February 2002
Volume 43, Issue 2
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Physiology and Pharmacology  |   February 2002
New Insight into the Functional Role of Acetylcholine in Developing Embryonic Rat Retinal Neurons
Author Affiliations
  • Hiroki Yasuyoshi
    From the Departments of Ophthalmology and Visual Sciences, Graduate School of Medicine and
  • Satoshi Kashii
    From the Departments of Ophthalmology and Visual Sciences, Graduate School of Medicine and
  • Masashi Kikuchi
    From the Departments of Ophthalmology and Visual Sciences, Graduate School of Medicine and
  • Shen Zhang
    From the Departments of Ophthalmology and Visual Sciences, Graduate School of Medicine and
  • Yoshihito Honda
    From the Departments of Ophthalmology and Visual Sciences, Graduate School of Medicine and
  • Akinori Akaike
    Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Investigative Ophthalmology & Visual Science February 2002, Vol.43, 446-451. doi:
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      Hiroki Yasuyoshi, Satoshi Kashii, Masashi Kikuchi, Shen Zhang, Yoshihito Honda, Akinori Akaike; New Insight into the Functional Role of Acetylcholine in Developing Embryonic Rat Retinal Neurons. Invest. Ophthalmol. Vis. Sci. 2002;43(2):446-451.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To examine the effects of acetylcholine (ACh) on glutamate-induced neurotoxicity in embryonic rat retinal neurons.

methods. Primary cultures were obtained from rat retinas at embryonic days 17 to 19. Cultured cells were exposed to glutamate for 10 minutes, followed by incubation in glutamate-free medium for 1 hour. Drugs were added to the incubation medium for 1 to 24 hours until immediately before glutamate exposure and were removed from culture medium during glutamate exposure and the postincubation period. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method.

results. Cell viability was markedly reduced by 10-minute exposure to 500 μM glutamate followed by a 1-hour incubation in glutamate-free medium. Incubating the cultures with 1 μM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 μM). The protective effect of ACh against glutamate neurotoxicity was inhibited by a nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine (0.5 μM), whereas a muscarinic acetylcholine receptor (mAChR) antagonist, atropine (0.5 μM) did not affect ACh-induced protection. In addition, a similar protection was induced by application of nicotine (1 μM), but not by muscarine (1 μM). Pretreatment with nicotine induced a protective effect in a time-dependent manner, ranging from 1 to 12 hours. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 μM induced dose-dependent protection against glutamate neurotoxicity. Furthermore, the protective action of nicotine was inhibited by simultaneous application of dopamine D1 receptor antagonist, SCH23390 (1 μM), with nicotine, whereas a dopamine D2 receptor antagonist, domperidone (1 μM), did not affect nicotine-induced protection.

conclusions. These results suggest that pretreatment of cultured rat retinal neurons with ACh or the nAChR agonists, nicotine and carbachol, has a protective action against glutamate neurotoxicity through nAChRs and that the dopamine release induced by nicotinic stimulation subsequently protects the retinal neurons by way of dopamine D1 receptors.

