lo) and medulla (Me) of the optic lobes are visible. (b-c) Dark-field autoradiographs of total 125I-[Tyr26]-pGAL1-29 binding to frontal sections of the blowfly brain. Refer to Fig. 1 for anatomical details of brain organization. (b) Midregion of the brain, at the level of the central body (CB) and posterior portion of the antennal lobes (AL), both of which are labeled. Also in the basal portion of the medulla a weak labeling can be seen. (c) Anterior portion of the brain with labeling in the antennal lobes (AL). Scale bar 200 lam.
128 60,000 A 50,000 40,000
5O
5 30,000 20,000
1251[Tyr26]-pGAL1-29 > > ~25I-[Tyr9]-pGAL1-29. In contrast to the other GAL-ligands, no specific binding of rat GAL, 125I-[Tyr9]-rGAL1-29, could be demonstrated on sections of the blowfly brain at the conditions described. It should be noted that unspecific binding to membranes of air sacks surrounding the brain was seen (Figs. 4 and 5) and a weak residual non-specific background remained in brain neuropil at displacement with high concentrations of ligand (Fig. 4d). With the use of the C-terminus fragment of porcine GAL ~25I-[TyrZ6]-pGAL15-29, additional specific binding sites were revealed in the medulla of the optic lobe, the pars intercerebralis of the protocerebrum and in the subesophageal ganglion (Fig. 5). This additional binding, as well as that in the central body and antennal lobes, could gradually be displaced by pGAL15-29 (over a range of 0.1-1 ~tM). Also pGAL1-29, which was tested only at 1 ~tM, totally displaced the binding. Discussion
Although the distribution and action ofneuropeptides in insects has been extensively studied [27-31], there are until now only a few reports on peptide binding sites in the nervous system. These concern binding kinetics of radiolabeled opioid peptide analogues and give no detailed information about the tissue distribution [32,33]. Thus, this is to our knowledge, the first visualization of peptide binding sites in the nervous system of insects. The distribution of 125I-pGAL1-29 binding-sites in the blowfly brain corresponds well with the distribution of processes of GAL-like immunoreactive (GAL-IR) neurons reported by Lundquist et al. [25]. Thus, immunocytochemistry showed that there is a dense supply of GAL-IR processes in the central body neuropil and a much sparser innervation in neuropil of the antennal lobes. Both these synaptic neuropils exhibit a distinct specific binding of 125I-pGAL1-29 and 125I-pGAL15-29. Likewise, neuropils of the optic lobes (the medulla), the pars intercerebralis and the subesophageal ganglion that bind 125I-pGAL15-29 are also innervated by GAL-IR neurons [25]. Hence, the similarities in distribution of GAL-IR processes and 125I-pGAL binding sites, suggest that a GAL-like peptide may act as a neurotransmitter or neuromodulator in these synaptic neuropil regions of the blowfly brain. The more extensive distribution of bound 125I-pGAL15-29, compared to 125I-pGAL1-29, may be due to superior ability to bind to the blowfly brain, i.e., the affinity for 125I-pGAL15-29 is possibly higher and hence this ligand may be easier to detect in areas containing a lower number of GAL binding sites. Alternatively ~2sI-
intercerebralis (PI) seen in b and c, in the subesophagealganglion(SEG) seen in a-c, and in the antennal lobes (AL) shown in d. The specificlabeling to these neuropil regions could be gradually displaced with pGALI5-29 and with 1 ~tM pGAL1-29. Also with 125I-pGAL15-29unspecificlabeling of air sack membranes (arrows in a) could be seen. Scale bar 200 lam.
