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Presence of light-responding neurons in the reaggregate cultures of rat retinae
Developmental Brain Research, 57 (1990) 143-145 Elsevier
143
BRESD 60383
Presence of light-responding neurons in the reaggregate cultures of rat retinae Kimio Akagawa Department of Neurochemistry, National Institute for Physiological Sciences, Aichi (Japan) (Accepted 7 August 1990) Key words,: Reaggregate culture; Rat retina; Light response
Since histological studies on retinal reaggregates have given evidence of lamination similar to that in the intact retina, the presence of retinal function, i.e. light response, in vitro has been expected as well. Therefore, neurons in the outermost layers of reaggregates, mostly consisting of amacrine cells, were studied with cell-attached recording. About one fifth of the neurons in that layer showed spontaneous discharges. Interestingly, the frequency of spontaneous discharges of some neurons increased with light stimulation but decreased to the basal level under dark conditions. They were reproducible and lasted more than 10 min in the best case. These results suggest that functional connections between photoreceptors which are exclusively present in the central region and amacrine cells in the outermost layer might be reconstituted in reaggregate cultures. Most neuronal functions are thought to be determined by distinct networks consisting of various neurons. In order to study genetic and/or epigenetic factors participating in the formation of these networks, culture systems have been widely used since they can provide good methods to investigate neuronal development and maturation in an artifically controlled environment. A m o n g culture methods, reaggregate cultures from single cell suspensions of various neuronal tissues revealed histotypic rearrangements and cell differentiation 7"9'11'~2, possibly because reaggregate cultures could allow many of the cell-cell interactions that might be important for neuronal maturations. The author has also reported that selective layer formations consisting of distinct neuronal types and neuronal processes were reconstituted in rat retinal reaggregate cultures 4,6. Since these histotypic characteristics observed in retinal reaggregates resembled those in vivo, the possible presence of neural networks, which might be rudimentary for a retinal function, had been expected in vitro as well. Therefore, electrophysiological experiments were carried out in order to study this possibility. This paper reports that some neurons in the outermost layer of the retinal reaggregates, most of which are amacrine cells, change their frequency of spontaneous discharges when light stimuli are given. These results also demonstrate the presence of functional networks in the reaggregate cultures as well as in intact retina.
Reaggregate cultures were prepared as described previously 2'4'6. In brief, retinae obtained from new born rats ( L o n g - E v a n s strain) without contamination of pigment epithelia were dissociated by trituration in calciumand magnesium-free Hanks' salt solution. Single-cell suspension was dispersed at a density of 4 × 105/ml in minimal essential medium (MEM) containing 10% rat serum, 2 m M glutamine, 0.6% glucose, 3 mM taurine, 1 /~g/ml vitamin B12 and 1 /~g/ml gentamycin. The cell suspension was rotated at 60 cycles/min in culture bottles and kept under 5% C O 2 in air at 37 °C. The reaggregates were cultured for at the most 15 days under dark conditions except for medium changes. Reaggregates of 300-500 /~m in diameter were chosen for experiments since cells inside the reaggregates larger than 600 /~m appeared to be dying after one week in culture. The details of the histotypic characteristics and the identification of cell types in the reaggregate were described elsewhere 2'4. For electrophysiological studies, the reaggregates were bathed in a solution containing 135 mM NaCI, 5 mM KCI, 1 mM MgCI2, 2 mM CaCI 2, 5 mM glucose and 8 mM H E P E S - N a O H (pH 7.4) and were recorded with a patch clamp amplifier (Nohon Koden, CEZ2100) under an inverted microscope (Nikon, TMD) as described earlier 5. Cell-attached configuration was achieved by a conventional method 8"1°. The electrode resistance was 5 - 7 MI2. The electrode solution was 0.14 M NaC1 containing 5 mM KCI and 0.1 mM E D T A .
