332
Brain Research, 534 (1990) 332-335 Elsevier
BRES 24405
Quantitative autoradiographic localization of [1251]a-bungarotoxin binding sites in the honeybee brain Angelika Scheidler 1, Peter Kaulen 2, Gerold Briining 3 and Joachim Erber 1 t Department of Biology, Technical University of Berlin, 2Departmentof Ophthalmology, Klinikum Rudolf Virchow, Free University of Berlin and 3Department of Anatomy, Free University of Berlin, Berlin (E R. G.)
(Accepted 21 August 1990) Key words: a-Bungarotoxin; Acetylcholinereceptor; Honeybee brain; Autoradiography
Binding sites for the nicotinic acetylcholine receptor antagonist, [~25I]a-bungarotoxin,were localized in the honeybee brain by in vitro autoradiography. Highest binding site densities were localized in the suboesophageal ganglion, the optic tubercles, optic lobes medulla and lobula, antennal lobes, dorsal lobes and the a-lobes of the mushroom bodies. The distribution pattern of these putative nicotinic acetylcholine receptors suggests that acetylcholine is involved in several sensory pathways and in central information processing in the honeybee brain.
The snake venom, a-bungarotoxin, is known to be a high-affinity, selective antagonist of nicotinic acetylcholine (ACh) receptors in vertebrates and insects. So far, most information about ACh receptors in insects is available for locusts, cockroaches, flies, moths, and crickets (for reviews see refs. 1, 3, 8, 9). In the present study the quantitative distribution of [tESI]a-bungarotoxin binding sites in the honeybee brain was investigated by in vitro autoradiography. Cryostat sections (20/zm) of 10 honeybee brains (Apis mellifera) were preincubated for 30 rain in Tris-buffer (0.17 M, pH 7.4, 20 °C) and subsequently for 120 min in the same buffer containing 0.7 nM [125I]a-bungarotoxin (3-[125I]iodotyrosylS4-a-bungarotoxin, 118.7 Ci/mmol, Amersham). Slides were washed in buffer (5 min, 0 °C) twice, dipped into cold distilled water and dried rapidly. Non-specific binding, determined in the presence of I mM nicotine, was low (1.4-2.5 fmol/mm 2) without significant differences among brain regions. The sections were exposed to hyperfilm (7 days) and subsequently to photoemulsion (Ilford K2) coated coverslips (23 days). Quantitation was performed using an image-analyzing system (IBAS II, Kontron). The conversion of grey values (measured on hyperfilm autoradiograms) to fmol/ mm 2 was performed using calibration curves determined from coexposed standards of the same thickness ([125I]microscales, Amersham). [125I]a-Bungarotoxin binding was confined to neuropil areas, in particular to those with primary and higher order sensory projections. Somata regions were not
labelled. The highest density of binding sites (211.0 + 21.1 fmol/mm 2) was observed in the suboesophageal ganglion (Figs. ld, e and 2). In the antennal pathway binding sites were concentrated in the glomeruli of the antennal lobes (130.3 + 4.8 fmol/mm 2) (Figs. la, b and 2) and in the dorsal lobes (139.6 + 14.1 fmol/mm 2) (Figs. ld and 2). Second-order neurons of the antenno-glomerular tracts project from the antennal lobes to the mushroom bodies. The input regions of this neuropil, the lip (92.5 + 33.6 fmol/mm 2) and the basal ring (98.9 + 31.8 fmol/mm 2) of the calyces (Figs. l b - e and 2), were enriched in [125I]a-bungarotoxin binding sites. This also applies to the output regions of the mushroom bodies, the a- (Figs. lb and 2) and r-lobes (93.6 + 5.2 fmol/mm 2) (Figs. lc and 2). The a-lobe showed a strong labelling (157.3 + 20.1 fmoi/mm 2) in layers 3, 4 and 5 (ref. 6), mainly at the level of the a-exit point. The pedunculi of the mushroom bodies displayed only few binding sites (24.0 + 5.6 fmol/mm2), located mostly in the area where the K-cells bifurcate into the a- and r-lobes (Fig. lc). In the visual pathway (Figs. l b - e and 2) the lamina, the first optic ganglion, displayed the lowest concentration of binding sites located mainly in the C-layer (90.3 + 5.3 fmol/mm2). In the medulla, the second optic ganglion, a clear stratification was visible (182.3 + 18.9 fmol/mm 2 in the distal and proximal part) with less binding sites in the area of the serpentine layer (104.4 + 4.7 fmol/mm2). In the lobula, the third optic ganglion, two main compartments of high receptor density could be differentiated (168.7 + 21.2 fmol/mm 2 in the distal, 111.8
Correspondence: A. Scheidler, Department of Biology, Technical University, Franklinstrasse 28/29, D-1000 Berlin 10, F.R.G.
