Current Eye Research
SHORT COMMUNICATION Volume 11 number 7 1992, 71 1-715
Localization of vasoactive intestinal peptide (VIP) messenger RNA (mRNA) in amacrine cells of rat retina Shonosuke Okamoto, Hitoshi Okamura', Hirofumi Terubayashi, Yoshio Akagi, Hiroshi Okamoto2 and Yasuhiko Ibata'
Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
Departments of Ophthalmology and 'Anatomy, Kyoto Prefectural University of Medicine, Kyoto 602 and 2Department of Biochemistry, Tohoku University School of Medicine, Sendai 980, Miyagi, Japan
ABSTRACT We detected vasoactive intestinal peptide (VIP) messenger RNA (mRNA) i n t h e r a t retina using an i n s i t u hybridization technique and a 35S-labelled cDNA probe. VIP mRNA was present i n t h e cells of t h e inner nuclear layer (INL). The VIP mRNApositive cells showed a d i s t r i b u t i o n smilar t o that of t h e VIP-like immunoreactive amacrine cells. This observation suggests t h a t VIP mRNA undergoes transcription i n t h e V I P-immunoreactive amacri ne cells.
o f t h e r a t using an i n s i t u hybridization technique, and compared t h e f i n d i n g w i t h those determined b y immunocytochemistry o f VIP. Eight male Wistar r a t s (180-200 g) were housed under a daily l i g h t - d a r k cycle (12 hours l i g h t - 12 hours d a r k ) and given free access t o food and water. A f t e r inducing deep anesthesia w i t h sodium
The retina, an integral p a r t of t h e central
pentobarbital, t h e animals were sacrifaced at 12.00
nervous system, i s r i c h i n several kinds of
h - 14.00 h and perfused w i t h ice-cold 0.1 M
neuropeptides. Irnmunocytochemical analyses have
phosphate b u f f e r saline (pH 7.5), followed b y
shown the localization o f a v a r i e t y o f neuropeptides
perfusion w i t h 4 % paraformaldehyde i n 0.1 M
i n t h e amacrine cell of t h e r e t i n a (1-3). However
phosphate b u f f e r (PB) (pH 7.5). The eyes were
t h e i r functions as neurotransmitters or
immediatl y removed and t h e anterior segments were
neuromodulators have not been described.
resected. A l l procedure on animals were carried o u t
Vasoactive intestinal peptide (VIP), a 28-amino acid
in accordance with t h e Guiding Principles i n t h e
peptide t h a t was f i r s t isolated from the porcine
Care and use o f Animals (DHEW Publication, NIH
duodenum b y Said and Mutt in 1970 (4), i s widely
86-23).
d i s t r i b u t e d i n t h e central and peripheral nervous
For i n s i t u hybridization study, t h e retina was
systems (5). Considering t h e organs involved i n
carefully removed whole from t h e choroidal
vision, immunocytochemical studies f o r VIP had
membrane and was immersed i n 4 % para-
been reported in t h e visual cortex, t h e superior
formaldehyde in 0.1 M PB (pH 7.5) f o r 3 hours. Each
colliculus (6), hypothalamus as well as r e t i n a of t h e
, g/ml proteinase K r e t i n a was deproteinated i n 1.O u
r a t (7,8). Recently, t h e cDNA coding sequence of VIP
(Sigma Chemical Co., St. Louis, MO.) f o r 30 min and
has been clarified i n r a t s and humans (9) and an i n
was then delipidated in a graded alcohol series.
s i t u hybridization technique showed t h e
After prehybridization f o r 2 hours, t h e whole
localization of VIP mRNA i n t h e central and
retinas were incubated i n a hybridization solution
peripheral nervous systems. We attempted t o
consisting of formamide (50 % vol/vol), dextran
examine t h e localization of VIP mRNA i n t h e retina
sulfate (10 % vol/voi), 3 x standard saline c i t r a t e
Received on September 9, 1991; accepted on June 22, 1992
71 1
Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
Current Eye Research
Figure 1 VIP mRNA positive grains are seen i n the cell on t h e vitreous side of the inner nucleus layer (INL) (A). Labelled cells are spase in both t h e central and peripheral regions of t h e retina. Populations of positive cells are similar i n those regions. No positive grains are observed on t h e sections t h a t
are incubated with a pBR322 cDNA probe as a control (B). Bar = 50 prn. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: o u t e r plexiform layer, ONL: outer nuclear layer.
