- Email: [email protected]
Avian retinal cells express enkephalin-like immunoreactivity in culture
Developmental Brain Research, 31 (1987) 147-150 Elsevier
147
BRD 60189
Avian retinal cells express enkephalin-Iike immunoreactivity in culture M a s a k a t s u F u k u d a 1'* , H e r m e s H. Y e h 2 a n d D o n a l d G. P u r o 3 1Laboratory of Vision Research, National Eye Institute, National Institutes of Health, Bethesda, MD (U.S.A.), 2Departmentof Neurobiology and Anatomy, Universityof Rochester School of Medicine and Dentistry, Rochester NY (U.S.A.) and 3Department of Ophthalmology, Bascom Palmer Eye Institute and Department of Physiology and Biophysics, University of Miami School of Medicine, MiamL FL (U.S.A.) (Accepted 2 September 1986) Key words: Retina; Cell culture; Leu-enkephalin; Immunocytochemistry; Development
A population of retinal ceils derived from the embryonic chick acquire Leu-enkephalin-like immunoreactivity as they develop in cell culture.
The retinas of a variety of species contain a subset of enkephalin-containing amacrine cells 1'7. The development of enkephalinergic amacrine cells has been studied chiefly in the avian retina where enkephalins are first detected by radioimmunoassay at embryonic day 11 and by immunocytochemistry at embryonic day 131'3. Although the enkephalinergic system of the avian retina has been studied extensively, little is known about the regulation of the development of the enkephalin-containing amacrine cells. In ontogenetic studies of other types of neurons, the experimental advantages of cell culture systems have proven helpful in analyzing the regulation of neuronal maturation 4,s. Our working hypothesis is that the study of developing enkephalinergic retinal neurons can be facilitated by the use of a culture system. Clearly, a fundamental prerequisite for such an approach is that enkephalin-containing neurons be demonstrated to be present in culture. In this communication, we present immunocytochemical evidence for the expression of Leu-enkephalin in cultures of dissociated retinal cells derived from the embryonic chick retina. Retinal cell cultures were prepared as described
previously 6. In brief, retinas were dissected from the eyes of embryonic day-7 chicks, exposed to trypsin, mechanically dissociated and added (3 x 106 cells per 35 mm dish) to poly-D-lysine-coated plastic culture dishes in 1.5 ml of medium containing 90% basal medium of Eagle with Earle's salts (GIBCO) and 10% dialyzed fetal bovine serum (GIBCO). The growth
Fig. 1. A cultured retinal cell positively stained for enkephalinlike immunoreactivity. Dissociated retinal cells from embryonic day-7 chicks were maintained in culture for 3 days. The bar indicates 20/~m.
* Present address: Department of Ophthalmology, University of Osaka, Osaka, Japan. Correspondence: D.G. Puro, Bascom Palmer Eye Institute, University of Miami School of Medicine, P.O. Box 016880, Miami, FL 33101, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
148 medium was replaced every other day. l'wo to 11 days after plating, cultures were rinsed 3x with 2.0 ml of ice-cold picric acid paraformaldehyde at pH 7.4 and stored in the fixative for 24 h at 4 °C. Cultures were then rinsed with 0.02 M phosphate buffered saline (PBS) and stored in PBS for 18-24 h at 4 °C, then were washed for 10 rain at room temperature in 10% normal goat serum (GIBCO), diluted with PBS and then incubated at room temperature with a 1:1000 dilution of anti-Leu-enkephalin serum (Im-
munonuclear) for 24 h. After 3 10-min washes with PBS containing 0.1% Triton X-100, the cultures were incubated with a 1:100 dilution of fluorescein isothiocyanate-conjugated anti-rabbit goat serum (Miles) at 37 °C in a moist chamber for 60 rain. The cultures were air-dried after 3 10-rain washes with PBS. Cultures were examined by epifluorescence. In control experiments, fluorescence was virtually eliminated by preabsorption of the primary antiserum with 1 mM of synthetic Leu-enkephalin (Miles) or by
Fig. 2. Enkephalin-positive retinal cells at 4 (A) and 6 (B-D) days of culture. Bars = 20/~m.