Correspondence: K. Akagawa, Department of Neurochemistry, National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444, Japan. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144
Fig. 1. Whole view of a reaggregate being recorded. The reaggregate is settled down on the bottom of a recording dish and the electrode (lower left) is attached to the surface of a neuron in the outermost layer. The reaggregate is semi-transparent. For the histological details, see ref. 4. Bar = 200/~m.
Recording was obtained from the outer surface of neurons in the outermost layer of 10- to 15-day-old reaggregates. With these preparations, it was possible to establish a cell attached configuration without any enzymatic pretreatment (Fig. 1). After the cell-attached configuration was established, the culture was kept in the
t iiil
i ;i, iiiii ,
i
........
trwi:iii
dark for 5 min and the effect of light stimulation (white light of the microscope) given to the whole reaggregate was studied. It was found under dark conditions that about one fifth of cells in the outermost layer revealed spontaneous discharges. With the cell-attached configuration, spontaneous discharges were observed as large deflections. Interestingly, in a few cases the frequency of spontaneous discharges changed with light stimulation given to the whole reaggregate. A typical example is represented in Fig. 2. The frequency of spontaneous discharges increased with light stimulation but decreased to the basal level after light was ceased. These changes were reproducible and could be observed for more than 10 min in the best case. The proportion of cells revealing these light response-like reactions was 1.6% (6 out of 380 cells with spontaneous discharges). However, no light-responding cell was found in the one week cultures despite ceils with spontaneous discharges were present in the outermost layer. It has already been found by using a cell type-specific monoclonal antibody 4 or by biochemical properties 1"3 that most cells, if not all, in the outermost layer of the reaggregate were amacrine cells. These amacrine cells in vitro made many synaptic contacts with other cells 4. While the photoreceptor cells were exclusively localized in the central region of the reaggregates, they also formed centripetal cell alignments 4'6 in which rhodopsin
t
i
-
!t-
'
,ini,iiiifli!t
I
Fig. 2. A typical example of light response-like activity. Two-week-old cultures were used for the experiments. Action currents were observed as large deflections. Note that the frequency of discharges increasedduring light stimulation. The upper bars indicate the light stimulation. The recording continues from upper left to lower right. Holding potential was 0 mV to the extracellular side. Bar = 5 s and 50 pA.
145 and the o u t e r s e g m e n t - l i k e m e m b r a n e pile-ups were found. F u r t h e r m o r e , ribbon synapses characteristic of p h o t o r e c e p t o r s were also observed. These previous histological observations indicated that p h o t o r e c e p t o r s in vitro were w e l l - d e v e l o p e d and might be able to transmit light information to o t h e r cells. The overall view of l a y e r e d structures in the reaggregates also r e s e m b l e d that of retinae in vivo 4. Considering these previous findings and the electrophysiologicai results described herein, it is d e m o n s t r a t e d that in some reaggregates there are functional connections, though there are not so many, b e t w e e n p h o t o r e c e p t o r s in the central region and amacrine cells in the o u t e r m o s t layer. F u r t h e r m o r e , preliminary e x p e r i m e n t s have shown that addition of high m a g n e s i u m in the m e d i u m diminished this light responselike activity (data not shown), suggesting that this
response might be m e d i a t e d by synaptic interactions. T h e r e f o r e , it is interesting to know histologically if these connections are specifically m e d i a t e d by bipolar cells as in intact retina. A t present, however, there is no morphological information about these possible connections through which light response-like activity may be processed. Accordingly, further studies will be necessary to know the physiological and m o r p h o l o g i c a l meaning of these results. W h a t e v e r the m e a n i n g is, these results obviously d e m o n s t r a t e that reaggregate cultures could provide interesting models to study the d e v e l o p m e n t of neural networks in vitro.