(SSC; 0.15 M NaCI, 0.015 M trisodium citrate), 1 x
pBR322 DNA was nick- translated and used as
Denhart's solution, 100 p g/ml yeast t-RNA, 100 p
described for t h e VIP probe. After
g/ml salmon sperm DNA, 10 mM DTT, 1.0 rnM EDTA,
posthybridization i n 0.5 x SSC f o r 2 hours, whole
and 8.7 x l o 5 dprn/ml of denatured nick-translated
retinas were dehydrated in a graded alcohol series
VIP cDNA probe at 42 "C for 20 hours. The
and then embedded in methacrylate (JB-4
prehybridization and hybridization conditions were
Embedding K i t , Polyscience, Inc. Warrington, PA).
previously described ( 1 0 , l l ) . The constriction and
Serni-thin sections ( 2-5 p m) were made on a
characterization of t h e cloning method have also
microtome and then mounted on gelatin-coated
been described (9). The cDNA containing
slides. The slides were dipped i n NTBP (Eastman
approximately 700 base pairs of t h e entire coding
Kodak Co., Rochester, NY) nuclear t r a c k emulsion
sequence f o r r a t VIP was labelled b y
(dilution, 1:l distilled water) and exposed f o r 2-4
nick-translation w i t h [35S]dATP (Amersham UK.)
weeks. The emulsion-coated slides were developed
and used as the hybridization probe. As a control,
with Kodak D19 and fixed. The tissue sections were
71 2
Current Eye Research and treated w i t h anti-VIP antisera (dilution 1 :5000) for 7 days. After being reacted w i t h anti-rabbit IgG (dilution 1:200) f o r 2 h, they were allowed t o react w i t h PAP solution (dilution 1:200) f o r 90 min and final Iy exposed t o 3,3'-diaminobenzidine substrate solution containing HzOz.A l l sera were diluted w i t h 0.1 M PBS (pH 7.5) containing 0.3% T r i t o n X-100. The Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
characterization o f VIP antisera has been described elsewhere (12). Control sections were treated w i t h t h e anti-VIP serum absorbed w i t h synthetic VIP (0.1 - 1.0 p g/rnl). No immunoreactive substances
were detected in t h e control sections. Visible well-concentrated grains which were considered t o be VIP mRNA-positive grains, were localized on t h e cells in vitreous side o f t h e i n n e r nuclear layer (Figs. l A , 2A). The positive cells were morphologically similar t o t h e s u r r o u n d i n g nonpositive cells i n t h e inner layer. No concentration o f t h e g r a i n s could be found in t h e intermediate and o u t e r sides of t h e inner nuclear Figure 2 Dark field autoradiograph showing a VIP mRNA positive cell i n the inner nuclear layer (A). A VIP-like immunoreactivity cell i s also located i n t h e inner side of that layer. VIP-like immunoreactive processes are present i n t h e inner plexiform layer (6). Bar = 50 y m.
layer as well as i n t h e other layers o f t h e retina. The number o f labelled VIP mRNA cells was small, being only a few cells per cross section. The population of labelled VIP mRNA cells was similar in
GCL: ganglion cell layer, IPL: inner plexiform
t h e central and peripheral regions o f t h e retina.
layer, INL: i n n e r nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer.
When hybridization was carried o u t w i t h a pBR322 cDNA probe as a control, No concentration o f t h e labelled grains on t h e cell was present (Fig.1 B). I n
then counterstained with toluidine blue and
t h e immunocytochemical studies, VIP
observed b y both b r i g h t - and dark-field l i g h t
irnmunoreactive cells were also found i n t h e
m icroscop y .
innermost side o f t h e inner nuclear layer where t h e
F o r immunoc ytoc hemical s t u d y , t h e eyecups
labelled VIP mRNA cells were d i s t r i b u t e d (Fig. 28).
dissected from t h e anterior segment were fixed with
These f i n d i n g suggest t h a t t h e labelled VIP mRNA
t h e same fixative containing 0.2 % picric acid f o r 6
cells are likely t o be amacrine cells t h a t synthesize
hours and were cryoprotected w i t h 20 % sucrose i n
VIP.