132 pGAL15-29, may detect a subset of binding-sites with different structural requirements. However since this binding was displaced by both pGAL 1-29 and pGAL 15-29 the former hypothesis seems more likely. Since 125I-pGAL15-29 bound more extensively and intensively in the blowfly brain than 125I-pGAL1-29 and the unlabeled fragment pGAL15-29 displaces 125I-[TyrZ6]pGAL1-29 whereas pGAL1-22 does not, it may be suggested that the C-terminal portion of GAL is important for the binding. Moreover, 125I-[Tyr9]-pGAL1-29 exhibits only a weak binding whereas 125I-[Tyr9]-rGAL1-29 apparently does not bind at all. The difference between these two radioiodinated peptides resides in the Cterminal portion, in the amino acid residues at positions 23, 26 and 29 [34], further indicating the importance of the C-terminal part of GAL for binding in the blowfly. The circumstance that the binding of the N-terminally labeled fraction 125I-[Tyr9]-pGAL129 is weaker than that of the C-terminal labeled ~25I-[Tyr26]-pGAL1-29 is obscure. The introduction of an iodine molecule in the N-terminal part may, however, induce some structural changes affecting the binding. The GAL-like peptide(s) in the blowfly differs from pGAL by being more basic and by having other hydrophobic properties [25]. Since we have found that the C-terminal portion of the pGAL molecule seem to be important for binding in the blowfly brain, the major differences in structure of the blowfly GAL and pGAL may possibly reside in the N-terminal portion of the molecule. Interestingly, this would contrast to the finding that the vertebrate galanins are preserved in the N-terminal pentadecapeptide and the interspecies differences are seen in the C-terminal [ 1,34-38]. In the mammalian nervous system, different types of GAL receptors have been described. A preferentially N-terminal dependent type, where the species-conserved portion of the GAL-molecule is crucial for binding and actions has been described in the brain and pancreas [8,9,11,23,39,40]. Another receptor type, which is preferentially dependent on an intact, species specific C-terminal portion of the GAL-molecule, has also been shown to exist in the gastrointestinal tract [14,17,41]. Our findings in the present study indicate that the binding sites for a GAL-like peptide in the blowfly brain may have properties in common with the C-terminal specific receptor type in mammals. However, the binding site in the fly brain is apparently different from the mammalian receptor type since the C-terminal portion binds better than the holopeptide pGAL 129, whereas in the rat gastrointestinal tract only partial binding is obtained with similar C-terminal ligands [14]. Recently, two different receptors resembling mammalian tachykinin receptors were demonstrated in the fruitfly Drosophila melanogasterby recombinant DNA technique [42,43] indicating that certain neuropeptides and their receptors may have evolved early in phylogeny. The existence of GAL like peptide(s) and GAL binding sites in the insect brain may be an additional example of a preservation of an ancient ligandreceptor system.
Acknowledgements This study was supported by the Swedish Natural Science Research Council (BBU-1820-308) to D.R.N. and the Swedish Medical Research Council (Projects 7906
133
and 8997), Magnus Bergvalls Foundation, Nordic Insulin Fund and the Karolinska Institute to A.R. We thank Anne Karlsson for technical assistance.
