- Email: [email protected]
Synaptic connections of a type of cone bipolar cell in the inner plexiform layer of the rabbit retina
Friday, 5:30-7:00 P.M., Sep 25, 1992 Palazzo Dei Congressi
X ICER Abstracts 840
16 BINDING OF THIORIDAZINE AND ITS HETABOLITES TO PHOTORECEPTOR RETINOlD BINDING PROTEIN (IREPI Grant. K.R. and Converse. C.A. Dept. of Pharmaceutical Sciences, University Strathclyde. Glasgow, Scotland.
INTER-
IS TH-
MPI f. Himforschung, Deutschordenstr. Dpto. de Anatomia y Embtiologia, E-26040 Madrid, Spain (J.G.-s.)
of
IRBP is a high HU glycoprotein located in the interphotoreceptor matrix (IPH) and thought to be involved in retinoid movement during the visual CYCle. IRBP has been shorn to bind a variety of I igands and it is postulated that it may also bind drug substances. Such binding interfere with IREP’s ability to bind may endogenous ligands.
opsin
is expressed
develping
(BrdU) in cells that
mice
injected
with
IN FiODENT RETINAE ?
46, W-6000 Frenkfwl 71, Germany (L.P& Fat. de Veterinaria, Universidad Complotense,
LY injections of a large number of horizontal cells revealed A- end B-type cells in guinea pig, but only B-type cells in rat end gerbil. LY injected tissue wes counterstained with aNF. In guinea pig, identified A-type cells end AT% of B-type cells were aNF-positive. In rat and gerbil, no A-type cells were revealed, but identified B-AT% were aNF-positive. Newofilement immunoreactii of EATS’s has not been described before. Previous observations of aNF-positive processes in the outer plexiform layer of rodents have been interpreted as indicating A-type horizontal cells. We show that this is a misinterpretation. a&BP stained the Aand B-type populations in guinea pig, but only a homogeneous regular mosaic of cells with B-type features in rat and moose. Additional Nissl staining showed that &sBP stained all horizontal cells present. Our results suggest that only one type of horizontal cell, the axon-bearing B-type, is present in ret, gerbil end mouse retina. The guinea pig, on the other hand, contains both A- and B-type cells. This would support a recent claim that guinea pigs are not rodents (Grew et al., Netwe [Supported by a grant of the D.G.I.C.Y.T., Spain, to J.G.-S.] 351: 649, 1991).
THE REGULATION OF THE MOUSE GENE ENCODING INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN. G. I. Lieu Department of Ophthalmology, Medical College of Georgia, Augusta, GA Interphotoreceptor retinoid-binding protein (IRBP) is a retinal photoreceptorand pinealocyte-specific lipo-glycoprotein that is involved in the visual process. The timing of IRBP and opsin expression during photoreceptor development was studied by antigen detection using mono-clonal antibodies with double-labeling immune-fluorescence from
CELL
Msmmalisn retinae generally contain two morpMogical types of horizontal cell. lhe A-type has a larger, more sparsely branched dendritic tree end no axon. lho Btype has a smaller, mom densely branched dendrftic tree and an axon with en axon terminal system (Al’s). We studiid horizontal cells of four rodent species, rat, gerbil, mouse and guinea pig, using intracellular injections of Lucifer Yellow (Lv) end immv nohistochemistry with antibodies against neurofilaments (aNfl and against s calcium binding protein (arCaBP).
