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Distribution of visinin-like protein (VILIP) immunoreactivity in the hippocampus of the Mongolian gerbil (Meriones unguiculatus)
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N[UROSCIENC[ LETTHS
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ELSEVIER
Neuroscience Letters 206 (1996) 133-136
Distribution of visinin-like protein (VILIP) immunoreactivity in the hippocarnpus of the Mongolian gerbil (Meriones unguiculatus) Stefan E. Lenz, Werner Zuschratter, Eckart D. Gundelfinger* Federal Institute for Neurobiology, P.O. Box 1860, 39008 Magdeburg, Germany
Received 10 January 1996; revised version received 7 February 1996; accepted 7 February 1996
Abstract
Visinin-like protein (V~LIP) is a neuronal EF-hand Ca2÷-binding protein. In the chick brain, it is widely expressed, e.g. in neurons of the visual pathway and the cerebellum. In the cerebellum, a presynaptic localization of VILIP in glutamatergic parallel- and climbing-fiber terminals has been observed. Here, we describe the distribution of immunoreactivity (IR) detected by antibodies against chick VILIP in the gerbil hippocampus at the light and electron microscopic level. VILIP antibodies stain neurons in the whole hippocampal formation including pyramidal cells in the CA1 and CA3 region of the Ammon's horn and granule cells of the dentate gyrus. In CA3 neurons, VILIP-IR is localized in dendrites and dendritic spines. Keywords: Visinin-like protein (VILIP); Ca2÷-binding protein; Neuronal expression; Dendrite; Synapse; Hippocampus; Immunohistochemistry; Mongolian gerbil
Visinin-like protein (VILIP) is a member of the visinin/recoverin family e f EF-hand Ca2÷-binding proteins found in different neuronal subsets of all parts of the chick central nervous system [3,6,7]. Most prominent expression is observod in the tecto-fugal and tectothalamic visual pathways and the cerebellum [7]. V I L I P is extremely conserved among vertebrates. For example, neural visinin.-like protein 1 (NVP-1) [5], its rat homolog, is 100% identical, indicating that the protein has crucial functions in neurons. In vitro, VILIP mimics the effect of the retinal proteins S-modulin and recoverin on rhodopsin phosphorylation, suggesting a role in the regulation of G-protein-coupled receptors [2]. Frequenin, a Drosophila homolog of VILIP, can facilitate synaptic transmission at the neuromuscular junction [9]. VILIPimmunoreactivity (IR) has been found in axon terminals of parallel- and climbing-fibers [7] suggesting that VILIP, like frequenin, could be involved in the modulation of synaptic functions. The mammalian hippocampus is considered as a key structure in distinct forms of learning (e.g. [8]). The Mongolian gerbil is an established animal model for ischemia and learning behaviour [1,13]. As many of the * Corresponding author. Tel.: +49 391 6263228; fax: +49 391 6263229; e-mail: gundelfinge.'[email protected].
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Fig. 1. Immunoblot analysis of gerbil VILIP. Affinity-purifiedantibodies raised against chick VILIP were tested on Western blots of 5/~g GST-VILIP (lane 1) and 50/~g of soluble protein fractions (100 000 × g supernatants) of chick (lane 2) and gerbil (lane 3) brain homogenates [7]. Immunoreactivitywas visualized using the AMPPDSystem (Oncogene Science, Cambridge, MA). Sizes, in kDa, of two marker proteins are indicated.