0.1 M PB. Frozen sections (30 y m t h i c k ) were made
The investigation o f VIP mRNA expression i n t h e
71 3
Current Eye Research CNS b y i n s i t u hybridization technique has
CORRESPONDING AUTHOR
reported previously (1 3-15) b u t there are no
Shonosuke Okamoto, M.D.
r e p o r t s o f t h e detection of VIP mRNA i n t h e r a t
Department o f Ophthalmology, Kyoto Prefectural
retina. I n the present study, we f i r s t detected VIP
U n i v e r s i t y o f Medicine, Kyoto 602, Japan.
mRNA positive cells. Since VIP immunoreactivity and
VIP mRNA levels were low compared with those o f VIP synthesizing cells i n t h e brain. We assume t h a t
Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
t h e VIP-containing cells i n t h e retina have low mRNA level and synthesize only a small amount of VIP. I n t h e CNS, t h e highest concentration of VIP i s found i n the suprachiasmatic nucleus (SCN) o f t h e hypothalamus. The VIP-containing neurons i n t h e SCN showed an increased immunoreactivity
following enucleation of the eye (16). Although t h e role o f retinal VIP is unknown, a recent investigation demonstrated an increased VIP immunoreactivity i n t h e retina o f t h e primate myopic eye (17). Another study showed that VIP induced increase i n intracellular concentration of cyclic AMP i n cultured glial cells o f t h e r a t r e t i n a (18). These observations suggest that VIP may play a role i n regulation o f ocular morphogenesis between t h e amacrine and glial cells of t h e dynamic changes i n VIP processing i s required. The i n s i t u hybridization technique can make an important contribution t o revealing the function of retinal VIP.
ACKNOWLEDGEMENT We thank Dr. Noboru Yanaihara f o r a k i n d g i f t o f t h e antiserum t o VIP (R.502). This work was supported i n p a r t b y a g r a n t (60480105, 01480112) from t h e Ministry o f Education, Science and Culture, Japan.
714
REFERENCES l.Brecha, N., Karten, H.J. and Laverack, C. (1979) Enkephalin-containing amacrine cells in t h e avian retina: imrnunohistochemical localization. Proc. Natl. Acad. Sci. U.S.A. 76, 3010-3014. 2.Karten, H.J. and Brecha, N. (1980) Localization o f substance P irnmunoreactivity in amacrine cells o f t h e retina. Nature 238, 87-88. 3.Brecha, N., Karten, H.J. and Schenker, C. (1981) Neurotensin-li ke and somatostatin-li k e immunoreactivity w i t h in amacrine cells o f t h e retina. Neuroscience 5, 1329-1 340. 4.Said, S.I. and Mutt, V. (1970) Polypeptide w i t h broad biological a c t i v i t y : isolation from small o , 1217-1218. intestine. Science N.Y. l S.Larsson, L.I., Fahrenkrug, J., Schaffalitzky de Muckadell, O.B., Sunder, F., Hakanson, R. and Rehfeld, J.F. (1 976) Localization o f vasoactive intestinal polypeptide (VIP) t o central and peripheral neurons. Proc. Natl. Acad. U.S.A. 73, 31 97-3200. 6.0kamot0, S., Okamura, H., Takahashi, Y., Akagi, Y., Yanaihara, N. and Ibata, Y. (1990) Contrary effect o f eye enucleation on VIP-immunoreactive neurons i n t h e suprachiasmatic nucleus and t h e superior colliculus of t h e rat. Neurosci. Lett. 112, 137-1 42. 7.Loren, I., Tornqvist, K. and Alumets, J. (1980) VIP (vasoactive intestinal polypeptide)immunoreactive neurons i n t h e retina o f t h e rat. Cell Tissue Res. 210, 167-1 70. 8.Terubayashi, H., Tsuto, T., Fukui, K., Obata, H.L., Okamura, H., Fujisawa, H., Itoi, M., Yanaihara, C., Yanaihara, N. and Ibata, Y. (1983) VIP (vasoactive intestinal polypeptide)-li ke immunoreactive amacrine cells in t h e r e t i n a of t h e rat. Exp. Eye Res. 2,143-749. S.Nishizawa, M., Hayakawa, Y., Yanaihara, N. and Okamoto, H. (1 985) Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett. 183,55-59. lO.Mary, L.P., Hames, B.D. and Higgins, S.J. (1985) I n S i t u Hybridization in Nucleic Acid Hybridization. Ppl79-202 IRL Press Oxfqrd, Washington DC. l l . U h r , G.R. (1986) I n Situ Hybridization i n Brain, Pp259-290 Plenum. N.Y. lZ.Yanaihara, N., Sakagami, M., Sato, H., Yamamoto, K., Hashimoto, T., Yanaihara, C., Ito, Z., Yamaguchi, K. and Abe, K. (1977) Immunological aspects of secretin, substance P, and VIP. Gastroenterology 72, 803-810. ld.Gozes, I., Avidor, R., Biegon, A. and Baldino, F.Jr.
