339
Brain Research, 597 (1992) 339-342 © 1992 Elsevier Science Publishers B,V. All!rights reserved 0006-8993/92/$05.00
BRES 25451
Sarcoplasmic calcium-binding protein-immunoreactive material in the central nervous system of the snail, Helixpomatia H u b e r t H. K e r s c h b a u m , V e r e n a K a i n z a n d A n t o n H e r m a n n University of Salzburg, Institute of Zoology, Depa.~,mentof Physiology, Salzburg (Austria) (Accepted 8 September 1992)
Key words: Sarcoplasmic calcium-binding protein; Parvalbumin; Serotonin; Neuron; Helixpomatia; Mollusca
In Heli¢pomatia a 10 kDa sarcoplasmic calcium binding protein I (SCP l)-immunoreactive material was localized immunologicallyin neurons of the central nervous system, including the electrically silent serotonergic metacerebral giant neuron. Antisera against sareoplasmic calcium binding protein iI (SCP Ii) stained a 20 kDa protein in individual muscle cells of the epineurium. SCP- and parvalbumin (PV)-immunoreactive material were not co-localized.
Sarcoplasmic calcium binding proteins (SCPs), soluble sarcoplasmic proteins with high affinity for Ca 2+, were originally isolated from crayfish muscle. These proteins have been identified in muscles of several invertebrates, but not of vertebrates s'~9. SCPs are believed to transport Ca 2+ from the myofibrilles to the sarcoplasmic reticulum during the relaxation phase of the muscle contraction s. The two isoforms, SCP ! and SCP II, originally isolated from amphioxus muscle, differ from each other in 7 amino acid substitutions~. In vertebrates parvalbumins (PVs) have been identified immunologically, as well as on the basis of their amino acid and nucleic acid sequences6. SCP and PV are members of the EF.hand proteins characterized by a Ca2+-binding loop flanked by two helices. SCP and PV are suggested to have a similar function in Ca 2+activated processes s'tg. Due to the structural and functional similarities and the unique distribution of SCP and PV in the animal kingdom, Wnuk et ai. Is postulated that PVs evolved from an invertebrate SCP stem. PV is assumed to be involved in discharge activity of GABAergic neurons in mammals by buffering free Ca 2+4. A correlation between SCP and electrical discharge activity has not been reported. Furthermore, a neurotransmitter co-localized with SCP is not known. Recently, PV-like material, characterized immuno-
logically by cross-reactivity with antisera against vertebrate parvalbumin, has been described in the nervous system of invertebrates 3'7'~°. In the present study, we examined the distribution of SCP-like proteins in the central nervous system of Helix pomatia, the relationship between SCP- and PV-immunoreactive material, the possible co-existence of SCP-like protein(s) with serotonin, and the electrical behaviour of SCP-immunoreactive neurons. Animals. Helix pomatia, purchased from a commercial source (Stein, Lauingen, Germany), were kept either in a terrarium and fed with lettuce, or in a refrigerator hibernating at 4°C. Antisera. Polyclonal rabbit antisera to amphioxus SCP 1 and SCP II were a gift from Prof. Cox (Geneve), polyclonal antiserum to frog PV (Sigma) was raised in rabbits, and serotonin antiserum was purchased from Chemicon, USA. lmmunocytochemistry. Circumesophageal ganglia and buccal ganglia were removed from the animal, fixed in Bouin's solution or in a solution of 1% paraformaldehyde and 0.25% glutaraldehyde, washed in PBS (phosphate buffered solution, pH 7.4), dehydrated in a graded series of ethanol and embedded in paraplast using benzene as an intermedium. Sections (7 ~m) were mounted on slides coated with chrome-alum
Correspondence: H.H. Kerschbaum, University of Salzburg, Department of Physiolo~, Institute of Zoology, Hellbrunnerstr. 34, A-5020 Salzburg, Austria. Fax: (43) (662) 8044-5698.
340
Mr xlOa 5 -
i
39: iii~!