149 substituting preimmune serum for the primary antiserum. In a series of 4 experiments, no reactivity was detected in chick retinal neurons that had been maintained in culture for two days. However, by the third day of culture, enkephalin-immunopositive neurons were detected (Fig. 1). At this time in culture, immunopositive retinal neurons displayed relatively bright fluorescence in their somas, but only faint immunofluorescence along their neuronal processes. Between 3 and 6 days of culture, as retinal neurons developed more elaborate branching of their processes, the intensity of staining along neurites increased dramatically (Fig. 2). At 6 days in culture, intensely fluorescent neurites from immunopositive retinal neurons frequently could be traced for several hundred pm. Often these processes were in close proximity to non-staining retinal cells (Fig. 3). At all ages examined, positively staining cells typically had ovid somata of ca. 8 p m in diameter. Only a small percentage of the retinal cells were reactive. In the second week of culture, there was little apparent change in the num-
ber or characteristics of cells that stained positively for Leu-enkephalin. However, the marked tendency for the retinal cells in the older cultures to aggregate into multilevel clumps made it difficult to study individual neurons in their entirety. This is the first report of cultured retinal neurons expressing neuropeptide-like immunoreactivity. Our observations of an initial absence and subsequent emergence in vitro of enkephalin-positive retinal cells derived from embryonic day-7 chicks is consistent with the in vivo findings of no detectable enkephalin-like immunoreactivity prior to embryonic day 111'2. The emergence in cell culture of enkephalin-like immunoreactivity indicates that the ability of retinal neurons to synthesize this class of peptides can be expressed in the absence of the 3-dimensional cellular organization found in vivo. Also, the finding that a population of embryonic retinal neurons in culture acquires enkephalin-like immunoreactivity provides the opportunity to systematically manipulate growth conditions to investigate the developmental regulation of these enkephalinergic neurons.
Fig. 3. A phase-contrast (A) and an epifluorescence (B) view of a retinal cell culture 6 days after plating. The longer arrow in A points to the immunopositive cell seen in B. The shorter arrow in A shows a cell that appears to have enkephalin-positive fibers around it (arrow in B). Bar = 20pm,
150 This work was supported in part by the Walter G.
partment of Health and H u m a n Services, National
Ross F o u n d a t i o n , Washington, DC, and by Public Health Service Research G r a n t 5P30 EY0218(), De-
Institutes of Health, National Eve Institute, Bethesda, MD 20205.
1 Brecha, N., Retinal neurotransmitters: histochemical and biochemical studies. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, 1983, pp. 85-129. 2 Humbert, J., Paradelles, P., Gros. C. and Dray, F., Enkephalin-like products in embryonic chick retina, Neurosci. Lett., 12 (1979) 259-263. 3 Ishimoto, I., Shiosaka, S., Shimizu, Y., Kuwayamma, Y., Fukuda, M., Inagaki, S., Takagi, H., Sakanaka, M., Sasaoka, A., Seneba, E., Sakiyama, T. and Tohyama, M., Leucine-enkephalin-like immunoreactivity in the chicken retina with a special reference to its fine structures, Invest. Oph-
thalmol. Vis. Sci., 24 (1984) 879-885. 4 Patterson, P.H., Environmental determination of autonomic neurotransmitter functions, Annu. Rev. Neurosci., (1977) 1-17. 5 Puro, D.G. and Agardh, E., Insulin-mediated regulation of neuronal maturation, Science, 225 (1984) 1170-1172. 6 Puro, D.G. and Yeh, H.H., Development of synaptic transmission by cholinergic neurons in culture, J. Neurosci. Res., 10 (1983) 241-250. 7 Watt, C.B., Su, Y.-Y.T. and Lam, D.M.-K., Enkephalins in the vertebrate retina, Prog. Retinal Res., 4 (1985) 221-242.