Bovine IRBP was prepared according to the method of AlHahdari and colleagues and its ability to bind retinal assessed by fluorimetry. The binding of thioridarine to IRBP was investigated by equilibrium dialysis. 1RBP (lo-6tl) was equilibrated at 4*C against thioridazine in phosphate buffered saline at varying concentrations and the amount of free drug assessed by absorbance. There “as no apparent binding but high drug binding to the dialysis membrane could have obscured low affinity binding. IRBP was also incubated with thioridazine and three of its q etabolites (mesoridazine, sulforidkzine. and its ring sulfoxidal and the interaction monitored by both absorbance and fluorescence. The preliminary of energy transfer between ligand and observation I RBP is indicative of direct binding to IRBP. Ue are grateful to Ltd. for the gift of Sandoz thioridarine and its metabolites. Al-Hahdawi, S. et al, (1990). J Clin Lab Immunol, 32: 21
on retina cells isolated analog bromodeoxy-uridine
ONLY ONE TYPE OF HOFUZONTAL
SYNAPTIC CONNECTIONS OF A ‘lWE OF CONE BIPOLAR CELL IN THE lNNER PLEXIFORM LAYER OF THE RABBIT KEWINA. Strettoi. & Dacheux, R.F.. Kaviola E. Istituto di Neurofisiologia de1 CNR, Pisa, Ital , and Department of Anatomy and Cellular Biology, Harvard Medical B hool, Boston, USA.
thymidine
. It was found from these mice that are post mitotic whereas IRBP is
expressed during or immediately after the last S phase of the photoreceptor precursor cells (Wang et al, Invest Ophth & Vis Sci, Suppl, 1992). This suggests that IRBP is implicated in the differentiation of the photoreceptor cells. The regulation of the IRBP gene has been studied in trsnsgenic mice harboring human IRBP gene promoter conjugated with either chloramphenicol acetyl-trsnsfersse
(CAT) nene or lac-Z enc. reporter gene expression
A uhotoreceotor-soecific was demonstrated
reeulation of the in t&agenic mice harboring a 1.3 kb upstream fragment of the human IRBP gene, and the position of this tissue-specific &-element was defined by CAT assays to be within 212 bp upstream fragment of the IRBP gene. Further defining of the cis-element within 150 bp upstream fragment is being conducted in transgenic mice using RNase protection and PCR analyses. An expression-correlated, tissue-specific hypomethylation of both the endogenous gene and the human IRBP-CAT tra&gene has been demonstrated in trsnsnenic mice (Lieu et al. Invest Ouhth & Vis Sci. Suppl, 1992). In these genes thd CpG island associa&d with tissue: specific hypomethylation included the upstream 212 bn. -rSupported by EY03829 and RPB, Inc
15
flanking
region
within
presynaptic
to any amacrine
MO3011
and EY01344).
839
AN EARLY DECREASE IN IRBP GENE EXPRESSION IN ABYSSlNlAN CATS HOM,OZYGOUS FOR PRG$RJ?WE TAL f?OPHY B. . Lee. L.‘. Ku@. G. . N&&on. J. . Si. J S. . CJ&J. G.J.‘, w ‘NM, Bethesda, MD USA, *Emory University, Atlanta, GA USA, 3University of Agricultural Sciences, Uppsala, Sweden.
COLOR PATHWAYS IN THE INNER PLEXIFORM Ammermtiller, J.. Kolb. H.. Normann, R.A.
LAYER OF THE TURTLE RETINA.
University City, USA
University
Oldenburg.
Oldenburg.
FRG.