10-
la
lb
Fig. 1. lmmunoblots after one-dimensionalgel electrophoresis of Helix brain homogenates,a: immunoreactiveSCP I; b: immunoreactire SCP II. gelatin (0.5% gelatin and 0.005% chrome-alum in aqueous solution). For immunostaining the peroxidase anti-peroxidase method was used 14. Sections were incubated in normal goat serum for 30 rain, followed by
incubation with the primary antibody (1 : 800) for 24 h. Subsequeiltly, sections were rinsed 3 x 5 min in PBS, incubated in goat anti-rabbit antiserum for 30 rain, washed 3 x 5 min in PBS and incubated in horseradish peroxidase-conjugated antibody. Peroxidase was visualized with diaminobenzidine (Aldrich) and H202. For control purposes sections were incubated with antiserum preabsorbed with 1 /zM of the respective compound for 24 h at 4°C or were incubated with normal serum. Tissue extracts. Ganglia were dissected out from the animal, frozen in liquid nitrogen and pulverized under liquid nitrogen in a motar. The pulver was suspended in extraction buffer (4 mM EDTA disodium salt (ethylendiaminetetraacetic acid) buffer containing 100 mM pepstatin A, 1.5 mM TPCK (N-tosyl-t.-phenyl alanyl chlormethan ketone), 10 mM PMSF (phenyl methylsulfonylfluoride), 10/~M leupeptin, 100 ~tM trasylol; pH 7.0) 2'15 and homogenized. The suspension was sonicated twice for 10 s and stored on ice for 1 h. After centrifugation (8,000 x g; 15 min) the pellet was resuspended in extraction buffer, sonicated and centrifuged as described. The pooled supernatants were heated for
Fig. 2. Consecutivesectionsthroughthe cerebral gangliain the regionof the metacerebralgiant neuron, a: localizationof SCP I-like materialin the cytoplasmof the metacerebral giant neuron ( x 760). b: adjacent section to (a) revealed no PV staining in the cytoplasmof the giant metacerebral neuron ( x 760).
341 30 min at 85°C and centrifuged (8,000 × g, 15 min). Immunoblotting. Extracts were diluted 1 : 1 with sample buffer (100 mM DDT, 5% SDS, 10% glycine, 0.06 M Tris; pH 6.8) and heated for 5 min at 95°C. Proteins were separated on a SDS-polyacrylamide gel (7.5%20%) H and transferred onto a nitrocellulose membrane 17 at constant voltage of 40 V for 2 h. Proteins were identified immunologically ts. Electrophysiology. Isolated ganglia were mounted in a chamber perfused with Helve saline (80 mM NaCl, 4 mM KCI, 10 mM CaCl 2, 5 mM MgCl, 5 mM Tris-HCl, 10 mM sucrose; pH 7.5). After application of protease XIV (Sigma) for 5-10 min the connective tissue was removed using fine scissors. Membrane potential was recorded between an intracellular electrode filled with 3 M KCI (8 M ~ ) and a reference KCl-agar electrode outside the cell. Western blot analysis. Antisera against SCP l and SCP II recognized two different proteins in the brain homogenate of Helix pomatia. Anti-SCP II stained a protein at 20 kDa (Fig. lb) whereas anti-SCP I indentifled a protein at 10 kDa (Fig. la).
Immunocytochemistry. SCP l-positive neurons were restricted to the cerebral, pedal and bueeal ganglia, and were not found in the pleural, parietal and visceral ganglia. In the cerebral ganglia the metacerebral giant neurons, identified by their location, size and electrical behaviour, exhibited a SCP I-positive reaction product (6 animals; Fig. 2A). Antisera against SCP I labelled the cytoplasm but not the nucleoplasm of neurons. In addition, both ~erotonin- and SCP I-immunoreactive material w e s ~ o u n d on adjacent sections of the metacerebral giant cell. Identzfiable giant cells of the subesophageal ganglia and buccal ganglia 9'12'~3 were SCP I negative. Antisera against SCP-II did not stain neurons. Antisera against SCP II labelled a population of muscle cells in the connective tissue enveloping the central nervous system. These cells were SCP I negative. Therefore, SCP ll-positive staining of ganglia using immunoblotting is likely to be due to the co-ectraction of the connective tissue surrounding the ganglia. SCP and PV are reported to have a species-dependent distribution 5'18. However, antisera against SCP .f'
,
•
•
Fig. 3. Consecutivesections through the cerebral gansiia (x 760). a: section treated with antisera agains SCP-|. b: neuron stained with antisera against PV (x 760).