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII: S0304-3940(96) 12444-5
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m o l e c u l a r m e c h a n i s m s underlying neuronal function and plasticity are Ca2+-dependent [4], it is o f challenging importance to establish the distribution o f Ca2+-binding proteins in the hippocampus. For the detection o f gerbil VILIP, affinity-purified an-
tibodies raised against r e c o m b i n a n t c h i c k V I L I P were used [7]. On Western blots the antibodies r e c o g n i z e glutathione S-transferase ( G S T ) - V I L I P fusion protein, and single i m m u n o r e a c t i v e bands of 25 k D a in crude cytoplasmic fractions of chick and gerbil brain h o m o g e n a t e s
Fig. 2. Distribution of VILIP-like IR in the gerbil hippocampus. Photomicrographs of horizontal vibratome sections are shown. (A) General view of IR in the hippocampus; neurons and neuropil in the CA1 and CA3 regions, the dentate gyrus (DG) and the hilus (h) are immunostalned. (B) VILIP-IR in granule cells and the adjacent neuropil of the DG. (C) Distribution of gerbil VILIP in hilus neurons and neuropil. (D) VILIP-IR in the pyramidal cell layer (Py) and the adjacent stratum radiatum (Sr) of the CA 1 region. (E) VILIP-IR in the CA3 region. Note the unstained regions between the intensely stained proximal pyramidal cell dendrites. Arrows point to neurons with strongly immunoreactive nuclei, white arrowheads to neurons with unstained nuclei. (F) High magnification of CA3 pyramidal cells. The asterisk marks an unstained cell. Size bars: (A) 0.5 mm; (B,C,E) 100/tm; (D,F) 25/am.
S.E. Lenz et al. / Neuroscience Letters 206 (1996) 133-136
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(Fig. 1). The antibodies do not cross-react with Drosophila frequenin, rat NCS-1 and rat NVP-3 which are 53, 57 and 69% identical with VILIP, respectively [7], nor with hippocalcin which shares 67% identity with VILIP (see below). Immunohistochemistry and electron microscopy were performed as described[ previously [12]. Generally, immunoreaction product is localized exclusively in cells which by shape, neuritic organization and localization are identifiable as neurons. In all hippocampal regions, including the Ammon's horn and the dentate gyrus, cell somata are strongly immunoreactive (Fig. 2A). In many cases nuclear staining L,; observed, whereas in other cases cell nuclei are devoid of VILIP-IR. This is consistent with the distribution of VILIP observed in neurons of the chick brain. In the dentate gyrus, most granule cells are immunoreactive (Fig. 2B). A punctate distribution of IR is found in the neuropil on either side of the granule cell layer. The hilus contains large, intensely stained neurons with immunoreactive processes (Fig. 2C). The lightly stained hilar neuropil is traversed by a network of immunopositive neurites. Within the neuropil unstained cells appear as white holes (Fig. 2C). In the CA1 region, pyramidal cells with relatively small perikarya show intense staining (Fig. 2A,D). They have strongly stained long and barely arborized dendrites running into the stratum radiatum. Between these dendrites an evenly distributed punctate staining is observed (Fiig. 2D). The majority of CA3 pyramidal cells are also intensely stained (Fig. 2E,F). Their immunoreactive dendrites arborize in the stratum radiaturn (Fig. 2E). The area between the cell bodies and the arborizing processes is essentially unstained. Interspersed in the pyramidal layer some small and completely unstained cells can be detected (Fig. 2F). IRs in the stratum radiatum and the stratum lacunosum moleculare of the CA3 region are stronger than those of CA1 (Fig. 2A). The strong punctate IR in CA3 is due to a strong postsynaptic expression of VILIP at mossy fiber synapses on CA3 pyramidal cell dendrites (compare Fig. 3). The more stringy distribution of VILIP-IR in CA1 appears to represent protein localized in dendrites of CA1 pyramidal cells contacted by Schaffer collateral fibers. Towards the dentate gyrus the stained cell layer of the Ammon's horn becomes broader (Fig. 2A); only a subpopulation of these cells is stained. Immunoreactive neurons of this region have intensely stained neurites (not shown). At the ultrastructural level synaptic structures of the CA3 region were analyzed. VILIP-IR can be detected in dendritic and postsynaptic structures. Both putatively excitatory shaft (Fig. 3A) and spine synapses (Fig. 3B) are stained. Axon terminals are devoid of IR (Fig. 3A,B). Immunoreaction product is detected throughout the dendrites. It appears, however, enriched in subsynaptic areas. This is in contrast to the situation in the chick cerebellum where we have found glutamatergic VILIP-positive nerve