Current Eye Research I
(1 989) Lactation elevates vasoactive intestinal peptide messenger ribonucleic acid i n r a t supra181-189. chiasmatic nucleus. Endocrinology 14.Stopa, E.G., Minamitani, N., Jonassen, J.A., King, J.C., Wolfe, H., Mobtaker, H. and Alber, H.E. (1988) Localization of vasoactive intestinal peptide and peptide histidine isoleucine irnmunoreactivity and mRNA within t h e r a t suprachiasmatic nucleus, Mol. Brain Res. 4, 319-325. 15.0kamot0, S., Okamura, H., Miyake, M., Takagi, S., Fukui, K., Akagi, Y., Okarnoto, H. and Ibata, Y. (1 991) A d i u r n a l variation o f vasoactive intestinal peptide (VIP) mRNA under a daily l i g h t - d a r k cycle in t h e r a t suprachiasmatic nucleus. Histochemistry. 95, 525-528. 16.0kamura, H., Takahashi, Y., Terubayashi, H., Hamada, S., Yanaihara, N. and Ibata, Y. (1987) VIP-like immunoreactive neurons and retinal projections i n t h e r a t suprachiasmatic nucleus, Biomed. Res. 8, 253-262. 17.Stone, R.A., Laties, A.M., Ravila, E. and Wiesel, T.N. (1988) Increase i n retinal vasoactive intestinal polypeptide after eyelid fusion i n primates. Proc. Natl. Sci. U.S.A. 85, 257-260. 18.Koh, SW.M. and Roberge, f.G. (1989) V I P modulation of c u l t u r e d glial cells of the r a t retina. C u r r . Eye Res. 8, 1207-1210.
Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
=,
71 5
SHORT COMMUNICATION Volume 11 number 7 1992, 71 1-715
Localization of vasoactive intestinal peptide (VIP) messenger RNA (mRNA) in amacrine cells of rat retina Shonosuke Okamoto, Hitoshi Okamura', Hirofumi Terubayashi, Yoshio Akagi, Hiroshi Okamoto2 and Yasuhiko Ibata'
Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
Departments of Ophthalmology and 'Anatomy, Kyoto Prefectural University of Medicine, Kyoto 602 and 2Department of Biochemistry, Tohoku University School of Medicine, Sendai 980, Miyagi, Japan
ABSTRACT We detected vasoactive intestinal peptide (VIP) messenger RNA (mRNA) i n t h e r a t retina using an i n s i t u hybridization technique and a 35S-labelled cDNA probe. VIP mRNA was present i n t h e cells of t h e inner nuclear layer (INL). The VIP mRNApositive cells showed a d i s t r i b u t i o n smilar t o that of t h e VIP-like immunoreactive amacrine cells. This observation suggests t h a t VIP mRNA undergoes transcription i n t h e V I P-immunoreactive amacri ne cells.
o f t h e r a t using an i n s i t u hybridization technique, and compared t h e f i n d i n g w i t h those determined b y immunocytochemistry o f VIP. Eight male Wistar r a t s (180-200 g) were housed under a daily l i g h t - d a r k cycle (12 hours l i g h t - 12 hours d a r k ) and given free access t o food and water. A f t e r inducing deep anesthesia w i t h sodium
The retina, an integral p a r t of t h e central
pentobarbital, t h e animals were sacrifaced at 12.00
nervous system, i s r i c h i n several kinds of
h - 14.00 h and perfused w i t h ice-cold 0.1 M
neuropeptides. Irnmunocytochemical analyses have
phosphate b u f f e r saline (pH 7.5), followed b y
shown the localization o f a v a r i e t y o f neuropeptides
perfusion w i t h 4 % paraformaldehyde i n 0.1 M
i n t h e amacrine cell of t h e r e t i n a (1-3). However
phosphate b u f f e r (PB) (pH 7.5). The eyes were
t h e i r functions as neurotransmitters or
immediatl y removed and t h e anterior segments were
neuromodulators have not been described.