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134 regulation of corticotropin and thyrotropin secretion in the rat, Endocrinology, 127 (1991) 2281-2289. 21 McDonald, T.J., Dupre, J., Tatemoto K., Greenberger, G. R., Radziuk, J. and Mutt. V., G alanin inhibits insulin secretion and induces hyperglycemia in dogs. Diabetes, 34 (1985) 192-196. 22 Rattan, S., Role of galanin in the gut, Gastroenterology, 100 (1991) 1762-1768. 23 Crawley, J.N., Austin, M.C., Fiske, S.M., Martin, B., Consolo, S., Berthold, M., Langel, U., Fisone, G. and Bartfai, T., Activity of centrally administered galanin fragments on stimulation of feeding behaviour and on galanin receptor binding in the rat hypothalamus. J. Neurosci., 10 (1990) 3695-3700. 24 Roberts, M.H., Speh, J.C. and Moore, R.Y., The central nervous system of Bulla gouldiana: peptide localization, Peptides, 9 (1989) 1323-1334. 25 Lundquist, C.T., ROkaeus, ~.. and N~issel, D.R., Galanin immunoreactivity in the blowfly nervous system: localization and chromatographic analysis. J. Comp. Neurol. 312 (1991) 77-96. 26 De Loof, A. and Schools, L., Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated form invertebrate sources. Comp. Biochem. Physiol., 95B (1990)459468. 27 Holman, G.M., Nachman, R.J. and Wright, M.S., Insect neuropeptides. Annu. Rev. Entomol., 35 (1990) 201-217. 28 Raabe, M., Recent developments in insect neurohormones, Plenum, New York, 1989, 503 pp. 29 O'Shea, M. and Schaffer, M., Neuropeptide function: the invertebrate contribution, Annu. Rev. Neurosci., 8 (1985) 171-198. 30 N~tssel, D. R., Neurotransmitters and neuromodulators in the insect visual system, Progr. Neurobiol., 37 (1991) 179-254. 31 N~issel, D.R., Cantera, R., Johard, H.A.D., Lundquist, C.T., Muren, E. and Shiga, S., Organization of peptidergic pathways in insects. In R.N. Singh (Ed.), Nervous systems: Principles of design and function. Wiley Eastern, New Delhi, 1992, pp. 189-212 (in press). 32 Stefano, G.B. and Scharrer, B., High affinity binding of an enkephalin analog in the cerebral ganglia of an insect Leucophaea maderae. Brain Res., 225 (1981) 107-114. 33 Stefano, G. B., Scharrer, B. and Assanah, P., Demonstration, characterization and localization of opioid binding sites in the midgut of the insect Leucophaea maderae (Blattaria), Brain Res., 253 (1982) 205-212. 34 Vrontakis, M. E., Peden, L.M., Duckworth, M. L. and Friesen, H.G., Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA, J. Biol. Chem., 262 (1987) 16755-16758. 35 Bersani, M., Johnsen, A.J., Hojrup, P., Dunning, B. E., Andreasen, J.J. and Hoist, J.J., Human galanin: primary structure and identification of two molecular forms, FEBS Lett., 283 (1991) 189-194. 36 Norberg, A., Sillard, R., Carlquist, M., JOrnvall, H. and Mutt, V., Chemical detection of natural peptides by specific structures: Isolation of chicken galanin by monitoring for its N-terminal dipeptide, and determination of the amino acid sequence, FEBS Lett., 288 (1991) 151-153. 37 ROkaeus, A.. and Carlquist, M., Nucleotide sequence analysis of cDNAs encoding a bovine galanin precursor protein in the adrenal medulla and chemical isolation of bovine gut galanin, FEBS Lett., 234 (1988) 400-406. 38 Sillard, R., Langel, f]. and JOrnvall, H., Isolation and characterization of galanin from sheep brain, Peptides, 12 (1991) 855-859. 39 Lagny-Pourmir, I., Lorinet, A. M., Yanaihara, N. and Laburthe, M., Structural requirements for galanin interaction with receptors from pancreatic beta cells and from brain tissue of the rat, Peptides, 10 (1989) 757-761. 40 Xu, X.-J., Wiesenfeld-Hallin, Z., Fisone, G., Bartfai, T. and HOkfelt, T., The N-terminal 1-16, but not the C-terminal 17-29, galanin fragment affects the flexor reflex in rats, Eur. J. Pharmacot., 182 (1990) 137-414. 41 Katsoulis, S., Schmidt, W.E., SchwOrer, H. and Creutzfeldt, W., Effects of galanin, its analogues and fragments on rat isolated fundus strips. Br. J. Pharmacol., 101 (1990) 297-300. 42 Li, X.J., Wolfgang, W., Wu, Y.N., North, R.A. and Forte, M., Cloning, heterologous expression and developmental regulation of a Drosophila receptor for tachykinin-like peptides, EMBO J., I0 (1991) 3221-3229. 43 Monnier, D., Colas, J.F., Rosay, P., Hen, R., Borrelli, E. and Maroteaux, L., NKD, a developmentally regulated tachykinin receptor in Drosophila, J. Biol. Chem., 267 (1992) 1298-1302.