Fig. 3. Ultrastructural localization of VILIP-IR in the CA3 region. Electron microscopic micrographs of ultrathin sections of gerbil hippoeampal region CA3. (A) Axon terminal (AT) making synaptic contact with a dendritic shaft of a pyramidalcell (D). Arrowheadspoint to imrnunoreaction product facing the presynaptic terminal. (B) Axon terminal (AT) making synaptic contact with a dendritic spine (s) of a cross-sectioned dendrite (D). Immunoreactionproduct in the spine is indicated by arrowheads. Size bars: (A) 0.4/zm; (B) 0.3 gin. terminals faced to VILIP-negative Purkinje cell dendrites [7]. The expression pattern of the IR detected by an affinity-purified antibody against chick VILIP in the hippocampus of the gerbil most likely reflects an immunoreaction with a homolog very similar to VILIP/NVP-1 [5,6]. This is concluded from a comparison of the specific transcript distribution detected by in situ hybridization and the distribution of IR in the rat hippocampus (unpublished observation). NVP-2, a protein closely related to VILIP, is also expressed in the hippocampus [10]. In rats, NVP-2 expression is high in neurons of CA1 and the dentate gyrus and low in CA3. This is in contrast to VILIP expression which is very strong in CA3. Some cross-reaction of VILIP antisera with NVP-2 can, however, not be excluded. Hippocalcin is another member of the visinin/recoverin family that is strongly expressed in the hippocampus [ 11 ]. It has a distribution very similar to VILIP. A cross-reaction of VILIP antibodies with hippocalcin is very unlikely, as the IRs of two the proteins
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display a complementary distribution in the cerebellum. While hippocalcin is exclusively expressed in Purkinje cells [11], VILIP does not occur in these cells in chick, rat and gerbil ([7], and unpublished data). The localization of VILIP in synaptic structures, both in postsynapses, e. g., pyramidal neurons of the CA3 region, and in presynapses, as shown for glutamatergic terminals in the cerebellum, suggests an involvement of VILIP in the modulation of Ca2+-dependent synaptic processes. This is reminiscent of Drosophila frequenin which is implicated in synaptic facilitation [9]. The distribution of both VILIP and frequenin is, however, not restricted to synaptic structures, suggesting that these proteins may have additional roles in the regulation of Ca 2+dependent processes in neurons. We thank Anja Bolz for excellent technical assistance. Supported by the Deutsche Forschungsgemeinschaft, the BMBF and the Fonds der Chemischen Industrie. [1] Araki, H., Nojiri, M., Kimura, M., and Aihara, H., Effects of minaprimine on 'delayed neuronal death' in Mongolian gerbils with occluded common carotid arteries, J. Pharmacol. Exp. Ther., 242 (1987) 686-691. [2] De Castro, E., Nef, S., Fiumelli, H., Lenz, S.E., Kawamura, S., and Nef, P., Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors, Biochem. Biophys. Res. Commun., 216 (1995) 133-140. [3] De Raad, S., Comte, M., Nef, P., Lenz, S.E., Gundelfinger, E.D., and Cox, J.A., Distribution pattern of three neural calciumbinding proteins - NCS-1, VILIP and recoverin - in chicken, bovine and rat retinas, Histochem. J., 27 (1995) 524-535. [4] Kennedy, M., Regulation of neuronal function by calcium, Trends