resected. A l l procedure on animals were carried o u t
Vasoactive intestinal peptide (VIP), a 28-amino acid
in accordance with t h e Guiding Principles i n t h e
peptide t h a t was f i r s t isolated from the porcine
Care and use o f Animals (DHEW Publication, NIH
duodenum b y Said and Mutt in 1970 (4), i s widely
86-23).
d i s t r i b u t e d i n t h e central and peripheral nervous
For i n s i t u hybridization study, t h e retina was
systems (5). Considering t h e organs involved i n
carefully removed whole from t h e choroidal
vision, immunocytochemical studies f o r VIP had
membrane and was immersed i n 4 % para-
been reported in t h e visual cortex, t h e superior
formaldehyde in 0.1 M PB (pH 7.5) f o r 3 hours. Each
colliculus (6), hypothalamus as well as r e t i n a of t h e
, g/ml proteinase K r e t i n a was deproteinated i n 1.O u
r a t (7,8). Recently, t h e cDNA coding sequence of VIP
(Sigma Chemical Co., St. Louis, MO.) f o r 30 min and
has been clarified i n r a t s and humans (9) and an i n
was then delipidated in a graded alcohol series.
s i t u hybridization technique showed t h e
After prehybridization f o r 2 hours, t h e whole
localization of VIP mRNA i n t h e central and
retinas were incubated i n a hybridization solution
peripheral nervous systems. We attempted t o
consisting of formamide (50 % vol/vol), dextran
examine t h e localization of VIP mRNA i n t h e retina
sulfate (10 % vol/voi), 3 x standard saline c i t r a t e
Received on September 9, 1991; accepted on June 22, 1992
71 1
Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
Current Eye Research
Figure 1 VIP mRNA positive grains are seen i n the cell on t h e vitreous side of the inner nucleus layer (INL) (A). Labelled cells are spase in both t h e central and peripheral regions of t h e retina. Populations of positive cells are similar i n those regions. No positive grains are observed on t h e sections t h a t
are incubated with a pBR322 cDNA probe as a control (B). Bar = 50 prn. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: o u t e r plexiform layer, ONL: outer nuclear layer.
(SSC; 0.15 M NaCI, 0.015 M trisodium citrate), 1 x
pBR322 DNA was nick- translated and used as
Denhart's solution, 100 p g/ml yeast t-RNA, 100 p
described for t h e VIP probe. After
g/ml salmon sperm DNA, 10 mM DTT, 1.0 rnM EDTA,
posthybridization i n 0.5 x SSC f o r 2 hours, whole
and 8.7 x l o 5 dprn/ml of denatured nick-translated
retinas were dehydrated in a graded alcohol series
VIP cDNA probe at 42 "C for 20 hours. The
and then embedded in methacrylate (JB-4
prehybridization and hybridization conditions were
Embedding K i t , Polyscience, Inc. Warrington, PA).
previously described ( 1 0 , l l ) . The constriction and
Serni-thin sections ( 2-5 p m) were made on a
characterization of t h e cloning method have also
microtome and then mounted on gelatin-coated
been described (9). The cDNA containing
slides. The slides were dipped i n NTBP (Eastman
approximately 700 base pairs of t h e entire coding
Kodak Co., Rochester, NY) nuclear t r a c k emulsion
sequence f o r r a t VIP was labelled b y
(dilution, 1:l distilled water) and exposed f o r 2-4
nick-translation w i t h [35S]dATP (Amersham UK.)
weeks. The emulsion-coated slides were developed
and used as the hybridization probe. As a control,
with Kodak D19 and fixed. The tissue sections were
71 2
Current Eye Research and treated w i t h anti-VIP antisera (dilution 1 :5000) for 7 days. After being reacted w i t h anti-rabbit IgG (dilution 1:200) f o r 2 h, they were allowed t o react w i t h PAP solution (dilution 1:200) f o r 90 min and final Iy exposed t o 3,3'-diaminobenzidine substrate solution containing HzOz.A l l sera were diluted w i t h 0.1 M PBS (pH 7.5) containing 0.3% T r i t o n X-100. The Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
characterization o f VIP antisera has been described elsewhere (12). Control sections were treated w i t h t h e anti-VIP serum absorbed w i t h synthetic VIP (0.1 - 1.0 p g/rnl). No immunoreactive substances
were detected in t h e control sections. Visible well-concentrated grains which were considered t o be VIP mRNA-positive grains, were localized on t h e cells in vitreous side o f t h e i n n e r nuclear layer (Figs. l A , 2A). The positive cells were morphologically similar t o t h e s u r r o u n d i n g nonpositive cells i n t h e inner layer. No concentration o f t h e g r a i n s could be found in t h e intermediate and o u t e r sides of t h e inner nuclear Figure 2 Dark field autoradiograph showing a VIP mRNA positive cell i n the inner nuclear layer (A). A VIP-like immunoreactivity cell i s also located i n t h e inner side of that layer. VIP-like immunoreactive processes are present i n t h e inner plexiform layer (6). Bar = 50 y m.