0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
333
Fig. 1. [lzSI]a-Bungarotoxin(0.7 nM) binding in frontal sections of the honeybee brain. Binding sites are indicated by accumulations of silver grains, a-e: total binding, (a,b) corresponding to Fig. 2, left hemisphere, (c-e) corresponding to Fig. 2, right hemisphere, f: non-specific binding. Scale bar = 200/~m.
+ 14.5 fmol/mm 2 the proximal part). An extremely high density of binding sites (203.5 + 14.8 fmol/mm 2) appeared in the optic tubercles (Figs. la and 2). An accumulation of silver grains (61.3 + 4.8 fmol/mm 2) was also found in the ocellar neuropil (Fig. ld, e). Within the central complex only the protocerebral bridge was enriched in a-bungarotoxin binding sites (62.8 + 4.9 fmol/mm2). The anterior median protocerebrum, which is located around the a-lobes, was intensely
labelled in a ring-shaped manner (128.0 + 1.0 fmol/mm 2) (Figs. lb and 2). Neurons in this non-glomerular neuropil process higher order information from the mushroom bodies and other central neuropils. The non-glomerular neuropil in the posterior ventral parts of the brain showed a high concentration of binding sites (113.2 + 4.0 fmol/mm 2) in areas where projections from the optic lobes, the antennal lobes, and the ocellar pathway are found 7 (Figs. l c - e and 2). In contrast, prominent
334 d
lip
co
Fig. 2. Schematic illustration of [125I]a-bungarotoxin binding sites in the honeybee brain (frontal view). Left hemisphere: level corresponding to Fig. 1 (a,b); right hemisphere: level corresponding to Fig. 1 (c-e). Binding site densities are indicated in 4 categories of grey values from white (~150 fmol/mm2), a = anterior; a = a-lobe; al = antennal lobe; an = antennal nerve; fl = fl-lobe; br = basal ring; c = C-layer of the lamina; cb = central body; co = collar; d = dorsal; d l = dorsal lobe; ioc = inferior optic commissure; la = lamina; lca = lateral calyx; 1o = lobula; mb = mushroom bodies, indicated by their contours; mca = median calyx; me = medulla; ngn = non-glomerular neuropil; of = oesophageal foramen; ot = optic tubercle; p = posterior; pb = protocerebral bridge; r = ring-shaped labelling in the median protocerebrum around the a-lobe; sl = serpentine layer; sog = suboesophageal ganglion; v = ventral. Scale bar = 200/~m.
commissures and tracts were devoid of binding sites. A close correlation exists between the autoradiographic localization of [12sI]a-bungarotoxin binding sites and the distribution of putative cholinergic neurons d e m o n s t r a t e d by A C h - e s t e r a s e staining 5. This is obvious in the optic lobes, optic tubercle, lip region of the calyces, antennal lobes and the region of the sensory afferents to the dorsal lobes. The distribution of [125I]a-bungarotoxin binding sites overlaps with the immunocytochemical staining of putative A C h receptors 5 in the optic lobes, ocellar neuropil, antennal lobes, the lips of the calyces as well as in the a- and fl-lobes of the m u s h r o o m bodies. However, the dorsal lobes and the optic tubercles are rich in [125I]a-bungarotoxin binding sites but display only weak immunolabelling. Conversely, the pedunculi, central body,
inner and outer optic chiasms, and the antennal nerve show A C h receptor immunolabelling but are almost free of [lzsI]a-bungarotoxin binding sites. Species differences may contribute to this mismatch, as the antibody was developed for locust A C h receptors. The antibody might also recognize subclasses of nicotinic A C h receptors which do not bind a-bungarotoxin at low concentrations. The spatial pattern of [125I]a-bungarotoxin binding in the honeybee brain resembles that in Drosophila 2"1° and Manduca sexta4. The distribution of these putative nicotinic A C h receptors suggests that A C h is involved in several sensory pathways and in central information processing in the bee brain as described for other arthropods 3'8'9.