Neurosci., 12 (1989) 417--420. 15] Kuno, T., Kajimoto, Y., Hashimoto, T., Mukai, H., Shirai, Y., Saheki, S., and Tanaka, C., cDNA cloning of a neural visinin-like Ca2+-binding protein, Biochem. Biophys. Res. Commun., 184 (1992) 1219-1225. [6] Lenz, S.E., Henschel, Y., Zopf, D., Voss, B., and Gundelfinger, E.D., VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain, Mol. Brain Res., 15 (1992) 133-140. [7] Lenz, S.E., Jiang, S., Braun, K., and Gundelfinger, E.D., Localization of the neural calcium-binding protein VILIP (visinin-like protein) in neurons of the chick visual system and cerebellum, Cell Tissue Res., (1995) in press. [8] Matthies, H.J., In search of cellular mechanisms of memory, Prog. Neurobiol., 32 (1989) 277-349. [9] Pongs, O., Lindemeier, J., Zhu, X.R., Theil, T., Engelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K.W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J., Canal, I., Barbas, J.A., and Ferrus, A., Frequenin, a novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system, Neuron, 11 (1993) 15-28. [10] Saitoh, S., Takamatsu, K., Kobayashi, M., and Noguchi, T., Immunohistochemical localization of neural visinin-like Ca 2+binding protein 2 in adult rat brain, Neurosci. Lett., 171 (1994) 155-158. [11] Saitoh, S., Takamatsu, K., Kobayashi, M., and Nogushi, T., Distribution of hippocalcin mRNA and immunoreactivity in rat brain, Neurosci. Lett., 157 (1993) 107-110. [12] Seidel, B., Zuschratter, W., Wex, H., Garner, C.C., and Gundelfinger, E.D., Spatial and sub-cellular localization of the membrane cytoskeleton-associated protein alpha-adducin in the rat brain, Brain Res., 700 (1995) 13-24. [13] Yamamoto, M., Takahashi, K., Ohyama, M., Yamaguchi, T., Saitoh, S., Yatsugi, S., and Kogure, K., Behavioral and histological changes after repeated brief ischemia by carotid artery occlusion in gerbils, Brain Res., 608 (1993) 16-20.
,'
N[UROSCIENC[ LETTHS
i 'J
ELSEVIER
Neuroscience Letters 206 (1996) 133-136
Distribution of visinin-like protein (VILIP) immunoreactivity in the hippocarnpus of the Mongolian gerbil (Meriones unguiculatus) Stefan E. Lenz, Werner Zuschratter, Eckart D. Gundelfinger* Federal Institute for Neurobiology, P.O. Box 1860, 39008 Magdeburg, Germany
Received 10 January 1996; revised version received 7 February 1996; accepted 7 February 1996
Abstract
Visinin-like protein (V~LIP) is a neuronal EF-hand Ca2÷-binding protein. In the chick brain, it is widely expressed, e.g. in neurons of the visual pathway and the cerebellum. In the cerebellum, a presynaptic localization of VILIP in glutamatergic parallel- and climbing-fiber terminals has been observed. Here, we describe the distribution of immunoreactivity (IR) detected by antibodies against chick VILIP in the gerbil hippocampus at the light and electron microscopic level. VILIP antibodies stain neurons in the whole hippocampal formation including pyramidal cells in the CA1 and CA3 region of the Ammon's horn and granule cells of the dentate gyrus. In CA3 neurons, VILIP-IR is localized in dendrites and dendritic spines. Keywords: Visinin-like protein (VILIP); Ca2÷-binding protein; Neuronal expression; Dendrite; Synapse; Hippocampus; Immunohistochemistry; Mongolian gerbil
Visinin-like protein (VILIP) is a member of the visinin/recoverin family e f EF-hand Ca2÷-binding proteins found in different neuronal subsets of all parts of the chick central nervous system [3,6,7]. Most prominent expression is observod in the tecto-fugal and tectothalamic visual pathways and the cerebellum [7]. V I L I P is extremely conserved among vertebrates. For example, neural visinin.-like protein 1 (NVP-1) [5], its rat homolog, is 100% identical, indicating that the protein has crucial functions in neurons. In vitro, VILIP mimics the effect of the retinal proteins S-modulin and recoverin on rhodopsin phosphorylation, suggesting a role in the regulation of G-protein-coupled receptors [2]. Frequenin, a Drosophila homolog of VILIP, can facilitate synaptic transmission at the neuromuscular junction [9]. VILIPimmunoreactivity (IR) has been found in axon terminals of parallel- and climbing-fibers [7] suggesting that VILIP, like frequenin, could be involved in the modulation of synaptic functions. The mammalian hippocampus is considered as a key structure in distinct forms of learning (e.g. [8]). The Mongolian gerbil is an established animal model for ischemia and learning behaviour [1,13]. As many of the * Corresponding author. Tel.: +49 391 6263228; fax: +49 391 6263229; e-mail: gundelfinge.'[email protected].