Cells are closely connected with surrounding cells including neurons. Neurons respond to neurotransmitters released by adjacent neurons and take part in the formation of neuronal networks, whereas neurons maintain homeostasis and survive within the networks among surrounding neurons. 
In our previous study, we showed the involvement of glutamate in ischemia-reperfusion-induced retinal injury in vivo. 1 Glutamate is an excitatory neurotransmitter in the retina, 2 3 4 yet it has a toxic action 5 6 7 on postsynaptic neurons by stimulating its receptors when it is present in excess under pathologic conditions such as retinal ischemia. 8 9 10 The N-methyl-d-aspartate (NMDA) receptor, a subtype of glutamate receptors, plays a predominant role in the delayed retinal neuronal death induced by glutamate. 7 11 12 13 14 15 16 Furthermore, we have demonstrated that dopamine, one of the chemical neuromodulators in the retina, has a protective action on cultured embryonic rat retinal neurons against NMDA receptor–mediated glutamate neurotoxicity through dopamine D1 receptors. 15  
Based on these findings, we suggest that neurotransmitters such as glutamate and dopamine may contain some signals affecting neuronal cells’ survival and death in addition to their signal informations of the neuronal network. Acetylcholine (ACh), which is released from cholinergic amacrine cells, is one of the major endogenous neurotransmitters in the retina. ACh receptors (AChRs) are subdivided into two main types, nicotinic AChRs (nAChRs) and muscarinic AChRs (mAChRs), and both nAChRs and mAChRs are prevalently distributed in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the rat retina. 17 18 Therefore, to elucidate whether or not ACh is involved in neuronal cells’ survival and death in the retina, we examined the effect of ACh on glutamate-induced neurotoxicity mediated through NMDA receptors in cultured rat retinal neurons. 
Materials and Methods
Cell Culture
Primary cultures were obtained from Wistar rat retinas (embryonic days 17–19). The procedures have been described previously. 15 16 19 20 21 In brief, retinal tissues were mechanically dissociated and single-cell suspensions were plated on plastic coverslips (1.0 × 106 cells/mL). Ten coverslips were placed in a 60-mm dish (Falcon Labware, Oxnard, CA). Approximately 15 to 20 dishes were obtained and used in a single experiment. Retinal cultures were incubated with Eagle’s minimal essential medium (Eagle’s salts; Nissui, Tokyo, Japan) containing 2 mM glutamine, 11 mM glucose (total), 24 mM sodium bicarbonate, and 10 mM HEPES with 10% heat-inactivated fetal calf serum added during the first week and supplemented with 10% horse serum for the remaining 9 to 10 days. Ten micromolar cytosine arabinoside (ara-C) was added to the culture on the sixth day to eliminate proliferating cells. We used only those cultures maintained for 9 to 10 days in vitro and used only isolated cells in this study. Clusters of cells were excluded from the results, because cells located in the clusters could not be used for histologic experiments. 15 A previous immunocytochemical study revealed that these isolated cells mainly consist of amacrine cells. 15 All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Drug Application
In a previous study using cultured rat retinal neurons, we demonstrated that cell viability was markedly reduced by exposure to glutamate (0.5 and 1 mM) for 10 minutes followed by postincubation in glutamate-free medium for more than 1 hour, 7 15 16 and we showed that there was no significant difference between the reduction in cell viability during 1- and 24-hour incubations. 7 Therefore, in this study, cultures were exposed to drugs as follows: Glutamate neurotoxicity was assessed by 10-minute exposure to 500 μM glutamate, followed by a 1-hour incubation in glutamate-free medium. Effects of drugs were assessed by pretreatment with drugs before glutamate exposure and by simultaneous application of the drugs with glutamate. To assess the effects of pretreatment with the drugs, the drugs were added to the incubation medium for 1 to 24 hours until immediately before glutamate exposure and removed from the culture medium during glutamate exposure and the postincubation period. To investigate the effects of simultaneous drug application, drugs were added to the incubation medium during glutamate exposure and removed from culture medium during the postincubation period. 
The following drugs were used: monosodium l-glutamate (Nakalai Tesque, Kyoto, Japan), acetylcholine chloride (Research Biochemicals, Natick, MA), carbachol (Research Biochemicals), (−)-nicotine (Sigma, St. Louis, MO), muscarine (Research Biochemicals), MK-801 (Research Biochemicals), mecamylamine hydrochloride (Sigma), atropine sulfate monohydrate (Wako, Osaka, Japan), SCH23390 hydrochloride (Research Biochemicals), and domperidone (Research Biochemicals). 
Measurement of Neurotoxicity