1 Breer, H. and Sattelle, D.B., Molecular properties and functions of insect acetylcholine receptors, J. Insect. Physiol., 33 (1987) 771-790. 2 Dudai, Y. and Amsterdam, A., Nicotinic receptors in the brain of Drosophila melanogaster demonstrated by autoradiography with [125I]a-bungarotoxin, Brain Research, 130 (1977) 551-555.
3 Eldefrawi, A.T. and Eldefrawi, M.E., Acetylcholine. In G.G. Lunt and R.W. Olsen (Eds.), Comparative Invertebrate Neurochemistry, Croom Helm, London, 1988, pp. 1-41. 4 Hildebrand, J.G., Hall, L.M. and Osmond, B.C., Distribution of binding sites for 12SI-labeled a-bungarotoxin in normal and deafferented antennal lobes of Manduca sexta, Proc. Natl. Acad.
Supported by the Deutsche Forschungsgemeinschaft (Er 79/3).
335
Sci. U.S.A., 76 (1979) 499-503. 5 Kreissl, S. and Bicker, G., Histochemistry of acetyicholinesterase and immunocytochemistry of an acetylcholine receptor-like antigen in the brain of the honeybee, J. Comp. Neurol., 286 (1989) 71-84. 6 Mobbs, P.G., The brain of the honeybee Apis mellifera. I. The connections and spatial organization of the mushroom bodies, Phil. Trans. R. Soc. Lond. B, 298 (1982) 309-354. 7 Mobbs, P.G., Brain structure. In G.A. Kerkut and L.I. Gilbert (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 5, Pergamon Press, Oxford, 1985, pp. 299-370.
8 Sattelle, D.B., Acetyicholine receptors of insects. In M.J. Berridge, J.E. Treherne and V.B. Wigglesworth (Eds.), Advances in Insect Physiology, Vol. 15, Academic Press, London, 1980, pp. 215-315. 9 Sattelle, D.B., Acetylcholine receptors. In G.A. Kerkut and L.J. Gilbert (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 11, Pergamon Press, Oxford, 1985, pp. 395-434. 10 Schmidt-Nielsen, B.K., Gepner, J.I., Teng, N.N.H. and Hail, L.M., Characterization of an a-bungarotoxin binding component from Drosophila melanogaster, J. Neurochem., 29 (1977) 1013-1029.
Brain Research, 534 (1990) 332-335 Elsevier
BRES 24405
Quantitative autoradiographic localization of [1251]a-bungarotoxin binding sites in the honeybee brain Angelika Scheidler 1, Peter Kaulen 2, Gerold Briining 3 and Joachim Erber 1 t Department of Biology, Technical University of Berlin, 2Departmentof Ophthalmology, Klinikum Rudolf Virchow, Free University of Berlin and 3Department of Anatomy, Free University of Berlin, Berlin (E R. G.)
(Accepted 21 August 1990) Key words: a-Bungarotoxin; Acetylcholinereceptor; Honeybee brain; Autoradiography
Binding sites for the nicotinic acetylcholine receptor antagonist, [~25I]a-bungarotoxin,were localized in the honeybee brain by in vitro autoradiography. Highest binding site densities were localized in the suboesophageal ganglion, the optic tubercles, optic lobes medulla and lobula, antennal lobes, dorsal lobes and the a-lobes of the mushroom bodies. The distribution pattern of these putative nicotinic acetylcholine receptors suggests that acetylcholine is involved in several sensory pathways and in central information processing in the honeybee brain.