123
© 1992Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00 REGPEP 01239
Autoradiographic localization of 125I-galanin binding sites in the blowfly brain Helena A. D. Johard
a, C. Tomas Lundquist a, Ake ROkaeus b and Dick R. N~ssel a
aDepartment of Zoology, Stockholm University, Stockholm (Sweden) and b Department of Biochemistry I. Karolinska Institute, Stockholm (Sweden)
(Received 4 May 1992; revisedversion receivedand accepted 13 August 1992) K e y words." Galanin; Neuropeptide; Insect nervous system; Peptide receptor;
Autoradiography
Summary The localization of porcine galanin (pGAL) binding sites in the brain of the blowfly Phormia terraenovae was investigated by autoradiography using the following radioiodinated ligands: pGAL 1-29 (two isoforms), pGAL 15-29 and rat (r) GAL 129. The different porcine radioligands bound specifically with the following intensity: 125I-[Tyr26]-pGAL 15-29 > > 125I-[Tyr26]-pGAL 1-29 > > 125I-[Tyr9]-pGAL 1-29. With rat galanin 125I-[Tyr9]-rGAL1-29 no specific binding could be shown. In addition, displacement of 125I-[TyrZ6]-pGAL1-29 was tested with pGAL 1-29, pGAL 1-22 and pGAL 15-29 (at 0.1 n M - 1 laM). A gradual displacement was achieved with increasing concentrations of pGAL 1-29 and pGAL15-29, whereas no displacement with p G A L 1-22 was detected. The results indicate that the C-terminal portion of pGAL is important for binding in the blowfly. The pGAL binding sites were localized in synaptic neuropils of the central body, the antennal lobes, the optic lobes, the pars intercerebralis and the subesophageal ganglion, all of which contain GAL-like immunoreactive neural processes.
Correspondence to: D.R. Nassel, Department of Zoology, Stockholm University, S-106 91 Stockholm,
Sweden.
124 Introduction
Galanin (GAL) is an amidated 29 amino acid residue peptide, originally isolated from pig intestine [ 1], which is widely distributed in the central and peripheral nervous system of several mammalian [2-4] and submammalian species [5-7]. In close association with GAL-immunoreactive (GAL-IR) neurons, high affinity GAL binding sites have been visualized by autoradiography and characterized by binding assays, using radioiodinated GAL [8-16]. As functional correlate to the localization of GAL-IR neurons and receptors, a number of biological actions of GAL have been described, e.g., modulation of neurotransmitter release [ 17,18 ], regulation of hormone secretion [ 19-21 ], regulation of smooth muscle activity [ 3,17,22 ] and control of feeding behavior [23]. GAL-IR neurons have also been demonstrated in two invertebrate species. In the mollusc, Bulla gouldiana, a single pair of GAL-IR neurons was detected in the cerebral ganglion [24]. A more extensive distribution of GAL-IR neurons was demonstrated in the central nervous system of the blowfly, Phorrnia terraenovae [25]. Chromatographical analysis of the GAL-IR indicated the presence of several forms of GAL-like peptide in the blowfly [25]. Quite a number of other neuropeptides have been demonstrated in neurons and neurosecretory cells in the brain of insects [26-28]. Most of these insect peptides were isolated as neurohormones and their action has mainly been studied at peripheral targets [27-29]. Considering the large number of peptidecontaining interneurons in the insect brain it is to be expected that different neuropeptides have a variety of central modulatory actions. One strategy to search for central sites of action of neuropeptides is to analyze neuropeptide binding. In the present study we have thus investigated the presence of 125I-GAL binding sites in sections of the blowfly brain by autoradiography.
Materials and Methods
Tissue preparation Brains of the blowfly, Phormia terraenovae, were rapidly dissected in cold 0.1 M sodium phosphate buffer solution (pH 7.4) and immediately frozen in Tissue-Tek ® (Miles, Elkhart, IN). Frontal 20 lam-thick sections of the brain were cut on a cryostat ( - 14 ° C) and thaw-mounted on chrome-alun coated slides. Subsequently, the sections were dried at 35 °C for 30 min and stored dessicated at -80°C until used for autoradiography (within 2 weeks). Preparation of radioligands Synthetic porcine (p) G A L l - 2 9 (Peninsula, UK), rat (r) G A L l - 2 9 and pGAL15-29 (gift from P. Padgett, Laboratory of Cell Biology, NIMH-NIH, Bethesda, USA) were labeled by the chloramine T method using 1251 (Amersham, UK). Following radioiodination, peptides were rapidly purified from unincorporated iodine and other salts on a Sep Pak C18 cartridge (Waters, Millipore). A further purification of the labeled peptides was performed by reverse phase HPLC, using a linear gradient of 20-38~o
125 acetonitrile with 0.1 ~o trifluoroacetic acid over 70 rain at a flow rate of 0.5 ml/min. These conditions allowed separation of unlabeled peptides from labeled peptides as well as separation of two iodinated pGAL isoforms (125I-[TyrZ6]-pGAL1-29 and 125I-[Tyr9]-pGAL1-29; ROkaeus, unpublished data). The radioactivity of the separated fractions was measured in a gamma counter. The specific activity of the iodinated peptides was estimated to be higher than 2000 Ci/mmol.