342 and PV stained different populations of neurons, indicating SCP- and PV-like proteins in Helix pomatia (Figs. 2 and 3). Electrophysiology. Recordings of the membrane potential of giant metacerebrai neurons for about 1 h (n - 7) revealed that the metacerebral giant neurons were silent. Our immunological investigations indicate that there are at least two different SCPs in the snail, Helix pomatia. Antisera against amphioxus SCP I stained individual neurons in the central nervous system, including the metacerebral gian' r~euron, and identified a protein of at 10 kDa. SCP II antisera labelled a population of muscle cells within the connective tissue enveloping the brain and cross-reacted with a protein of about 20 kDa. SCP with a molecular weight ,,f approximately 20 kDa has also been found in muscle tissue of invertebrates, including sandworm, earthworm, clam, scallop, crayfish, shrimps, amphioxus and Aplysia californica 7"8:~. Since the first isolation of SCP from crayfish muscle ~, investigations on SCP have been focussed primarly on muscle tissue. However, SCP-immunoreactive material has also been found in the brain of Drosophila 3 and Aplysia c:,lifornica ~. Our results show that there are at least two different types of SCP-like materials specifically expressed in neurons and muscles, respectively. Wnuk et al. ~ postulated that PV evolved from an invertebrate SCP. Testing this hypothesis, we were able to show that antisera against SCP and PV labelled different populations of neurons. Furthermore, PV-like material in Helix pomatia has a molecular weight of 40 kDa (unpublished results), whereas SCP 1 and SCP II have molecular weights of 10 and 20 kDa, respectively. These findings are in agreement with those for the marine mollusc, Aplysia californica, where a PV-like material could be identified at 45 kDa and a SCP-like material at 23 kDa 7. Conclusions about the structural identity of PV- and SCP-like material in snails and their evolutionary relationship can only be drawn after elucidation of the amino acid or nucleic acid sequence in snails and other invertebrates. However, our findings point to the possibility that SCP is not the evolutionary precursor of PV and that a PV-like material already exists in invertebrates. In the nervous system of mammals PV is expressed in GABAergic neurons of high electrical activity 4. However, our findings show that SCP-I-like protein is expressed in electrically silent neurons containing the excitatory transmitter serotonin and thereby indicating
a further difference between PV- and SCP-containing neurons. This work was supported by the Austrian Forschungs-FiirderungsFonds, Projekt P 8050-Med. 1 Bcnzonana, G., Cox, J.A., Kohler, L.G. and Stein, E.A., Characerisation d'unde nouvelle metalloprot~inecalcique du myogene de certains crustaces, C.R. Hebd. Seances Adad. Sci.. 279 (1974) 1491-1493. 2 Berchtold, M.W.,Cello, M.R. and Heizmann,C.W., Parvalbumin in non-muscle tissue of the rat: quantitation and ~mmunohisto. chemical localization,J. Biol. (.;hem.,259 (1984) 51~9-5196. 3 Buchner, E., Bader, R., Buchner, S., Cox, J.A., Emson, P.C., FIory, E., Heizmann,C.W., Heroin, S., Hofbauer, A. and Oertel, W.H., Cell specific immuno-probes for the brain of normal and mutant Drosophila melanogastcr.I. Wild type system.Cell. Tissue Res., 253 (1988) 357-370. 4 Cello, M.R., Parvalbuminin most gamma-aminobutfficacid containing neurons of the rat cerebral cortex, Science, 231 (1986) 995 -997. 5 Cox, J'.,Unique calcium-binding-proteinsin invertebrates.In R. Pochet, D.E.M. Lawson, C.W. Heizmann (Eds.),CaMum-binding Proteins in Normal and Transfp~med Cells.Advances in ~tperimental Medicine and Biology, V~;" 23, Plenum Press,New York, 1990, pp 67-72. 6 Heizmann, C.W., Parvalbumin, an intracellularcalcium binding protein',propertiesand possible rolesin mammalian cells,Experientia, 40 (1984)'910-921. 7 Hermaan, A., Pauls,T.L. and Heizmann, C.W.. Calcium.binding proteins in Aplysia neurons, Cell. Mol. :Veurobiol., II (1991) 371-386. 8 Huch, R., Haese, J.D. and Gerdzy, Ch., A soluble calcium-binding protein from the terrestrialannelid Lumbricus terresMs L., J. Comp. Physiol.B. 158 (1988) 325-343. 9 Johansen, J., Jensen, L.H. and Holm, C., Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganlia of Helixpomatia, Comp. Biochem. Physiol., 71A (1982) 283-291. 10 Kerschbaum, H.H,, Holzinger, K and Hermann, A., Parvalbu.