1 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies, Brain Res., 383 (1986) 110-120. 2 Akagawa, K. and Barnstable, C.J., Selective localization of glycine-accumulating cells in reaggregate cultures of rat retina, Dev. Brain Res., 31 (1987) 124-128. 3 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types accumulating GABA in rat retinal cultures using cell type specific monoclonal antibodies, Brain Res., 408 (1987) 154-162. 4 Akagawa, K., Hicks, D. and Barnstable, C.J,, Histotypic organization and cell differentiation in rat retinal reaggregate cultures, Brain Res., 437 (1987) 298-308. 5 Akagawa, K., Takada, M., Hayashi, H. and Uyemura, K., Calcium- and voltage-dependent potassium channel in the rat retinal amacrine cells identified in vitro using a cell type specific monoclonal antibody, Brain Res., 518 (1990) 1-5. 6 Barnstable, C.J., Akagawa, K., Hofstein, R. and Horn, J.P., Monoclonal antibodies that label discrete cell types in the mammalian nervous system, Cold Spring Harbor Symp. Quant.
Biol., 48 (1983) 863-876. 7 Honegger, P., Biochemical differentiation in serum-free aggregating brain cell cultures, In J.E. Bottenstein, and G. Sato (Eds.), Cell cultures in Neurosciences, Plenum, New York, 1985, pp. 223-243. 8 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 9 Linser, P.J. and Moscona, A.A., Induction of glutamine synthetase in embryonic neural retina: Its suppression by the gliotoxic agent a-aminoadipic acid, Dev. Brain Res., 1 (1981) 103-119. 10 Marty, A. and Neher, E., Potassium channels in cultured bovine adrenal chromaffin cells, J. Physiol., 367 (1982) 117-141. 11 Moscona, A.A. and Degenstein, L., Lentoids in aggregates of embryonic neural cells, Cell Diff., 10 (1981) 39-46. 12 Whener, J.M., Feinmann, R.D. and Sheppard, LR., fl-adrenergic response in mouse CNS reaggregate cultures., Brain Res., 255 (1982) 207-217.
This study was partly supported by Grant-in-Aid 63570064 for Scientific Research from The Ministry of Education, Science and Culture of Japan.
143
BRESD 60383
Presence of light-responding neurons in the reaggregate cultures of rat retinae Kimio Akagawa Department of Neurochemistry, National Institute for Physiological Sciences, Aichi (Japan) (Accepted 7 August 1990) Key words,: Reaggregate culture; Rat retina; Light response
Since histological studies on retinal reaggregates have given evidence of lamination similar to that in the intact retina, the presence of retinal function, i.e. light response, in vitro has been expected as well. Therefore, neurons in the outermost layers of reaggregates, mostly consisting of amacrine cells, were studied with cell-attached recording. About one fifth of the neurons in that layer showed spontaneous discharges. Interestingly, the frequency of spontaneous discharges of some neurons increased with light stimulation but decreased to the basal level under dark conditions. They were reproducible and lasted more than 10 min in the best case. These results suggest that functional connections between photoreceptors which are exclusively present in the central region and amacrine cells in the outermost layer might be reconstituted in reaggregate cultures. Most neuronal functions are thought to be determined by distinct networks consisting of various neurons. In order to study genetic and/or epigenetic factors participating in the formation of these networks, culture systems have been widely used since they can provide good methods to investigate neuronal development and maturation in an artifically controlled environment. A m o n g culture methods, reaggregate cultures from single cell suspensions of various neuronal tissues revealed histotypic rearrangements and cell differentiation 7"9'11'~2, possibly because reaggregate cultures could allow many of the cell-cell interactions that might be important for neuronal maturations. The author has also reported that selective layer formations consisting of distinct neuronal types and neuronal processes were reconstituted in rat retinal reaggregate cultures 4,6. Since these histotypic characteristics observed in retinal reaggregates resembled those in vivo, the possible presence of neural networks, which might be rudimentary for a retinal function, had been expected in vitro as well. Therefore, electrophysiological experiments were carried out in order to study this possibility. This paper reports that some neurons in the outermost layer of the retinal reaggregates, most of which are amacrine cells, change their frequency of spontaneous discharges when light stimuli are given. These results also demonstrate the presence of functional networks in the reaggregate cultures as well as in intact retina.