Autoradiographic analysis of t25I-GAL binding Frozen sections of the fly brains were allowed to thaw for 15 min at room temperature before preincubation in 50 mM Tris buffer (pH 7.4) containing 5 mM MgC12, 2 mM EGTA, 0.1 ~o bovine serum albumin (B SA; Sigma) and 0.01 ~o bacitracin (Sigma) for 15 min at room temperature. Thereafter slides were incubated for 60 min in the same buffer as above, including 125I-[Tyr26]-pGAL1-29 at a concentration of 0.2-0.4 nM (500 cpm/~tl). Preliminary binding experiments were performed for 30, 60 and 120 min and 60 min was found to yield maximal specific binding. Incubations were terminated by rinsing four times 30 s in 50 mM Tris buffer containing 5 mM MgC1z (at 4°C) and 30 s in double distilled water (at 4°C). The sections were subsequently dried under a cold stream of air and fixed for 30 rain in paraformaldehyde vapor at 80°C. The slides were coated with Kodak NTB-2 emulsion, exposed for 14-21 days at 4°C, and developed in Kodak D-19 developer. Thereafter the sections were dehydrated in a series of alcohol/xylene steps, mounted with Permount and finally analyzed under light- and darkfield illumination in a Zeiss Axiophot microscope. A set of autoradiography slides were counterstained with Giemsa stain for more accurate localization of silver grains. In order to investigate the specificity of the GAL binding and to reveal which portion of the GAL molecule that was of importance for displacement of 125I-[Tyr26]pGAL 1-29 binding, unlabeled pGAL 1-29, pGAL 1-22 and pGAL 15-29, respectively, were added to the incubation buffer at six concentrations ranging from 0.1 nM to 10 I~M. To further analyze the structural requirements of the GAL binding sites, we investigated the ability of different iodinated GAL ligands to specifically visualize such sites at the incubation conditions described above. The following four radioligands were tested at a concentration of 0.2-0.4 nM: 125I-[TyrZ6]-pGAL1-29, ~25I-[Tyr9]pGAL1-29, 125I-[TyrZ6]-pGAL15-29 and rat galanin, 125I-[Tyr9]-rGAL1-29. Nonspecific binding was estimated by including 1 ~tM of pGAL1-29 in the incubation buffer. However, since 12sI-[Tyr26]-pGAL15-29 gave the best visualization of binding sites, the ability of unlabeled pGAL15-29 to displace this binding was tested in one experiment at five concentrations ranging from 0.1 nM to 1 ~tM. Triplets of brains were used for each condition described above and all experiments were repeated at least twice (except the displacement with pGAL15-29). The grain density on the brain sections was analyzed microscopically by several investigators, using six serially sectioned brains for each experimental condition. While the well delineated synaptic neuropil termed the central body is easy to distinguish and had the highest density of specific 125I-GAL-binding, it was used in all analysis of ligand displacement (see Fig. 4). To estimate the radioligand degradation during the incubation, the ~25I-[TyrZ6]pGAL1-29 in incubation buffer was retrieved from the sections for a comparison with
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fresh ligand. This analysis was performed with reversed phase HPLC at the same conditions as used for the radioligand purification.