147
BRD 60189
Avian retinal cells express enkephalin-Iike immunoreactivity in culture M a s a k a t s u F u k u d a 1'* , H e r m e s H. Y e h 2 a n d D o n a l d G. P u r o 3 1Laboratory of Vision Research, National Eye Institute, National Institutes of Health, Bethesda, MD (U.S.A.), 2Departmentof Neurobiology and Anatomy, Universityof Rochester School of Medicine and Dentistry, Rochester NY (U.S.A.) and 3Department of Ophthalmology, Bascom Palmer Eye Institute and Department of Physiology and Biophysics, University of Miami School of Medicine, MiamL FL (U.S.A.) (Accepted 2 September 1986) Key words: Retina; Cell culture; Leu-enkephalin; Immunocytochemistry; Development
A population of retinal ceils derived from the embryonic chick acquire Leu-enkephalin-like immunoreactivity as they develop in cell culture.
The retinas of a variety of species contain a subset of enkephalin-containing amacrine cells 1'7. The development of enkephalinergic amacrine cells has been studied chiefly in the avian retina where enkephalins are first detected by radioimmunoassay at embryonic day 11 and by immunocytochemistry at embryonic day 131'3. Although the enkephalinergic system of the avian retina has been studied extensively, little is known about the regulation of the development of the enkephalin-containing amacrine cells. In ontogenetic studies of other types of neurons, the experimental advantages of cell culture systems have proven helpful in analyzing the regulation of neuronal maturation 4,s. Our working hypothesis is that the study of developing enkephalinergic retinal neurons can be facilitated by the use of a culture system. Clearly, a fundamental prerequisite for such an approach is that enkephalin-containing neurons be demonstrated to be present in culture. In this communication, we present immunocytochemical evidence for the expression of Leu-enkephalin in cultures of dissociated retinal cells derived from the embryonic chick retina. Retinal cell cultures were prepared as described
previously 6. In brief, retinas were dissected from the eyes of embryonic day-7 chicks, exposed to trypsin, mechanically dissociated and added (3 x 106 cells per 35 mm dish) to poly-D-lysine-coated plastic culture dishes in 1.5 ml of medium containing 90% basal medium of Eagle with Earle's salts (GIBCO) and 10% dialyzed fetal bovine serum (GIBCO). The growth
Fig. 1. A cultured retinal cell positively stained for enkephalinlike immunoreactivity. Dissociated retinal cells from embryonic day-7 chicks were maintained in culture for 3 days. The bar indicates 20/~m.
* Present address: Department of Ophthalmology, University of Osaka, Osaka, Japan. Correspondence: D.G. Puro, Bascom Palmer Eye Institute, University of Miami School of Medicine, P.O. Box 016880, Miami, FL 33101, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
148 medium was replaced every other day. l'wo to 11 days after plating, cultures were rinsed 3x with 2.0 ml of ice-cold picric acid paraformaldehyde at pH 7.4 and stored in the fixative for 24 h at 4 °C. Cultures were then rinsed with 0.02 M phosphate buffered saline (PBS) and stored in PBS for 18-24 h at 4 °C, then were washed for 10 rain at room temperature in 10% normal goat serum (GIBCO), diluted with PBS and then incubated at room temperature with a 1:1000 dilution of anti-Leu-enkephalin serum (Im-
munonuclear) for 24 h. After 3 10-min washes with PBS containing 0.1% Triton X-100, the cultures were incubated with a 1:100 dilution of fluorescein isothiocyanate-conjugated anti-rabbit goat serum (Miles) at 37 °C in a moist chamber for 60 rain. The cultures were air-dried after 3 10-rain washes with PBS. Cultures were examined by epifluorescence. In control experiments, fluorescence was virtually eliminated by preabsorption of the primary antiserum with 1 mM of synthetic Leu-enkephalin (Miles) or by
Fig. 2. Enkephalin-positive retinal cells at 4 (A) and 6 (B-D) days of culture. Bars = 20/~m.