rain-positive neurons in the brain of Helixpomatia, Gen. Comp. Endocrinol., 74 (1989) 319-320. 11 Laemmli, U.K., Cleavageof strucutral proteins during the assembly of the head of bacteria phage T4, Nature, 227 (1970) 680. 12 Schulze, H., Speckmann, E.J., Kuhlmann, D. and Caspers, H., Topography and bioelectrical properties of identifiable neurons in Helixpomatia, Neursct. Lett., 1 (1975)277-281, 13 Steffens, H., The buccal gangliaof Helixpomatia L. (Gastropoda, Pulmonata), Zoomorphologie, 95 (1980) 195-212 14 Sternbergcr, L.A., lmmanocytochemistry, John Wiley and Sons, New York, 1979. 15 Stichel, C.C., Kaegi,U. and Heizmann,C.W., Parvalbuminin rat brain: isolation,characterizationand localization,J. Neurochem., 47 (1986), 46-53. 16 Takagi, T. and Cox, J.A., Amino acid sequences ot four isoforms of amphioxus sarcoplasmic calcium-binding proteins, Fur. J. Biochem., 192 (1990) 387-399. 17 Towbin, H. and Gordon, J., lmmunoblotting and dot immnnobinding - current status and outlook, J. lmmunol. Methods, 72 (1984) 313. 18 Tsang, V.C.W., Pesalta, J.M. and Simon, A.R., Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies by ~el electrophorsis, Methods Enzymol., 92 (1983) 377. 19 Wnuk, W., Cox,J. and Stein, E.A. Parvalbuminand other soluble high-affinitycalcium-bindingproteins from muscle. In W. Cheung (Ed.), Calcium and Cell Function, Academic Press, 1982, pp. 243-278.
Brain Research, 597 (1992) 339-342 © 1992 Elsevier Science Publishers B,V. All!rights reserved 0006-8993/92/$05.00
BRES 25451
Sarcoplasmic calcium-binding protein-immunoreactive material in the central nervous system of the snail, Helixpomatia H u b e r t H. K e r s c h b a u m , V e r e n a K a i n z a n d A n t o n H e r m a n n University of Salzburg, Institute of Zoology, Depa.~,mentof Physiology, Salzburg (Austria) (Accepted 8 September 1992)
Key words: Sarcoplasmic calcium-binding protein; Parvalbumin; Serotonin; Neuron; Helixpomatia; Mollusca
In Heli¢pomatia a 10 kDa sarcoplasmic calcium binding protein I (SCP l)-immunoreactive material was localized immunologicallyin neurons of the central nervous system, including the electrically silent serotonergic metacerebral giant neuron. Antisera against sareoplasmic calcium binding protein iI (SCP Ii) stained a 20 kDa protein in individual muscle cells of the epineurium. SCP- and parvalbumin (PV)-immunoreactive material were not co-localized.