Results
Binding of 12sI-[ Tyr26]-pGAL1-29 In Fig. 1 a schematic diagram of the blowfly brain is shown for reference to the distribution of binding sites. It is important to note that the insect brain is organized with all the neuronal cell bodies in a superficial rind and with axonal tracts and synaptic neuropil regions in the core. The radioligand binding described below is in synaptic neuropil regions, as determined from counterstained sections (Fig. 2a). Specific binding of 1251-[TyrZ6]-pGAL1-29 was most apparent in the central body, a distinct synaptic neuropil region in the protocerebrum of the blowflybrain (Fig. 2b). Specific binding of radiolabeled GAL, albeit at lower density, could also be observed in the neuropil of the antennal lobes in the deutocerebrum of the brain, and at a barely detectable level in the medulla of the optic lobe (Fig. 2b,c). The integrity of the radioiodinated ligand ~25I-[Tyr26]-pGAL 1-29, in the incubation buffer retrieved from sections, incubated during standard conditions is demonstrated in Fig. 3. It was found that a major portion of the radioligand eluted as a single peak, although approx. 10~o of the total radioactivity eluted at other positions (Fig. 3). However, as apparent in the chromatogram, some degradation of the radioligand had
Fig. 1. Schematic diagram of the blowfly brain with some of the neuropil regions of interest in this account indicated (frontal compressed view). The galanin-like immunoreactive (GAL-IR) cell bodies are indicated in black. Note that all these cell bodies actually are located superficially in a rind and that all neuropils are located in the core. The central body neuropil (stippled) is supplied by a dense innervation of GAL-IR fibers. Also in the antennal lobes (AL), medulla (Me) of the optic lobe and subesophageal ganglion (SEG), GAL-IR fibers can be found. The pars intercerebralis is the region above the central body, between the clusters of small and the large GAL-IR cell bodies. Lo, lobula; M, mushroom bodies.
127
a
Fig. 2. (a) Bright field micrograph of autoradiograph of frontal section of fly brain that has been Giemsa counterstained. In bright field illumination labeling can barely be seen in central body neuropil (CB). All other dark-staining structures are neuronal and glial cell bodies surrounding brain neuropils. The synaptic neuropils of the lobula (Lo) and medulla (Me) of the optic lobes are visible. (b-c) Dark-field autoradiographs of total 125I-[Tyr26]-pGAL1-29 binding to frontal sections of the blowfly brain. Refer to Fig. 1 for anatomical details of brain organization. (b) Midregion of the brain, at the level of the central body (CB) and posterior portion of the antennal lobes (AL), both of which are labeled. Also in the basal portion of the medulla a weak labeling can be seen. (c) Anterior portion of the brain with labeling in the antennal lobes (AL). Scale bar 200 lam.
128 60,000 A 50,000 40,000
5O
5 30,000 20,000
1251[Tyr26]-pGAL1-29 > > ~25I-[Tyr9]-pGAL1-29. In contrast to the other GAL-ligands, no specific binding of rat GAL, 125I-[Tyr9]-rGAL1-29, could be demonstrated on sections of the blowfly brain at the conditions described. It should be noted that unspecific binding to membranes of air sacks surrounding the brain was seen (Figs. 4 and 5) and a weak residual non-specific background remained in brain neuropil at displacement with high concentrations of ligand (Fig. 4d). With the use of the C-terminus fragment of porcine GAL ~25I-[TyrZ6]-pGAL15-29, additional specific binding sites were revealed in the medulla of the optic lobe, the pars intercerebralis of the protocerebrum and in the subesophageal ganglion (Fig. 5). This additional binding, as well as that in the central body and antennal lobes, could gradually be displaced by pGAL15-29 (over a range of 0.1-1 ~tM). Also pGAL1-29, which was tested only at 1 ~tM, totally displaced the binding. Discussion
Although the distribution and action ofneuropeptides in insects has been extensively studied [27-31], there are until now only a few reports on peptide binding sites in the nervous system. These concern binding kinetics of radiolabeled opioid peptide analogues and give no detailed information about the tissue distribution [32,33]. Thus, this is to our knowledge, the first visualization of peptide binding sites in the nervous system of insects. The distribution of 125I-pGAL1-29 binding-sites in the blowfly brain corresponds well with the distribution of processes of GAL-like immunoreactive (GAL-IR) neurons reported by Lundquist et al. [25]. Thus, immunocytochemistry showed that there is a dense supply of GAL-IR processes in the central body neuropil and a much sparser innervation in neuropil of the antennal lobes. Both these synaptic neuropils exhibit a distinct specific binding of 125I-pGAL1-29 and 125I-pGAL15-29. Likewise, neuropils of the optic lobes (the medulla), the pars intercerebralis and the subesophageal ganglion that bind 125I-pGAL15-29 are also innervated by GAL-IR neurons [25]. Hence, the similarities in distribution of GAL-IR processes and 125I-pGAL binding sites, suggest that a GAL-like peptide may act as a neurotransmitter or neuromodulator in these synaptic neuropil regions of the blowfly brain. The more extensive distribution of bound 125I-pGAL15-29, compared to 125I-pGAL1-29, may be due to superior ability to bind to the blowfly brain, i.e., the affinity for 125I-pGAL15-29 is possibly higher and hence this ligand may be easier to detect in areas containing a lower number of GAL binding sites. Alternatively ~2sI-
intercerebralis (PI) seen in b and c, in the subesophagealganglion(SEG) seen in a-c, and in the antennal lobes (AL) shown in d. The specificlabeling to these neuropil regions could be gradually displaced with pGALI5-29 and with 1 ~tM pGAL1-29. Also with 125I-pGAL15-29unspecificlabeling of air sack membranes (arrows in a) could be seen. Scale bar 200 lam.