Reaggregate cultures were prepared as described previously 2'4'6. In brief, retinae obtained from new born rats ( L o n g - E v a n s strain) without contamination of pigment epithelia were dissociated by trituration in calciumand magnesium-free Hanks' salt solution. Single-cell suspension was dispersed at a density of 4 × 105/ml in minimal essential medium (MEM) containing 10% rat serum, 2 m M glutamine, 0.6% glucose, 3 mM taurine, 1 /~g/ml vitamin B12 and 1 /~g/ml gentamycin. The cell suspension was rotated at 60 cycles/min in culture bottles and kept under 5% C O 2 in air at 37 °C. The reaggregates were cultured for at the most 15 days under dark conditions except for medium changes. Reaggregates of 300-500 /~m in diameter were chosen for experiments since cells inside the reaggregates larger than 600 /~m appeared to be dying after one week in culture. The details of the histotypic characteristics and the identification of cell types in the reaggregate were described elsewhere 2'4. For electrophysiological studies, the reaggregates were bathed in a solution containing 135 mM NaCI, 5 mM KCI, 1 mM MgCI2, 2 mM CaCI 2, 5 mM glucose and 8 mM H E P E S - N a O H (pH 7.4) and were recorded with a patch clamp amplifier (Nohon Koden, CEZ2100) under an inverted microscope (Nikon, TMD) as described earlier 5. Cell-attached configuration was achieved by a conventional method 8"1°. The electrode resistance was 5 - 7 MI2. The electrode solution was 0.14 M NaC1 containing 5 mM KCI and 0.1 mM E D T A .
Correspondence: K. Akagawa, Department of Neurochemistry, National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444, Japan. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144
Fig. 1. Whole view of a reaggregate being recorded. The reaggregate is settled down on the bottom of a recording dish and the electrode (lower left) is attached to the surface of a neuron in the outermost layer. The reaggregate is semi-transparent. For the histological details, see ref. 4. Bar = 200/~m.
Recording was obtained from the outer surface of neurons in the outermost layer of 10- to 15-day-old reaggregates. With these preparations, it was possible to establish a cell attached configuration without any enzymatic pretreatment (Fig. 1). After the cell-attached configuration was established, the culture was kept in the
t iiil
i ;i, iiiii ,
i
........
trwi:iii
dark for 5 min and the effect of light stimulation (white light of the microscope) given to the whole reaggregate was studied. It was found under dark conditions that about one fifth of cells in the outermost layer revealed spontaneous discharges. With the cell-attached configuration, spontaneous discharges were observed as large deflections. Interestingly, in a few cases the frequency of spontaneous discharges changed with light stimulation given to the whole reaggregate. A typical example is represented in Fig. 2. The frequency of spontaneous discharges increased with light stimulation but decreased to the basal level after light was ceased. These changes were reproducible and could be observed for more than 10 min in the best case. The proportion of cells revealing these light response-like reactions was 1.6% (6 out of 380 cells with spontaneous discharges). However, no light-responding cell was found in the one week cultures despite ceils with spontaneous discharges were present in the outermost layer. It has already been found by using a cell type-specific monoclonal antibody 4 or by biochemical properties 1"3 that most cells, if not all, in the outermost layer of the reaggregate were amacrine cells. These amacrine cells in vitro made many synaptic contacts with other cells 4. While the photoreceptor cells were exclusively localized in the central region of the reaggregates, they also formed centripetal cell alignments 4'6 in which rhodopsin
t
i
-
!t-
'
,ini,iiiifli!t
I
Fig. 2. A typical example of light response-like activity. Two-week-old cultures were used for the experiments. Action currents were observed as large deflections. Note that the frequency of discharges increasedduring light stimulation. The upper bars indicate the light stimulation. The recording continues from upper left to lower right. Holding potential was 0 mV to the extracellular side. Bar = 5 s and 50 pA.