Sarcoplasmic calcium binding proteins (SCPs), soluble sarcoplasmic proteins with high affinity for Ca 2+, were originally isolated from crayfish muscle. These proteins have been identified in muscles of several invertebrates, but not of vertebrates s'~9. SCPs are believed to transport Ca 2+ from the myofibrilles to the sarcoplasmic reticulum during the relaxation phase of the muscle contraction s. The two isoforms, SCP ! and SCP II, originally isolated from amphioxus muscle, differ from each other in 7 amino acid substitutions~. In vertebrates parvalbumins (PVs) have been identified immunologically, as well as on the basis of their amino acid and nucleic acid sequences6. SCP and PV are members of the EF.hand proteins characterized by a Ca2+-binding loop flanked by two helices. SCP and PV are suggested to have a similar function in Ca 2+activated processes s'tg. Due to the structural and functional similarities and the unique distribution of SCP and PV in the animal kingdom, Wnuk et ai. Is postulated that PVs evolved from an invertebrate SCP stem. PV is assumed to be involved in discharge activity of GABAergic neurons in mammals by buffering free Ca 2+4. A correlation between SCP and electrical discharge activity has not been reported. Furthermore, a neurotransmitter co-localized with SCP is not known. Recently, PV-like material, characterized immuno-
logically by cross-reactivity with antisera against vertebrate parvalbumin, has been described in the nervous system of invertebrates 3'7'~°. In the present study, we examined the distribution of SCP-like proteins in the central nervous system of Helix pomatia, the relationship between SCP- and PV-immunoreactive material, the possible co-existence of SCP-like protein(s) with serotonin, and the electrical behaviour of SCP-immunoreactive neurons. Animals. Helix pomatia, purchased from a commercial source (Stein, Lauingen, Germany), were kept either in a terrarium and fed with lettuce, or in a refrigerator hibernating at 4°C. Antisera. Polyclonal rabbit antisera to amphioxus SCP 1 and SCP II were a gift from Prof. Cox (Geneve), polyclonal antiserum to frog PV (Sigma) was raised in rabbits, and serotonin antiserum was purchased from Chemicon, USA. lmmunocytochemistry. Circumesophageal ganglia and buccal ganglia were removed from the animal, fixed in Bouin's solution or in a solution of 1% paraformaldehyde and 0.25% glutaraldehyde, washed in PBS (phosphate buffered solution, pH 7.4), dehydrated in a graded series of ethanol and embedded in paraplast using benzene as an intermedium. Sections (7 ~m) were mounted on slides coated with chrome-alum
Correspondence: H.H. Kerschbaum, University of Salzburg, Department of Physiolo~, Institute of Zoology, Hellbrunnerstr. 34, A-5020 Salzburg, Austria. Fax: (43) (662) 8044-5698.
340
Mr xlOa 5 -
i
39: iii~!
10-
la
lb
Fig. 1. lmmunoblots after one-dimensionalgel electrophoresis of Helix brain homogenates,a: immunoreactiveSCP I; b: immunoreactire SCP II. gelatin (0.5% gelatin and 0.005% chrome-alum in aqueous solution). For immunostaining the peroxidase anti-peroxidase method was used 14. Sections were incubated in normal goat serum for 30 rain, followed by
incubation with the primary antibody (1 : 800) for 24 h. Subsequeiltly, sections were rinsed 3 x 5 min in PBS, incubated in goat anti-rabbit antiserum for 30 rain, washed 3 x 5 min in PBS and incubated in horseradish peroxidase-conjugated antibody. Peroxidase was visualized with diaminobenzidine (Aldrich) and H202. For control purposes sections were incubated with antiserum preabsorbed with 1 /zM of the respective compound for 24 h at 4°C or were incubated with normal serum. Tissue extracts. Ganglia were dissected out from the animal, frozen in liquid nitrogen and pulverized under liquid nitrogen in a motar. The pulver was suspended in extraction buffer (4 mM EDTA disodium salt (ethylendiaminetetraacetic acid) buffer containing 100 mM pepstatin A, 1.5 mM TPCK (N-tosyl-t.-phenyl alanyl chlormethan ketone), 10 mM PMSF (phenyl methylsulfonylfluoride), 10/~M leupeptin, 100 ~tM trasylol; pH 7.0) 2'15 and homogenized. The suspension was sonicated twice for 10 s and stored on ice for 1 h. After centrifugation (8,000 x g; 15 min) the pellet was resuspended in extraction buffer, sonicated and centrifuged as described. The pooled supernatants were heated for
Fig. 2. Consecutivesectionsthroughthe cerebral gangliain the regionof the metacerebralgiant neuron, a: localizationof SCP I-like materialin the cytoplasmof the metacerebral giant neuron ( x 760). b: adjacent section to (a) revealed no PV staining in the cytoplasmof the giant metacerebral neuron ( x 760).