123
43--
18--
Fig. 1. Immunoblot analysis of gerbil VILIP. Affinity-purifiedantibodies raised against chick VILIP were tested on Western blots of 5/~g GST-VILIP (lane 1) and 50/~g of soluble protein fractions (100 000 × g supernatants) of chick (lane 2) and gerbil (lane 3) brain homogenates [7]. Immunoreactivitywas visualized using the AMPPDSystem (Oncogene Science, Cambridge, MA). Sizes, in kDa, of two marker proteins are indicated.
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII: S0304-3940(96) 12444-5
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s.E. Lenz et al. / Neuroscience Leners 206 (1996) 133-136
m o l e c u l a r m e c h a n i s m s underlying neuronal function and plasticity are Ca2+-dependent [4], it is o f challenging importance to establish the distribution o f Ca2+-binding proteins in the hippocampus. For the detection o f gerbil VILIP, affinity-purified an-
tibodies raised against r e c o m b i n a n t c h i c k V I L I P were used [7]. On Western blots the antibodies r e c o g n i z e glutathione S-transferase ( G S T ) - V I L I P fusion protein, and single i m m u n o r e a c t i v e bands of 25 k D a in crude cytoplasmic fractions of chick and gerbil brain h o m o g e n a t e s
Fig. 2. Distribution of VILIP-like IR in the gerbil hippocampus. Photomicrographs of horizontal vibratome sections are shown. (A) General view of IR in the hippocampus; neurons and neuropil in the CA1 and CA3 regions, the dentate gyrus (DG) and the hilus (h) are immunostalned. (B) VILIP-IR in granule cells and the adjacent neuropil of the DG. (C) Distribution of gerbil VILIP in hilus neurons and neuropil. (D) VILIP-IR in the pyramidal cell layer (Py) and the adjacent stratum radiatum (Sr) of the CA 1 region. (E) VILIP-IR in the CA3 region. Note the unstained regions between the intensely stained proximal pyramidal cell dendrites. Arrows point to neurons with strongly immunoreactive nuclei, white arrowheads to neurons with unstained nuclei. (F) High magnification of CA3 pyramidal cells. The asterisk marks an unstained cell. Size bars: (A) 0.5 mm; (B,C,E) 100/tm; (D,F) 25/am.