and
of Utah,
Salt
Lake
Wavelength dependent responses of neurons contributing to the inner retina of the turtle Pseudemys scriptal were studied by intracellular recording and staining techniques. We found color coding in bipolar cells (BCs). amacrine cells (ACs) and ganglion cells (GCs). BCs were simple color-opponent and of three types: red-ON/green-OFF red-ON/blue-OFF and red-OFF/green-ON. A type similar to 83 was the only one to be recovered morphologically. Five different AC types were either simpleopponent or double-opponent. Al4 responded in ON-OFF fashion and showed differential efficiancy at light-ON and light-OFF to blue and red respectively. Al ard A3 were red-OFF/blue-ON. A32 was red-OFF/ green-ON, while A33 was the reverse color type i.e. red-ON/green-OFF. The latter two types were double-opponent with the opposite color responses to their surrounds. Color coded GCs consisted of an incompletely stained, and thus unknown morphological type which was red-ON/
By 8-12 weeks of age, Abyssinian cats homozygous for the progressive retinal atrophy gene can be differentiated from normal cats by electroretinography (ERG) (Narfstr6m et al., 1988), and by age 5 weeks, electron microsoopic studies show alterations in the retinas of affected animals (Narfstr6m and Nilsson, 1989). In the present study, immunocbemical quantitation of JRBP in retinas from young cats demons&at& levels of IRBP in normal retinas at 10 weeks and 17 months to be approximately 1 ~8 IRBP/mg toIal soluble protein. In contrast, IRBP levels in affected cat retinas were only 0.22-0.53 ~8 lRBP/mg protein at 4-6 weeks and 0.12-0.23 pg at 14-17 months. When nor&era blots of RNA isolated from normal and affectad cat retinas were. probed using a 3.5 kb human IRBP cDNA probe @IRBP 18-3500) (Si et al., 1989), two bands, one at 6.6 kb (minor) and another at 4.6 kb (major) were observed. As assessed by B-scanning, the 4.6 kb band was reduced by 4049% and the 6.6 kb band by 20% in 4 week old affected cat retinas as compared with normal 4 week and 10 week old retinas. There was no difference in 18s RNA and 110reduction in opsin message in affected retinas. The reduction ia IRBP gene expression in affected cat retinas to levels
green-OFF,
and
G3 and
G18 types.
The latter
were
color-coded
surrounds being red-ON/blue-OFF and red-OFF/green-ON, A double-opponent GC classified as a G6 type has been viously (Muller et al., 19911. Single opponent GCs could either single opponent BPS and ACs while double-opponent need double-opponent ACs providing input in the turtle
significantly this could
below normal as early as 4 weeks of age raises the possibility that represent a primary defect in the disease at the molecular level. ‘Supported by National Retinitis Pigmentosa Foundation 5Supported by Swedish Medical Research Council (Proj. No. B92-19X-09938-01)
Supported
S.244
by DFG Am
70
to J A
and
in the -
respectively. reported prebe driven by GCs might retina. EY04855
to
H.K
X ICER Abstracts 840
16 BINDING OF THIORIDAZINE AND ITS HETABOLITES TO PHOTORECEPTOR RETINOlD BINDING PROTEIN (IREPI Grant. K.R. and Converse. C.A. Dept. of Pharmaceutical Sciences, University Strathclyde. Glasgow, Scotland.
INTER-
IS TH-
MPI f. Himforschung, Deutschordenstr. Dpto. de Anatomia y Embtiologia, E-26040 Madrid, Spain (J.G.-s.)
of
IRBP is a high HU glycoprotein located in the interphotoreceptor matrix (IPH) and thought to be involved in retinoid movement during the visual CYCle. IRBP has been shorn to bind a variety of I igands and it is postulated that it may also bind drug substances. Such binding interfere with IREP’s ability to bind may endogenous ligands.
opsin
is expressed
develping
(BrdU) in cells that
mice
injected
with
IN FiODENT RETINAE ?
46, W-6000 Frenkfwl 71, Germany (L.P& Fat. de Veterinaria, Universidad Complotense,
LY injections of a large number of horizontal cells revealed A- end B-type cells in guinea pig, but only B-type cells in rat end gerbil. LY injected tissue wes counterstained with aNF. In guinea pig, identified A-type cells end AT% of B-type cells were aNF-positive. In rat and gerbil, no A-type cells were revealed, but identified B-AT% were aNF-positive. Newofilement immunoreactii of EATS’s has not been described before. Previous observations of aNF-positive processes in the outer plexiform layer of rodents have been interpreted as indicating A-type horizontal cells. We show that this is a misinterpretation. a&BP stained the Aand B-type populations in guinea pig, but only a homogeneous regular mosaic of cells with B-type features in rat and moose. Additional Nissl staining showed that &sBP stained all horizontal cells present. Our results suggest that only one type of horizontal cell, the axon-bearing B-type, is present in ret, gerbil end mouse retina. The guinea pig, on the other hand, contains both A- and B-type cells. This would support a recent claim that guinea pigs are not rodents (Grew et al., Netwe [Supported by a grant of the D.G.I.C.Y.T., Spain, to J.G.-S.] 351: 649, 1991).