341 30 min at 85°C and centrifuged (8,000 × g, 15 min). Immunoblotting. Extracts were diluted 1 : 1 with sample buffer (100 mM DDT, 5% SDS, 10% glycine, 0.06 M Tris; pH 6.8) and heated for 5 min at 95°C. Proteins were separated on a SDS-polyacrylamide gel (7.5%20%) H and transferred onto a nitrocellulose membrane 17 at constant voltage of 40 V for 2 h. Proteins were identified immunologically ts. Electrophysiology. Isolated ganglia were mounted in a chamber perfused with Helve saline (80 mM NaCl, 4 mM KCI, 10 mM CaCl 2, 5 mM MgCl, 5 mM Tris-HCl, 10 mM sucrose; pH 7.5). After application of protease XIV (Sigma) for 5-10 min the connective tissue was removed using fine scissors. Membrane potential was recorded between an intracellular electrode filled with 3 M KCI (8 M ~ ) and a reference KCl-agar electrode outside the cell. Western blot analysis. Antisera against SCP l and SCP II recognized two different proteins in the brain homogenate of Helix pomatia. Anti-SCP II stained a protein at 20 kDa (Fig. lb) whereas anti-SCP I indentifled a protein at 10 kDa (Fig. la).
Immunocytochemistry. SCP l-positive neurons were restricted to the cerebral, pedal and bueeal ganglia, and were not found in the pleural, parietal and visceral ganglia. In the cerebral ganglia the metacerebral giant neurons, identified by their location, size and electrical behaviour, exhibited a SCP I-positive reaction product (6 animals; Fig. 2A). Antisera against SCP I labelled the cytoplasm but not the nucleoplasm of neurons. In addition, both ~erotonin- and SCP I-immunoreactive material w e s ~ o u n d on adjacent sections of the metacerebral giant cell. Identzfiable giant cells of the subesophageal ganglia and buccal ganglia 9'12'~3 were SCP I negative. Antisera against SCP-II did not stain neurons. Antisera against SCP II labelled a population of muscle cells in the connective tissue enveloping the central nervous system. These cells were SCP I negative. Therefore, SCP ll-positive staining of ganglia using immunoblotting is likely to be due to the co-ectraction of the connective tissue surrounding the ganglia. SCP and PV are reported to have a species-dependent distribution 5'18. However, antisera against SCP .f'
,
•
•
Fig. 3. Consecutivesections through the cerebral gansiia (x 760). a: section treated with antisera agains SCP-|. b: neuron stained with antisera against PV (x 760).
342 and PV stained different populations of neurons, indicating SCP- and PV-like proteins in Helix pomatia (Figs. 2 and 3). Electrophysiology. Recordings of the membrane potential of giant metacerebrai neurons for about 1 h (n - 7) revealed that the metacerebral giant neurons were silent. Our immunological investigations indicate that there are at least two different SCPs in the snail, Helix pomatia. Antisera against amphioxus SCP I stained individual neurons in the central nervous system, including the metacerebral gian' r~euron, and identified a protein of at 10 kDa. SCP II antisera labelled a population of muscle cells within the connective tissue enveloping the brain and cross-reacted with a protein of about 20 kDa. SCP with a molecular weight ,,f approximately 20 kDa has also been found in muscle tissue of invertebrates, including sandworm, earthworm, clam, scallop, crayfish, shrimps, amphioxus and Aplysia californica 7"8:~. Since the first isolation of SCP from crayfish muscle ~, investigations on SCP have been focussed primarly on muscle tissue. However, SCP-immunoreactive material has also been found in the brain of Drosophila 3 and Aplysia c:,lifornica ~. Our results show that there are at least two different types of SCP-like materials specifically expressed in neurons and muscles, respectively. Wnuk et al. ~ postulated that PV evolved from an invertebrate SCP. Testing this hypothesis, we were able to show that antisera against SCP and PV labelled different populations of neurons. Furthermore, PV-like material in Helix pomatia has a molecular weight of 40 kDa (unpublished results), whereas SCP 1 and SCP II have molecular weights of 10 and 20 kDa, respectively. These