layer as well as i n t h e other layers o f t h e retina. The number o f labelled VIP mRNA cells was small, being only a few cells per cross section. The population of labelled VIP mRNA cells was similar in
GCL: ganglion cell layer, IPL: inner plexiform
t h e central and peripheral regions o f t h e retina.
layer, INL: i n n e r nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer.
When hybridization was carried o u t w i t h a pBR322 cDNA probe as a control, No concentration o f t h e labelled grains on t h e cell was present (Fig.1 B). I n
then counterstained with toluidine blue and
t h e immunocytochemical studies, VIP
observed b y both b r i g h t - and dark-field l i g h t
irnmunoreactive cells were also found i n t h e
m icroscop y .
innermost side o f t h e inner nuclear layer where t h e
F o r immunoc ytoc hemical s t u d y , t h e eyecups
labelled VIP mRNA cells were d i s t r i b u t e d (Fig. 28).
dissected from t h e anterior segment were fixed with
These f i n d i n g suggest t h a t t h e labelled VIP mRNA
t h e same fixative containing 0.2 % picric acid f o r 6
cells are likely t o be amacrine cells t h a t synthesize
hours and were cryoprotected w i t h 20 % sucrose i n
VIP.
0.1 M PB. Frozen sections (30 y m t h i c k ) were made
The investigation o f VIP mRNA expression i n t h e
71 3
Current Eye Research CNS b y i n s i t u hybridization technique has
CORRESPONDING AUTHOR
reported previously (1 3-15) b u t there are no
Shonosuke Okamoto, M.D.
r e p o r t s o f t h e detection of VIP mRNA i n t h e r a t
Department o f Ophthalmology, Kyoto Prefectural
retina. I n the present study, we f i r s t detected VIP
U n i v e r s i t y o f Medicine, Kyoto 602, Japan.
mRNA positive cells. Since VIP immunoreactivity and
VIP mRNA levels were low compared with those o f VIP synthesizing cells i n t h e brain. We assume t h a t
Curr Eye Res Downloaded from informahealthcare.com by QUT Queensland University of Tech on 11/23/14 For personal use only.
t h e VIP-containing cells i n t h e retina have low mRNA level and synthesize only a small amount of VIP. I n t h e CNS, t h e highest concentration of VIP i s found i n the suprachiasmatic nucleus (SCN) o f t h e hypothalamus. The VIP-containing neurons i n t h e SCN showed an increased immunoreactivity
following enucleation of the eye (16). Although t h e role o f retinal VIP is unknown, a recent investigation demonstrated an increased VIP immunoreactivity i n t h e retina o f t h e primate myopic eye (17). Another study showed that VIP induced increase i n intracellular concentration of cyclic AMP i n cultured glial cells o f t h e r a t r e t i n a (18). These observations suggest that VIP may play a role i n regulation o f ocular morphogenesis between t h e amacrine and glial cells of t h e dynamic changes i n VIP processing i s required. The i n s i t u hybridization technique can make an important contribution t o revealing the function of retinal VIP.
ACKNOWLEDGEMENT We thank Dr. Noboru Yanaihara f o r a k i n d g i f t o f t h e antiserum t o VIP (R.502). This work was supported i n p a r t b y a g r a n t (60480105, 01480112) from t h e Ministry o f Education, Science and Culture, Japan.