THE REGULATION OF THE MOUSE GENE ENCODING INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN. G. I. Lieu Department of Ophthalmology, Medical College of Georgia, Augusta, GA Interphotoreceptor retinoid-binding protein (IRBP) is a retinal photoreceptorand pinealocyte-specific lipo-glycoprotein that is involved in the visual process. The timing of IRBP and opsin expression during photoreceptor development was studied by antigen detection using mono-clonal antibodies with double-labeling immune-fluorescence from
CELL
Msmmalisn retinae generally contain two morpMogical types of horizontal cell. lhe A-type has a larger, more sparsely branched dendritic tree end no axon. lho Btype has a smaller, mom densely branched dendrftic tree and an axon with en axon terminal system (Al’s). We studiid horizontal cells of four rodent species, rat, gerbil, mouse and guinea pig, using intracellular injections of Lucifer Yellow (Lv) end immv nohistochemistry with antibodies against neurofilaments (aNfl and against s calcium binding protein (arCaBP).
Bovine IRBP was prepared according to the method of AlHahdari and colleagues and its ability to bind retinal assessed by fluorimetry. The binding of thioridarine to IRBP was investigated by equilibrium dialysis. 1RBP (lo-6tl) was equilibrated at 4*C against thioridazine in phosphate buffered saline at varying concentrations and the amount of free drug assessed by absorbance. There “as no apparent binding but high drug binding to the dialysis membrane could have obscured low affinity binding. IRBP was also incubated with thioridazine and three of its q etabolites (mesoridazine, sulforidkzine. and its ring sulfoxidal and the interaction monitored by both absorbance and fluorescence. The preliminary of energy transfer between ligand and observation I RBP is indicative of direct binding to IRBP. Ue are grateful to Ltd. for the gift of Sandoz thioridarine and its metabolites. Al-Hahdawi, S. et al, (1990). J Clin Lab Immunol, 32: 21
on retina cells isolated analog bromodeoxy-uridine
ONLY ONE TYPE OF HOFUZONTAL
SYNAPTIC CONNECTIONS OF A ‘lWE OF CONE BIPOLAR CELL IN THE lNNER PLEXIFORM LAYER OF THE RABBIT KEWINA. Strettoi. & Dacheux, R.F.. Kaviola E. Istituto di Neurofisiologia de1 CNR, Pisa, Ital , and Department of Anatomy and Cellular Biology, Harvard Medical B hool, Boston, USA.
thymidine
. It was found from these mice that are post mitotic whereas IRBP is
expressed during or immediately after the last S phase of the photoreceptor precursor cells (Wang et al, Invest Ophth & Vis Sci, Suppl, 1992). This suggests that IRBP is implicated in the differentiation of the photoreceptor cells. The regulation of the IRBP gene has been studied in trsnsgenic mice harboring human IRBP gene promoter conjugated with either chloramphenicol acetyl-trsnsfersse
(CAT) nene or lac-Z enc. reporter gene expression
A uhotoreceotor-soecific was demonstrated
reeulation of the in t&agenic mice harboring a 1.3 kb upstream fragment of the human IRBP gene, and the position of this tissue-specific &-element was defined by CAT assays to be within 212 bp upstream fragment of the IRBP gene. Further defining of the cis-element within 150 bp upstream fragment is being conducted in transgenic mice using RNase protection and PCR analyses. An expression-correlated, tissue-specific hypomethylation of both the endogenous gene and the human IRBP-CAT tra&gene has been demonstrated in trsnsnenic mice (Lieu et al. Invest Ouhth & Vis Sci. Suppl, 1992). In these genes thd CpG island associa&d with tissue: specific hypomethylation included the upstream 212 bn. -rSupported by EY03829 and RPB, Inc
15
flanking
region
within
presynaptic
to any amacrine
MO3011
and EY01344).