The snake venom, a-bungarotoxin, is known to be a high-affinity, selective antagonist of nicotinic acetylcholine (ACh) receptors in vertebrates and insects. So far, most information about ACh receptors in insects is available for locusts, cockroaches, flies, moths, and crickets (for reviews see refs. 1, 3, 8, 9). In the present study the quantitative distribution of [tESI]a-bungarotoxin binding sites in the honeybee brain was investigated by in vitro autoradiography. Cryostat sections (20/zm) of 10 honeybee brains (Apis mellifera) were preincubated for 30 rain in Tris-buffer (0.17 M, pH 7.4, 20 °C) and subsequently for 120 min in the same buffer containing 0.7 nM [125I]a-bungarotoxin (3-[125I]iodotyrosylS4-a-bungarotoxin, 118.7 Ci/mmol, Amersham). Slides were washed in buffer (5 min, 0 °C) twice, dipped into cold distilled water and dried rapidly. Non-specific binding, determined in the presence of I mM nicotine, was low (1.4-2.5 fmol/mm 2) without significant differences among brain regions. The sections were exposed to hyperfilm (7 days) and subsequently to photoemulsion (Ilford K2) coated coverslips (23 days). Quantitation was performed using an image-analyzing system (IBAS II, Kontron). The conversion of grey values (measured on hyperfilm autoradiograms) to fmol/ mm 2 was performed using calibration curves determined from coexposed standards of the same thickness ([125I]microscales, Amersham). [125I]a-Bungarotoxin binding was confined to neuropil areas, in particular to those with primary and higher order sensory projections. Somata regions were not
labelled. The highest density of binding sites (211.0 + 21.1 fmol/mm 2) was observed in the suboesophageal ganglion (Figs. ld, e and 2). In the antennal pathway binding sites were concentrated in the glomeruli of the antennal lobes (130.3 + 4.8 fmol/mm 2) (Figs. la, b and 2) and in the dorsal lobes (139.6 + 14.1 fmol/mm 2) (Figs. ld and 2). Second-order neurons of the antenno-glomerular tracts project from the antennal lobes to the mushroom bodies. The input regions of this neuropil, the lip (92.5 + 33.6 fmol/mm 2) and the basal ring (98.9 + 31.8 fmol/mm 2) of the calyces (Figs. l b - e and 2), were enriched in [125I]a-bungarotoxin binding sites. This also applies to the output regions of the mushroom bodies, the a- (Figs. lb and 2) and r-lobes (93.6 + 5.2 fmol/mm 2) (Figs. lc and 2). The a-lobe showed a strong labelling (157.3 + 20.1 fmoi/mm 2) in layers 3, 4 and 5 (ref. 6), mainly at the level of the a-exit point. The pedunculi of the mushroom bodies displayed only few binding sites (24.0 + 5.6 fmol/mm2), located mostly in the area where the K-cells bifurcate into the a- and r-lobes (Fig. lc). In the visual pathway (Figs. l b - e and 2) the lamina, the first optic ganglion, displayed the lowest concentration of binding sites located mainly in the C-layer (90.3 + 5.3 fmol/mm2). In the medulla, the second optic ganglion, a clear stratification was visible (182.3 + 18.9 fmol/mm 2 in the distal and proximal part) with less binding sites in the area of the serpentine layer (104.4 + 4.7 fmol/mm2). In the lobula, the third optic ganglion, two main compartments of high receptor density could be differentiated (168.7 + 21.2 fmol/mm 2 in the distal, 111.8
Correspondence: A. Scheidler, Department of Biology, Technical University, Franklinstrasse 28/29, D-1000 Berlin 10, F.R.G.
0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
333
Fig. 1. [lzSI]a-Bungarotoxin(0.7 nM) binding in frontal sections of the honeybee brain. Binding sites are indicated by accumulations of silver grains, a-e: total binding, (a,b) corresponding to Fig. 2, left hemisphere, (c-e) corresponding to Fig. 2, right hemisphere, f: non-specific binding. Scale bar = 200/~m.
+ 14.5 fmol/mm 2 the proximal part). An extremely high density of binding sites (203.5 + 14.8 fmol/mm 2) appeared in the optic tubercles (Figs. la and 2). An accumulation of silver grains (61.3 + 4.8 fmol/mm 2) was also found in the ocellar neuropil (Fig. ld, e). Within the central complex only the protocerebral bridge was enriched in a-bungarotoxin binding sites (62.8 + 4.9 fmol/mm2). The anterior median protocerebrum, which is located around the a-lobes, was intensely
labelled in a ring-shaped manner (128.0 + 1.0 fmol/mm 2) (Figs. lb and 2). Neurons in this non-glomerular neuropil process higher order information from the mushroom bodies and other central neuropils. The non-glomerular neuropil in the posterior ventral parts of the brain showed a high concentration of binding sites (113.2 + 4.0 fmol/mm 2) in areas where projections from the optic lobes, the antennal lobes, and the ocellar pathway are found 7 (Figs. l c - e and 2). In contrast, prominent
334 d
lip
co
Fig. 2. Schematic illustration of [125I]a-bungarotoxin binding sites in the honeybee brain (frontal view). Left hemisphere: level corresponding to Fig. 1 (a,b); right hemisphere: level corresponding to Fig. 1 (c-e). Binding site densities are indicated in 4 categories of grey values from white (~150 fmol/mm2), a = anterior; a = a-lobe; al = antennal lobe; an = antennal nerve; fl = fl-lobe; br = basal ring; c = C-layer of the lamina; cb = central body; co = collar; d = dorsal; d l = dorsal lobe; ioc = inferior optic commissure; la = lamina; lca = lateral calyx; 1o = lobula; mb = mushroom bodies, indicated by their contours; mca = median calyx; me = medulla; ngn = non-glomerular neuropil; of = oesophageal foramen; ot = optic tubercle; p = posterior; pb = protocerebral bridge; r = ring-shaped labelling in the median protocerebrum around the a-lobe; sl = serpentine layer; sog = suboesophageal ganglion; v = ventral. Scale bar = 200/~m.