The neurotoxic effects of glutamate and the protective effects of drugs on the retinal cultures were quantitatively assessed by the trypan blue exclusion method, as described previously. 7 15 16 19 20 21 22 At each session of the experiment, we randomly chose five coverslips from different dishes, which constituted the number of samples (n = 5) for measurement of neurotoxicity. All experiments were performed in Eagle’s solution at 37°C. After the completion of drug treatment, cell cultures were stained with 1.5% trypan blue solution at room temperature for 10 minutes and then fixed with isotonic formalin (pH 7.0, 2–4°C). The fixed cultures were rinsed with physiological saline and examined under Hoffman modulation microscopy at ×400 (Hoffman Modulation Optics, Greenvale, NY). More than 200 cells on each of five coverslips were randomly counted to determine the viability of the cell culture. The cell counts were made by a blind observer. Viability of culture was calculated as the percentage of the ratio of the number of unstained cells (viable cells) to the total number of cells counted (viable cells plus nonviable cells). In each experiment, five coverslips were used to obtain mean values ± SEM of cell viability. The significance of data were determined by the Dunnett two-tailed test. 
Results
Effects of ACh on Glutamate-Induced Neurotoxicity
Figure 1 demonstrates an example of the effect of ACh on glutamate-induced neurotoxicity. Most cells in nontreated culture (control) were not stained by trypan blue (Fig. 1A) , which is normally excluded by living cells. However, numerous cells were stained by trypan blue, and cell viability was markedly reduced by 10-minute exposure to 500 μM glutamate followed by 1-hour incubation in glutamate-free medium (Fig. 1B) . Incubating the culture with 1 μM ACh for 12 hours before glutamate exposure reduced the number of cells stained by trypan blue, and cell death was markedly reduced (Fig. 1C) . Furthermore, as shown in the Methods section, cholinergic antagonists and ACh (1 μM) were added to the incubation medium for 12 hours until immediately before glutamate exposure and removed from culture medium during glutamate exposure, followed by a 1-hour incubation. Mecamylamine (0.5μ M), an nAChR antagonist, increased the number of stained cells, and cell viability was markedly reduced (Fig. 1D) , whereas atropine (0.5μ M), an mAChR antagonist, did not affect the number of stained cells, and cell death was reduced (Fig. 1E)
The quantitative assessment of the effect of ACh on glutamate-induced neurotoxicity and the effects of cholinergic antagonists on ACh-induced action against glutamate neurotoxicity is shown in Figure 2 . ACh has a protective effect on neurotoxicity induced by glutamate. A similar effect was induced by application of carbachol (1 μM). Carbachol is similar to ACh but is not decomposed by acetylcholinesterase. Mecamylamine (0.5 μM), an nAChR antagonist, reversed the protective effects of ACh against glutamate neurotoxicity; by contrast, atropine (0.5 μM), an mAChR antagonist, did not affect ACh-induced protection. Cell viability was not affected by a 12-hour exposure of the cells to cholinergic antagonists used in this study. 
Figure 3 shows the effects of the simultaneous application of drugs with glutamate for 10 minutes on glutamate-induced neurotoxicity. The neurotoxic effect of glutamate was greatly reduced by the use of a selective NMDA channel blocker, MK-801. By contrast, ACh did not inhibit glutamate neurotoxicity. 
Effects of Cholinergic Agonists against Glutamate Neurotoxicity
The following study was undertaken, using nicotine as a selective nAChR agonist and muscarine as a selective mAChR agonist, to elucidate whether indeed the protective effects of ACh against glutamate neurotoxicity are mediated by nAChRs in cultured rat retinal neurons. 
Figure 4 summarizes the effects of the cholinergic agonists, nicotine or muscarine, on glutamate-induced neurotoxicity. Incubating the cultures with 1 μM nicotine for 12 hours before glutamate exposure reduced glutamate-induced neurotoxicity, but 1 μM muscarine did not inhibit glutamate neurotoxicity. Cell viability was not affected by a 12-hour exposure of the cells to nicotine or muscarine. 
Figure 5A demonstrates the time dependence of the protective effects of nicotine against glutamate neurotoxicity in cultured retinal neurons. Cell viability was not affected by a 24-hour exposure of the cells to nicotine (1 μM) alone. Incubating the cultures with 1 μM nicotine for 1 to 12 hours before glutamate exposure reduced the glutamate-induced neurotoxicity. Pretreatment with nicotine induced a protective effect in a time-dependent manner ranging from 1 to 12 hours. A significant difference was noted between cell viability of cultures pretreated with nicotine for more than 2 hours and that of glutamate-treated cultures. Maximal protection was observed in the culture pretreated with nicotine for 12 hours before glutamate exposure. 
Figure 5B summarizes the dose–response relationship of the protective effects of nicotine against glutamate-induced neurotoxicity. The cells were treated with various concentrations of nicotine for 12 hours before glutamate exposure. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 μM induced dose-dependent protection against glutamate neurotoxicity. A significant difference was noted between cell viability of cultures pretreated with nicotine at concentrations ranging from 0.01 to 1 μM for 12 hours and that of glutamate-treated cultures. Maximal protection was observed in culture pretreated with nicotine at concentrations of 0.1 and 1 μM. 
Effects of Dopamine Receptor Antagonists on nAChR-Mediated Protection against Glutamate Neurotoxicity
Dopamine is known to be released by nicotinic stimulation from dopaminergic amacrine cells in the retina. 23 Therefore, we examined the interaction between nicotine and dopamine. 