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(Fig. 1). The antibodies do not cross-react with Drosophila frequenin, rat NCS-1 and rat NVP-3 which are 53, 57 and 69% identical with VILIP, respectively [7], nor with hippocalcin which shares 67% identity with VILIP (see below). Immunohistochemistry and electron microscopy were performed as described[ previously [12]. Generally, immunoreaction product is localized exclusively in cells which by shape, neuritic organization and localization are identifiable as neurons. In all hippocampal regions, including the Ammon's horn and the dentate gyrus, cell somata are strongly immunoreactive (Fig. 2A). In many cases nuclear staining L,; observed, whereas in other cases cell nuclei are devoid of VILIP-IR. This is consistent with the distribution of VILIP observed in neurons of the chick brain. In the dentate gyrus, most granule cells are immunoreactive (Fig. 2B). A punctate distribution of IR is found in the neuropil on either side of the granule cell layer. The hilus contains large, intensely stained neurons with immunoreactive processes (Fig. 2C). The lightly stained hilar neuropil is traversed by a network of immunopositive neurites. Within the neuropil unstained cells appear as white holes (Fig. 2C). In the CA1 region, pyramidal cells with relatively small perikarya show intense staining (Fig. 2A,D). They have strongly stained long and barely arborized dendrites running into the stratum radiatum. Between these dendrites an evenly distributed punctate staining is observed (Fiig. 2D). The majority of CA3 pyramidal cells are also intensely stained (Fig. 2E,F). Their immunoreactive dendrites arborize in the stratum radiaturn (Fig. 2E). The area between the cell bodies and the arborizing processes is essentially unstained. Interspersed in the pyramidal layer some small and completely unstained cells can be detected (Fig. 2F). IRs in the stratum radiatum and the stratum lacunosum moleculare of the CA3 region are stronger than those of CA1 (Fig. 2A). The strong punctate IR in CA3 is due to a strong postsynaptic expression of VILIP at mossy fiber synapses on CA3 pyramidal cell dendrites (compare Fig. 3). The more stringy distribution of VILIP-IR in CA1 appears to represent protein localized in dendrites of CA1 pyramidal cells contacted by Schaffer collateral fibers. Towards the dentate gyrus the stained cell layer of the Ammon's horn becomes broader (Fig. 2A); only a subpopulation of these cells is stained. Immunoreactive neurons of this region have intensely stained neurites (not shown). At the ultrastructural level synaptic structures of the CA3 region were analyzed. VILIP-IR can be detected in dendritic and postsynaptic structures. Both putatively excitatory shaft (Fig. 3A) and spine synapses (Fig. 3B) are stained. Axon terminals are devoid of IR (Fig. 3A,B). Immunoreaction product is detected throughout the dendrites. It appears, however, enriched in subsynaptic areas. This is in contrast to the situation in the chick cerebellum where we have found glutamatergic VILIP-positive nerve
Fig. 3. Ultrastructural localization of VILIP-IR in the CA3 region. Electron microscopic micrographs of ultrathin sections of gerbil hippoeampal region CA3. (A) Axon terminal (AT) making synaptic contact with a dendritic shaft of a pyramidalcell (D). Arrowheadspoint to imrnunoreaction product facing the presynaptic terminal. (B) Axon terminal (AT) making synaptic contact with a dendritic spine (s) of a cross-sectioned dendrite (D). Immunoreactionproduct in the spine is indicated by arrowheads. Size bars: (A) 0.4/zm; (B) 0.3 gin. terminals faced to VILIP-negative Purkinje cell dendrites [7]. The expression pattern of the IR detected by an affinity-purified antibody against chick VILIP in the hippocampus of the gerbil most likely reflects an immunoreaction with a homolog very similar to VILIP/NVP-1 [5,6]. This is concluded from a comparison of the specific transcript distribution detected by in situ hybridization and the distribution of IR in the rat hippocampus (unpublished observation). NVP-2, a protein closely related to VILIP, is also expressed in the hippocampus [10]. In rats, NVP-2 expression is high in neurons of CA1 and the dentate gyrus and low in CA3. This is in contrast to VILIP expression which is very strong in CA3. Some cross-reaction of VILIP antisera with NVP-2 can, however, not be excluded. Hippocalcin is another member of the visinin/recoverin family that is strongly expressed in the hippocampus [ 11 ]. It has a distribution very similar to VILIP. A cross-reaction of VILIP antibodies with hippocalcin is very unlikely, as the IRs of two the proteins