839
AN EARLY DECREASE IN IRBP GENE EXPRESSION IN ABYSSlNlAN CATS HOM,OZYGOUS FOR PRG$RJ?WE TAL f?OPHY B. . Lee. L.‘. Ku@. G. . N&&on. J. . Si. J S. . CJ&J. G.J.‘, w ‘NM, Bethesda, MD USA, *Emory University, Atlanta, GA USA, 3University of Agricultural Sciences, Uppsala, Sweden.
COLOR PATHWAYS IN THE INNER PLEXIFORM Ammermtiller, J.. Kolb. H.. Normann, R.A.
LAYER OF THE TURTLE RETINA.
University City, USA
University
Oldenburg.
Oldenburg.
FRG.
and
of Utah,
Salt
Lake
Wavelength dependent responses of neurons contributing to the inner retina of the turtle Pseudemys scriptal were studied by intracellular recording and staining techniques. We found color coding in bipolar cells (BCs). amacrine cells (ACs) and ganglion cells (GCs). BCs were simple color-opponent and of three types: red-ON/green-OFF red-ON/blue-OFF and red-OFF/green-ON. A type similar to 83 was the only one to be recovered morphologically. Five different AC types were either simpleopponent or double-opponent. Al4 responded in ON-OFF fashion and showed differential efficiancy at light-ON and light-OFF to blue and red respectively. Al ard A3 were red-OFF/blue-ON. A32 was red-OFF/ green-ON, while A33 was the reverse color type i.e. red-ON/green-OFF. The latter two types were double-opponent with the opposite color responses to their surrounds. Color coded GCs consisted of an incompletely stained, and thus unknown morphological type which was red-ON/
By 8-12 weeks of age, Abyssinian cats homozygous for the progressive retinal atrophy gene can be differentiated from normal cats by electroretinography (ERG) (Narfstr6m et al., 1988), and by age 5 weeks, electron microsoopic studies show alterations in the retinas of affected animals (Narfstr6m and Nilsson, 1989). In the present study, immunocbemical quantitation of JRBP in retinas from young cats demons&at& levels of IRBP in normal retinas at 10 weeks and 17 months to be approximately 1 ~8 IRBP/mg toIal soluble protein. In contrast, IRBP levels in affected cat retinas were only 0.22-0.53 ~8 lRBP/mg protein at 4-6 weeks and 0.12-0.23 pg at 14-17 months. When nor&era blots of RNA isolated from normal and affectad cat retinas were. probed using a 3.5 kb human IRBP cDNA probe @IRBP 18-3500) (Si et al., 1989), two bands, one at 6.6 kb (minor) and another at 4.6 kb (major) were observed. As assessed by B-scanning, the 4.6 kb band was reduced by 4049% and the 6.6 kb band by 20% in 4 week old affected cat retinas as compared with normal 4 week and 10 week old retinas. There was no difference in 18s RNA and 110reduction in opsin message in affected retinas. The reduction ia IRBP gene expression in affected cat retinas to levels
green-OFF,
and
G3 and
G18 types.
The latter
were
color-coded
surrounds being red-ON/blue-OFF and red-OFF/green-ON, A double-opponent GC classified as a G6 type has been viously (Muller et al., 19911. Single opponent GCs could either single opponent BPS and ACs while double-opponent need double-opponent ACs providing input in the turtle
significantly this could
below normal as early as 4 weeks of age raises the possibility that represent a primary defect in the disease at the molecular level. ‘Supported by National Retinitis Pigmentosa Foundation 5Supported by Swedish Medical Research Council (Proj. No. B92-19X-09938-01)
Supported
S.244
by DFG Am
70
to J A
and
in the -
respectively. reported prebe driven by GCs might retina. EY04855
to
H.K