Figure 6 summarizes the effects of the dopamine receptor antagonists, SCH23390 and domperidone, on nicotine-induced protection against glutamate neurotoxicity. As shown in the Methods section, dopamine receptor antagonists (1 μM) and nicotine (1 μM) were added to the incubation medium for 12 hours until immediately before glutamate exposure and removed from the culture medium during glutamate exposure, followed by 1-hour incubation. SCH23390, a dopamine D1 receptor antagonist, reversed the protective effects of nicotine against glutamate neurotoxicity, whereas domperidone, a dopamine D2 receptor antagonist, did not affect the nicotine-induced protection. Exposure of the cells to 1 μM dopamine receptor antagonists for 12 hours did not affect the cell viability of the cultures. 
Discussion
In the present study, we demonstrate that the neurotoxic effect of glutamate was greatly reduced by pretreatment of 1 μM ACh for 12 hours in cultured rat retinal neurons. ACh is one of the endogenous excitatory neurotransmitters in the retina. AChRs are subdivided into two main types, nAChRs and mAChRs. Furthermore, to investigate whether the ACh-induced protection against glutamate neurotoxicity depends on the specific receptor-mediated effects, the effects of selective cholinergic antagonists and agonists were examined. The results show that the protective effects of ACh on glutamate-induced neurotoxicity was mediated by nAChRs. 
The neurotoxic effect of glutamate was not reduced by the simultaneous application of ACh with glutamate. A long-term exposure is necessary for ACh to inhibit glutamate neurotoxicity. Therefore, it is not likely that the effect of ACh is caused by directly blocking NMDA receptors in the cell. Thus, it is tempting to speculate that some other mechanism must have been involved in this nAChR-mediated protection against glutamate neurotoxicity. 
Dopamine is known to be released by nicotinic stimulation from dopaminergic amacrine cells in the retina 23 and substantia nigra–derived dopaminergic nerve terminals in the striatum. 24 Furthermore, we have demonstrated that dopamine has a protective action on cultured rat retinal neurons against NMDA receptor–mediated glutamate neurotoxicity through dopamine D1 receptors. 15 To investigate whether the nAChR-mediated neuroprotection against glutamate neurotoxicity depends on dopamine, the effects of dopamine receptor antagonists were examined. The results suggest that nAChR stimulation induces a release of dopamine and subsequently protects the retinal neurons against glutamate neurotoxicity through dopamine D1 receptors. However, nAChR stimulation may not only facilitate dopamine release but may also upregulate the expression of dopamine D1 receptors. Further studies are needed to elucidate the possibility that nAChR stimulation is related to upregulation of dopamine D1 receptors. 
Activation of nAChRs may interact with NMDA receptors or downregulate the expression of NMDA receptors. As a result, calcium influx through the NMDA receptors may be decreased through a direct or indirect pathway. In previous reports, this possibility was suggested. Aizenman et al. 25 reported that certain nicotinic agonists can interact with the NMDA receptor and block its function. Wong and Gallagher 26 reported that the application of nicotinic agonists to rat dorsolateral septal neurons demonstrate a direct membrane hyperpolarization mediated by an increase in potassium conductance. In our cultured retinal neurons, however, main protective action of ACh appears to be exerted through D1 receptors, because its protective action was inhibited by simultaneous application of D1 receptor antagonist. Therefore, it is suggested that these possibilities play little role in the protective action of ACh against glutamate neurotoxicity in the retina. 
In this study, ACh protected retinal neurons against glutamate neurotoxicity by stimulating the nAChRs, and dopamine was involved in the nAChR-mediated protection. Glutamate, dopamine, and ACh are closely interrelated in the retina. Close linkage and interactions between ACh and dopamine in retinal glutamate-induced neurotoxicity mediated through NMDA receptors suggests that certain neurotransmitters and neuromodulators play crucial roles in neuronal cell viability in addition to their primary role as a neuronal signal transmitter. During development, neurons that constitute a neuronal network are kept alive, but those that do not participate in the network are eliminated. Recently, Kaczmarek et al. 27 demonstrated that the NMDA receptor plays an important role in neuronal development, plasticity, and cell death in the central nervous system. Receptor studies in the developing retina have demonstrated that nAChRs 28 and dopamine D1 receptors 29 appear at embryonic days 13 to 15 in the rat retina. Glutamate, ACh, and dopamine, therefore, may play important roles in the selection of which neurons are to live or die and the formation of neuronal networks in the developing embryonic retina. It is tempting to speculate that they deliver not only neuronal signals but signals concerning survival or death. However, careful consideration is required to generalize the results from cultured embryonic rat retinal neurons, because glutamate neurotoxicity does not seem to occur in embryonic rat retina. 30 Further studies are necessary to elucidate roles of the endogenous neurotransmitters in neuronal death and survival in the developing retina. 
 