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display a complementary distribution in the cerebellum. While hippocalcin is exclusively expressed in Purkinje cells [11], VILIP does not occur in these cells in chick, rat and gerbil ([7], and unpublished data). The localization of VILIP in synaptic structures, both in postsynapses, e. g., pyramidal neurons of the CA3 region, and in presynapses, as shown for glutamatergic terminals in the cerebellum, suggests an involvement of VILIP in the modulation of Ca2+-dependent synaptic processes. This is reminiscent of Drosophila frequenin which is implicated in synaptic facilitation [9]. The distribution of both VILIP and frequenin is, however, not restricted to synaptic structures, suggesting that these proteins may have additional roles in the regulation of Ca 2+dependent processes in neurons. We thank Anja Bolz for excellent technical assistance. Supported by the Deutsche Forschungsgemeinschaft, the BMBF and the Fonds der Chemischen Industrie. [1] Araki, H., Nojiri, M., Kimura, M., and Aihara, H., Effects of minaprimine on 'delayed neuronal death' in Mongolian gerbils with occluded common carotid arteries, J. Pharmacol. Exp. Ther., 242 (1987) 686-691. [2] De Castro, E., Nef, S., Fiumelli, H., Lenz, S.E., Kawamura, S., and Nef, P., Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors, Biochem. Biophys. Res. Commun., 216 (1995) 133-140. [3] De Raad, S., Comte, M., Nef, P., Lenz, S.E., Gundelfinger, E.D., and Cox, J.A., Distribution pattern of three neural calciumbinding proteins - NCS-1, VILIP and recoverin - in chicken, bovine and rat retinas, Histochem. J., 27 (1995) 524-535. [4] Kennedy, M., Regulation of neuronal function by calcium, Trends
Neurosci., 12 (1989) 417--420. 15] Kuno, T., Kajimoto, Y., Hashimoto, T., Mukai, H., Shirai, Y., Saheki, S., and Tanaka, C., cDNA cloning of a neural visinin-like Ca2+-binding protein, Biochem. Biophys. Res. Commun., 184 (1992) 1219-1225. [6] Lenz, S.E., Henschel, Y., Zopf, D., Voss, B., and Gundelfinger, E.D., VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain, Mol. Brain Res., 15 (1992) 133-140. [7] Lenz, S.E., Jiang, S., Braun, K., and Gundelfinger, E.D., Localization of the neural calcium-binding protein VILIP (visinin-like protein) in neurons of the chick visual system and cerebellum, Cell Tissue Res., (1995) in press. [8] Matthies, H.J., In search of cellular mechanisms of memory, Prog. Neurobiol., 32 (1989) 277-349. [9] Pongs, O., Lindemeier, J., Zhu, X.R., Theil, T., Engelkamp, D., Krah-Jentgens, I., Lambrecht, H.-G., Koch, K.W., Schwemer, J., Rivosecchi, R., Mallart, A., Galceran, J., Canal, I., Barbas, J.A., and Ferrus, A., Frequenin, a novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system, Neuron, 11 (1993) 15-28. [10] Saitoh, S., Takamatsu, K., Kobayashi, M., and Noguchi, T., Immunohistochemical localization of neural visinin-like Ca 2+binding protein 2 in adult rat brain, Neurosci. Lett., 171 (1994) 155-158. [11] Saitoh, S., Takamatsu, K., Kobayashi, M., and Nogushi, T., Distribution of hippocalcin mRNA and immunoreactivity in rat brain, Neurosci. Lett., 157 (1993) 107-110. [12] Seidel, B., Zuschratter, W., Wex, H., Garner, C.C., and Gundelfinger, E.D., Spatial and sub-cellular localization of the membrane cytoskeleton-associated protein alpha-adducin in the rat brain, Brain Res., 700 (1995) 13-24. [13] Yamamoto, M., Takahashi, K., Ohyama, M., Yamaguchi, T., Saitoh, S., Yatsugi, S., and Kogure, K., Behavioral and histological changes after repeated brief ischemia by carotid artery occlusion in gerbils, Brain Res., 608 (1993) 16-20.