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REFERENCES l.Brecha, N., Karten, H.J. and Laverack, C. (1979) Enkephalin-containing amacrine cells in t h e avian retina: imrnunohistochemical localization. Proc. Natl. Acad. Sci. U.S.A. 76, 3010-3014. 2.Karten, H.J. and Brecha, N. (1980) Localization o f substance P irnmunoreactivity in amacrine cells o f t h e retina. Nature 238, 87-88. 3.Brecha, N., Karten, H.J. and Schenker, C. (1981) Neurotensin-li ke and somatostatin-li k e immunoreactivity w i t h in amacrine cells o f t h e retina. Neuroscience 5, 1329-1 340. 4.Said, S.I. and Mutt, V. (1970) Polypeptide w i t h broad biological a c t i v i t y : isolation from small o , 1217-1218. intestine. Science N.Y. l S.Larsson, L.I., Fahrenkrug, J., Schaffalitzky de Muckadell, O.B., Sunder, F., Hakanson, R. and Rehfeld, J.F. (1 976) Localization o f vasoactive intestinal polypeptide (VIP) t o central and peripheral neurons. Proc. Natl. Acad. U.S.A. 73, 31 97-3200. 6.0kamot0, S., Okamura, H., Takahashi, Y., Akagi, Y., Yanaihara, N. and Ibata, Y. (1990) Contrary effect o f eye enucleation on VIP-immunoreactive neurons i n t h e suprachiasmatic nucleus and t h e superior colliculus of t h e rat. Neurosci. Lett. 112, 137-1 42. 7.Loren, I., Tornqvist, K. and Alumets, J. (1980) VIP (vasoactive intestinal polypeptide)immunoreactive neurons i n t h e retina o f t h e rat. Cell Tissue Res. 210, 167-1 70. 8.Terubayashi, H., Tsuto, T., Fukui, K., Obata, H.L., Okamura, H., Fujisawa, H., Itoi, M., Yanaihara, C., Yanaihara, N. and Ibata, Y. (1983) VIP (vasoactive intestinal polypeptide)-li ke immunoreactive amacrine cells in t h e r e t i n a of t h e rat. Exp. Eye Res. 2,143-749. S.Nishizawa, M., Hayakawa, Y., Yanaihara, N. and Okamoto, H. (1 985) Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett. 183,55-59. lO.Mary, L.P., Hames, B.D. and Higgins, S.J. (1985) I n S i t u Hybridization in Nucleic Acid Hybridization. Ppl79-202 IRL Press Oxfqrd, Washington DC. l l . U h r , G.R. (1986) I n Situ Hybridization i n Brain, Pp259-290 Plenum. N.Y. lZ.Yanaihara, N., Sakagami, M., Sato, H., Yamamoto, K., Hashimoto, T., Yanaihara, C., Ito, Z., Yamaguchi, K. and Abe, K. (1977) Immunological aspects of secretin, substance P, and VIP. Gastroenterology 72, 803-810. ld.Gozes, I., Avidor, R., Biegon, A. and Baldino, F.Jr.
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(1 989) Lactation elevates vasoactive intestinal peptide messenger ribonucleic acid i n r a t supra181-189. chiasmatic nucleus. Endocrinology 14.Stopa, E.G., Minamitani, N., Jonassen, J.A., King, J.C., Wolfe, H., Mobtaker, H. and Alber, H.E. (1988) Localization of vasoactive intestinal peptide and peptide histidine isoleucine irnmunoreactivity and mRNA within t h e r a t suprachiasmatic nucleus, Mol. Brain Res. 4, 319-325. 15.0kamot0, S., Okamura, H., Miyake, M., Takagi, S., Fukui, K., Akagi, Y., Okarnoto, H. and Ibata, Y. (1 991) A d i u r n a l variation o f vasoactive intestinal peptide (VIP) mRNA under a daily l i g h t - d a r k cycle in t h e r a t suprachiasmatic nucleus. Histochemistry. 95, 525-528. 16.0kamura, H., Takahashi, Y., Terubayashi, H., Hamada, S., Yanaihara, N. and Ibata, Y. (1987) VIP-like immunoreactive neurons and retinal projections i n t h e r a t suprachiasmatic nucleus, Biomed. Res. 8, 253-262. 17.Stone, R.A., Laties, A.M., Ravila, E. and Wiesel, T.N. (1988) Increase i n retinal vasoactive intestinal polypeptide after eyelid fusion i n primates. Proc. Natl. Sci. U.S.A. 85, 257-260. 18.Koh, SW.M. and Roberge, f.G. (1989) V I P modulation of c u l t u r e d glial cells of the r a t retina. C u r r . Eye Res. 8, 1207-1210.
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