Figure 1.
 
Photomicrographs showing the effect of ACh on glutamate-induced neurotoxicity in cultured rat retinal neurons. All cultures were photographed after trypan blue staining followed by formalin fixation, by using modulation microscopy. Cells stained with trypan blue dye were regarded as nonviable. (A) Nontreated cells (control). Cells showed almost no stain. (B) Cells treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. Marked cell death occurred. (CE) Cells pretreated with drugs for 12 hours until exposure to glutamate, then treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. (C) Cells pretreated with ACh (1 μM). Cell death was markedly reduced. (D) Cells pretreated with both ACh (1μ M) and mecamylamine (0.5 μM). The number of stained cells increased, and cell viability was markedly reduced. (E) Cells pretreated with both ACh (1 μM) and atropine (0.5 μM). The number of stained cells was reduced, and cell viability was increased. Bar, 50 μm.
Figure 1.
 
Photomicrographs showing the effect of ACh on glutamate-induced neurotoxicity in cultured rat retinal neurons. All cultures were photographed after trypan blue staining followed by formalin fixation, by using modulation microscopy. Cells stained with trypan blue dye were regarded as nonviable. (A) Nontreated cells (control). Cells showed almost no stain. (B) Cells treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. Marked cell death occurred. (CE) Cells pretreated with drugs for 12 hours until exposure to glutamate, then treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. (C) Cells pretreated with ACh (1 μM). Cell death was markedly reduced. (D) Cells pretreated with both ACh (1μ M) and mecamylamine (0.5 μM). The number of stained cells increased, and cell viability was markedly reduced. (E) Cells pretreated with both ACh (1 μM) and atropine (0.5 μM). The number of stained cells was reduced, and cell viability was increased. Bar, 50 μm.
Figure 2.
 
The quantitative assessment of the effect of ACh and carbachol on glutamate-induced neurotoxicity and effects of cholinergic antagonists on ACh-induced protection against glutamate neurotoxicity. Incubating the cultures with 1 μM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 μM). Simultaneous application of the nicotinic receptor antagonist, 0.5 μM mecamylamine, with ACh for 12 hours before glutamate exposure reversed the protective effects of ACh against glutamate neurotoxicity, whereas the muscarinic receptor antagonist, 0.5 μM atropine, did not affect the ACh-induced protection (**P < 0.01, compared with the glutamate-only group). Error bars in this and the subsequent figure represent the SEM (n = 5). CAR, carbachol; MEC, mecamylamine; AT, atropine.
Figure 2.
 