132 pGAL15-29, may detect a subset of binding-sites with different structural requirements. However since this binding was displaced by both pGAL 1-29 and pGAL 15-29 the former hypothesis seems more likely. Since 125I-pGAL15-29 bound more extensively and intensively in the blowfly brain than 125I-pGAL1-29 and the unlabeled fragment pGAL15-29 displaces 125I-[TyrZ6]pGAL1-29 whereas pGAL1-22 does not, it may be suggested that the C-terminal portion of GAL is important for the binding. Moreover, 125I-[Tyr9]-pGAL1-29 exhibits only a weak binding whereas 125I-[Tyr9]-rGAL1-29 apparently does not bind at all. The difference between these two radioiodinated peptides resides in the Cterminal portion, in the amino acid residues at positions 23, 26 and 29 [34], further indicating the importance of the C-terminal part of GAL for binding in the blowfly. The circumstance that the binding of the N-terminally labeled fraction 125I-[Tyr9]-pGAL129 is weaker than that of the C-terminal labeled ~25I-[Tyr26]-pGAL1-29 is obscure. The introduction of an iodine molecule in the N-terminal part may, however, induce some structural changes affecting the binding. The GAL-like peptide(s) in the blowfly differs from pGAL by being more basic and by having other hydrophobic properties [25]. Since we have found that the C-terminal portion of the pGAL molecule seem to be important for binding in the blowfly brain, the major differences in structure of the blowfly GAL and pGAL may possibly reside in the N-terminal portion of the molecule. Interestingly, this would contrast to the finding that the vertebrate galanins are preserved in the N-terminal pentadecapeptide and the interspecies differences are seen in the C-terminal [ 1,34-38]. In the mammalian nervous system, different types of GAL receptors have been described. A preferentially N-terminal dependent type, where the species-conserved portion of the GAL-molecule is crucial for binding and actions has been described in the brain and pancreas [8,9,11,23,39,40]. Another receptor type, which is preferentially dependent on an intact, species specific C-terminal portion of the GAL-molecule, has also been shown to exist in the gastrointestinal tract [14,17,41]. Our findings in the present study indicate that the binding sites for a GAL-like peptide in the blowfly brain may have properties in common with the C-terminal specific receptor type in mammals. However, the binding site in the fly brain is apparently different from the mammalian receptor type since the C-terminal portion binds better than the holopeptide pGAL 129, whereas in the rat gastrointestinal tract only partial binding is obtained with similar C-terminal ligands [14]. Recently, two different receptors resembling mammalian tachykinin receptors were demonstrated in the fruitfly Drosophila melanogasterby recombinant DNA technique [42,43] indicating that certain neuropeptides and their receptors may have evolved early in phylogeny. The existence of GAL like peptide(s) and GAL binding sites in the insect brain may be an additional example of a preservation of an ancient ligandreceptor system.
Acknowledgements This study was supported by the Swedish Natural Science Research Council (BBU-1820-308) to D.R.N. and the Swedish Medical Research Council (Projects 7906
133
and 8997), Magnus Bergvalls Foundation, Nordic Insulin Fund and the Karolinska Institute to A.R. We thank Anne Karlsson for technical assistance.
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