149 substituting preimmune serum for the primary antiserum. In a series of 4 experiments, no reactivity was detected in chick retinal neurons that had been maintained in culture for two days. However, by the third day of culture, enkephalin-immunopositive neurons were detected (Fig. 1). At this time in culture, immunopositive retinal neurons displayed relatively bright fluorescence in their somas, but only faint immunofluorescence along their neuronal processes. Between 3 and 6 days of culture, as retinal neurons developed more elaborate branching of their processes, the intensity of staining along neurites increased dramatically (Fig. 2). At 6 days in culture, intensely fluorescent neurites from immunopositive retinal neurons frequently could be traced for several hundred pm. Often these processes were in close proximity to non-staining retinal cells (Fig. 3). At all ages examined, positively staining cells typically had ovid somata of ca. 8 p m in diameter. Only a small percentage of the retinal cells were reactive. In the second week of culture, there was little apparent change in the num-
ber or characteristics of cells that stained positively for Leu-enkephalin. However, the marked tendency for the retinal cells in the older cultures to aggregate into multilevel clumps made it difficult to study individual neurons in their entirety. This is the first report of cultured retinal neurons expressing neuropeptide-like immunoreactivity. Our observations of an initial absence and subsequent emergence in vitro of enkephalin-positive retinal cells derived from embryonic day-7 chicks is consistent with the in vivo findings of no detectable enkephalin-like immunoreactivity prior to embryonic day 111'2. The emergence in cell culture of enkephalin-like immunoreactivity indicates that the ability of retinal neurons to synthesize this class of peptides can be expressed in the absence of the 3-dimensional cellular organization found in vivo. Also, the finding that a population of embryonic retinal neurons in culture acquires enkephalin-like immunoreactivity provides the opportunity to systematically manipulate growth conditions to investigate the developmental regulation of these enkephalinergic neurons.
Fig. 3. A phase-contrast (A) and an epifluorescence (B) view of a retinal cell culture 6 days after plating. The longer arrow in A points to the immunopositive cell seen in B. The shorter arrow in A shows a cell that appears to have enkephalin-positive fibers around it (arrow in B). Bar = 20pm,
150 This work was supported in part by the Walter G.
partment of Health and H u m a n Services, National
Ross F o u n d a t i o n , Washington, DC, and by Public Health Service Research G r a n t 5P30 EY0218(), De-
Institutes of Health, National Eve Institute, Bethesda, MD 20205.
1 Brecha, N., Retinal neurotransmitters: histochemical and biochemical studies. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, 1983, pp. 85-129. 2 Humbert, J., Paradelles, P., Gros. C. and Dray, F., Enkephalin-like products in embryonic chick retina, Neurosci. Lett., 12 (1979) 259-263. 3 Ishimoto, I., Shiosaka, S., Shimizu, Y., Kuwayamma, Y., Fukuda, M., Inagaki, S., Takagi, H., Sakanaka, M., Sasaoka, A., Seneba, E., Sakiyama, T. and Tohyama, M., Leucine-enkephalin-like immunoreactivity in the chicken retina with a special reference to its fine structures, Invest. Oph-
thalmol. Vis. Sci., 24 (1984) 879-885. 4 Patterson, P.H., Environmental determination of autonomic neurotransmitter functions, Annu. Rev. Neurosci., (1977) 1-17. 5 Puro, D.G. and Agardh, E., Insulin-mediated regulation of neuronal maturation, Science, 225 (1984) 1170-1172. 6 Puro, D.G. and Yeh, H.H., Development of synaptic transmission by cholinergic neurons in culture, J. Neurosci. Res., 10 (1983) 241-250. 7 Watt, C.B., Su, Y.-Y.T. and Lam, D.M.-K., Enkephalins in the vertebrate retina, Prog. Retinal Res., 4 (1985) 221-242.