commissures and tracts were devoid of binding sites. A close correlation exists between the autoradiographic localization of [12sI]a-bungarotoxin binding sites and the distribution of putative cholinergic neurons d e m o n s t r a t e d by A C h - e s t e r a s e staining 5. This is obvious in the optic lobes, optic tubercle, lip region of the calyces, antennal lobes and the region of the sensory afferents to the dorsal lobes. The distribution of [125I]a-bungarotoxin binding sites overlaps with the immunocytochemical staining of putative A C h receptors 5 in the optic lobes, ocellar neuropil, antennal lobes, the lips of the calyces as well as in the a- and fl-lobes of the m u s h r o o m bodies. However, the dorsal lobes and the optic tubercles are rich in [125I]a-bungarotoxin binding sites but display only weak immunolabelling. Conversely, the pedunculi, central body,
inner and outer optic chiasms, and the antennal nerve show A C h receptor immunolabelling but are almost free of [lzsI]a-bungarotoxin binding sites. Species differences may contribute to this mismatch, as the antibody was developed for locust A C h receptors. The antibody might also recognize subclasses of nicotinic A C h receptors which do not bind a-bungarotoxin at low concentrations. The spatial pattern of [125I]a-bungarotoxin binding in the honeybee brain resembles that in Drosophila 2"1° and Manduca sexta4. The distribution of these putative nicotinic A C h receptors suggests that A C h is involved in several sensory pathways and in central information processing in the bee brain as described for other arthropods 3'8'9.
1 Breer, H. and Sattelle, D.B., Molecular properties and functions of insect acetylcholine receptors, J. Insect. Physiol., 33 (1987) 771-790. 2 Dudai, Y. and Amsterdam, A., Nicotinic receptors in the brain of Drosophila melanogaster demonstrated by autoradiography with [125I]a-bungarotoxin, Brain Research, 130 (1977) 551-555.
3 Eldefrawi, A.T. and Eldefrawi, M.E., Acetylcholine. In G.G. Lunt and R.W. Olsen (Eds.), Comparative Invertebrate Neurochemistry, Croom Helm, London, 1988, pp. 1-41. 4 Hildebrand, J.G., Hall, L.M. and Osmond, B.C., Distribution of binding sites for 12SI-labeled a-bungarotoxin in normal and deafferented antennal lobes of Manduca sexta, Proc. Natl. Acad.
Supported by the Deutsche Forschungsgemeinschaft (Er 79/3).
335
Sci. U.S.A., 76 (1979) 499-503. 5 Kreissl, S. and Bicker, G., Histochemistry of acetyicholinesterase and immunocytochemistry of an acetylcholine receptor-like antigen in the brain of the honeybee, J. Comp. Neurol., 286 (1989) 71-84. 6 Mobbs, P.G., The brain of the honeybee Apis mellifera. I. The connections and spatial organization of the mushroom bodies, Phil. Trans. R. Soc. Lond. B, 298 (1982) 309-354. 7 Mobbs, P.G., Brain structure. In G.A. Kerkut and L.I. Gilbert (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 5, Pergamon Press, Oxford, 1985, pp. 299-370.
8 Sattelle, D.B., Acetyicholine receptors of insects. In M.J. Berridge, J.E. Treherne and V.B. Wigglesworth (Eds.), Advances in Insect Physiology, Vol. 15, Academic Press, London, 1980, pp. 215-315. 9 Sattelle, D.B., Acetylcholine receptors. In G.A. Kerkut and L.J. Gilbert (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 11, Pergamon Press, Oxford, 1985, pp. 395-434. 10 Schmidt-Nielsen, B.K., Gepner, J.I., Teng, N.N.H. and Hail, L.M., Characterization of an a-bungarotoxin binding component from Drosophila melanogaster, J. Neurochem., 29 (1977) 1013-1029.