findings are in agreement with those for the marine mollusc, Aplysia californica, where a PV-like material could be identified at 45 kDa and a SCP-like material at 23 kDa 7. Conclusions about the structural identity of PV- and SCP-like material in snails and their evolutionary relationship can only be drawn after elucidation of the amino acid or nucleic acid sequence in snails and other invertebrates. However, our findings point to the possibility that SCP is not the evolutionary precursor of PV and that a PV-like material already exists in invertebrates. In the nervous system of mammals PV is expressed in GABAergic neurons of high electrical activity 4. However, our findings show that SCP-I-like protein is expressed in electrically silent neurons containing the excitatory transmitter serotonin and thereby indicating
a further difference between PV- and SCP-containing neurons. This work was supported by the Austrian Forschungs-FiirderungsFonds, Projekt P 8050-Med. 1 Bcnzonana, G., Cox, J.A., Kohler, L.G. and Stein, E.A., Characerisation d'unde nouvelle metalloprot~inecalcique du myogene de certains crustaces, C.R. Hebd. Seances Adad. Sci.. 279 (1974) 1491-1493. 2 Berchtold, M.W.,Cello, M.R. and Heizmann,C.W., Parvalbumin in non-muscle tissue of the rat: quantitation and ~mmunohisto. chemical localization,J. Biol. (.;hem.,259 (1984) 51~9-5196. 3 Buchner, E., Bader, R., Buchner, S., Cox, J.A., Emson, P.C., FIory, E., Heizmann,C.W., Heroin, S., Hofbauer, A. and Oertel, W.H., Cell specific immuno-probes for the brain of normal and mutant Drosophila melanogastcr.I. Wild type system.Cell. Tissue Res., 253 (1988) 357-370. 4 Cello, M.R., Parvalbuminin most gamma-aminobutfficacid containing neurons of the rat cerebral cortex, Science, 231 (1986) 995 -997. 5 Cox, J'.,Unique calcium-binding-proteinsin invertebrates.In R. Pochet, D.E.M. Lawson, C.W. Heizmann (Eds.),CaMum-binding Proteins in Normal and Transfp~med Cells.Advances in ~tperimental Medicine and Biology, V~;" 23, Plenum Press,New York, 1990, pp 67-72. 6 Heizmann, C.W., Parvalbumin, an intracellularcalcium binding protein',propertiesand possible rolesin mammalian cells,Experientia, 40 (1984)'910-921. 7 Hermaan, A., Pauls,T.L. and Heizmann, C.W.. Calcium.binding proteins in Aplysia neurons, Cell. Mol. :Veurobiol., II (1991) 371-386. 8 Huch, R., Haese, J.D. and Gerdzy, Ch., A soluble calcium-binding protein from the terrestrialannelid Lumbricus terresMs L., J. Comp. Physiol.B. 158 (1988) 325-343. 9 Johansen, J., Jensen, L.H. and Holm, C., Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganlia of Helixpomatia, Comp. Biochem. Physiol., 71A (1982) 283-291. 10 Kerschbaum, H.H,, Holzinger, K and Hermann, A., Parvalbu.
rain-positive neurons in the brain of Helixpomatia, Gen. Comp. Endocrinol., 74 (1989) 319-320. 11 Laemmli, U.K., Cleavageof strucutral proteins during the assembly of the head of bacteria phage T4, Nature, 227 (1970) 680. 12 Schulze, H., Speckmann, E.J., Kuhlmann, D. and Caspers, H., Topography and bioelectrical properties of identifiable neurons in Helixpomatia, Neursct. Lett., 1 (1975)277-281, 13 Steffens, H., The buccal gangliaof Helixpomatia L. (Gastropoda, Pulmonata), Zoomorphologie, 95 (1980) 195-212 14 Sternbergcr, L.A., lmmanocytochemistry, John Wiley and Sons, New York, 1979. 15 Stichel, C.C., Kaegi,U. and Heizmann,C.W., Parvalbuminin rat brain: isolation,characterizationand localization,J. Neurochem., 47 (1986), 46-53. 16 Takagi, T. and Cox, J.A., Amino acid sequences ot four isoforms of amphioxus sarcoplasmic calcium-binding proteins, Fur. J. Biochem., 192 (1990) 387-399. 17 Towbin, H. and Gordon, J., lmmunoblotting and dot immnnobinding - current status and outlook, J. lmmunol. Methods, 72 (1984) 313. 18 Tsang, V.C.W., Pesalta, J.M. and Simon, A.R., Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies by ~el electrophorsis, Methods Enzymol., 92 (1983) 377. 19 Wnuk, W., Cox,J. and Stein, E.A. Parvalbuminand other soluble high-affinitycalcium-bindingproteins from muscle. In W. Cheung (Ed.), Calcium and Cell Function, Academic Press, 1982, pp. 243-278.