145 and the o u t e r s e g m e n t - l i k e m e m b r a n e pile-ups were found. F u r t h e r m o r e , ribbon synapses characteristic of p h o t o r e c e p t o r s were also observed. These previous histological observations indicated that p h o t o r e c e p t o r s in vitro were w e l l - d e v e l o p e d and might be able to transmit light information to o t h e r cells. The overall view of l a y e r e d structures in the reaggregates also r e s e m b l e d that of retinae in vivo 4. Considering these previous findings and the electrophysiologicai results described herein, it is d e m o n s t r a t e d that in some reaggregates there are functional connections, though there are not so many, b e t w e e n p h o t o r e c e p t o r s in the central region and amacrine cells in the o u t e r m o s t layer. F u r t h e r m o r e , preliminary e x p e r i m e n t s have shown that addition of high m a g n e s i u m in the m e d i u m diminished this light responselike activity (data not shown), suggesting that this
response might be m e d i a t e d by synaptic interactions. T h e r e f o r e , it is interesting to know histologically if these connections are specifically m e d i a t e d by bipolar cells as in intact retina. A t present, however, there is no morphological information about these possible connections through which light response-like activity may be processed. Accordingly, further studies will be necessary to know the physiological and m o r p h o l o g i c a l meaning of these results. W h a t e v e r the m e a n i n g is, these results obviously d e m o n s t r a t e that reaggregate cultures could provide interesting models to study the d e v e l o p m e n t of neural networks in vitro.
1 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types in monolayer cultures of rat retina using monoclonal antibodies, Brain Res., 383 (1986) 110-120. 2 Akagawa, K. and Barnstable, C.J., Selective localization of glycine-accumulating cells in reaggregate cultures of rat retina, Dev. Brain Res., 31 (1987) 124-128. 3 Akagawa, K. and Barnstable, C.J., Identification and characterization of cell types accumulating GABA in rat retinal cultures using cell type specific monoclonal antibodies, Brain Res., 408 (1987) 154-162. 4 Akagawa, K., Hicks, D. and Barnstable, C.J,, Histotypic organization and cell differentiation in rat retinal reaggregate cultures, Brain Res., 437 (1987) 298-308. 5 Akagawa, K., Takada, M., Hayashi, H. and Uyemura, K., Calcium- and voltage-dependent potassium channel in the rat retinal amacrine cells identified in vitro using a cell type specific monoclonal antibody, Brain Res., 518 (1990) 1-5. 6 Barnstable, C.J., Akagawa, K., Hofstein, R. and Horn, J.P., Monoclonal antibodies that label discrete cell types in the mammalian nervous system, Cold Spring Harbor Symp. Quant.
Biol., 48 (1983) 863-876. 7 Honegger, P., Biochemical differentiation in serum-free aggregating brain cell cultures, In J.E. Bottenstein, and G. Sato (Eds.), Cell cultures in Neurosciences, Plenum, New York, 1985, pp. 223-243. 8 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 9 Linser, P.J. and Moscona, A.A., Induction of glutamine synthetase in embryonic neural retina: Its suppression by the gliotoxic agent a-aminoadipic acid, Dev. Brain Res., 1 (1981) 103-119. 10 Marty, A. and Neher, E., Potassium channels in cultured bovine adrenal chromaffin cells, J. Physiol., 367 (1982) 117-141. 11 Moscona, A.A. and Degenstein, L., Lentoids in aggregates of embryonic neural cells, Cell Diff., 10 (1981) 39-46. 12 Whener, J.M., Feinmann, R.D. and Sheppard, LR., fl-adrenergic response in mouse CNS reaggregate cultures., Brain Res., 255 (1982) 207-217.
This study was partly supported by Grant-in-Aid 63570064 for Scientific Research from The Ministry of Education, Science and Culture of Japan.