The quantitative assessment of the effect of ACh and carbachol on glutamate-induced neurotoxicity and effects of cholinergic antagonists on ACh-induced protection against glutamate neurotoxicity. Incubating the cultures with 1 μM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 μM). Simultaneous application of the nicotinic receptor antagonist, 0.5 μM mecamylamine, with ACh for 12 hours before glutamate exposure reversed the protective effects of ACh against glutamate neurotoxicity, whereas the muscarinic receptor antagonist, 0.5 μM atropine, did not affect the ACh-induced protection (**P < 0.01, compared with the glutamate-only group). Error bars in this and the subsequent figure represent the SEM (n = 5). CAR, carbachol; MEC, mecamylamine; AT, atropine.
Figure 3.
 
Effects of simultaneous application of ACh with glutamate on glutamate-induced neurotoxicity. Simultaneous application of a selective NMDA channel blocker, MK-801 (1 μM), with glutamate markedly reduced the neurotoxic effect of glutamate. By contrast, simultaneous application of ACh (1 μM) with glutamate did not inhibit glutamate-induced neurotoxicity (**P < 0.01, compared with the glutamate-only group).
Figure 3.
 
Effects of simultaneous application of ACh with glutamate on glutamate-induced neurotoxicity. Simultaneous application of a selective NMDA channel blocker, MK-801 (1 μM), with glutamate markedly reduced the neurotoxic effect of glutamate. By contrast, simultaneous application of ACh (1 μM) with glutamate did not inhibit glutamate-induced neurotoxicity (**P < 0.01, compared with the glutamate-only group).
Figure 4.
 
The effects of the cholinergic agonists, nicotine and muscarine, on glutamate-induced neurotoxicity. Incubating the cultures with 1 μM nicotine for 12 hours before glutamate exposure reduced glutamate-induced neurotoxicity. However, 1 μM muscarine did not inhibit glutamate neurotoxicity. Cell viability was not affected by a 12-hour exposure of the cells to nicotine or muscarine (**P < 0.01, compared with the glutamate-only group).
Figure 4.
 
The effects of the cholinergic agonists, nicotine and muscarine, on glutamate-induced neurotoxicity. Incubating the cultures with 1 μM nicotine for 12 hours before glutamate exposure reduced glutamate-induced neurotoxicity. However, 1 μM muscarine did not inhibit glutamate neurotoxicity. Cell viability was not affected by a 12-hour exposure of the cells to nicotine or muscarine (**P < 0.01, compared with the glutamate-only group).
Figure 5.
 
(A) Time dependence of the protective effect of nicotine, an nAChR agonist, against glutamate neurotoxicity. Incubating the cultures with 1 μM nicotine for 1 to 12 hours before glutamate exposure reduced the glutamate-induced neurotoxicity in a time-dependent manner. The cultures pretreated with nicotine (1 μM) for more than 2 hours significantly inhibited the cell death induced by glutamate (500 μM). Maximal protection was observed in the culture pretreated with nicotine for 12 hours before glutamate exposure (**P < 0.01, compared with the glutamate-only group). (B) The dose–response relationship of the protective action of nicotine against glutamate neurotoxicity. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 μM for 12 hours before glutamate exposure demonstrated dose-dependent protection against glutamate-induced neurotoxicity. Maximal protection was observed in the culture pretreated with nicotine at concentrations of 0.1 and 1 μM (*P < 0.05, **P < 0.01, compared with the glutamate-only group).
Figure 5.
 
(A) Time dependence of the protective effect of nicotine, an nAChR agonist, against glutamate neurotoxicity. Incubating the cultures with 1 μM nicotine for 1 to 12 hours before glutamate exposure reduced the glutamate-induced neurotoxicity in a time-dependent manner. The cultures pretreated with nicotine (1 μM) for more than 2 hours significantly inhibited the cell death induced by glutamate (500 μM). Maximal protection was observed in the culture pretreated with nicotine for 12 hours before glutamate exposure (**P < 0.01, compared with the glutamate-only group). (B) The dose–response relationship of the protective action of nicotine against glutamate neurotoxicity. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 μM for 12 hours before glutamate exposure demonstrated dose-dependent protection against glutamate-induced neurotoxicity. Maximal protection was observed in the culture pretreated with nicotine at concentrations of 0.1 and 1 μM (*P < 0.05, **P < 0.01, compared with the glutamate-only group).
Figure 6.
 
The effects of dopamine receptor antagonists, SCH23390 and domperidone, on nicotine-induced protection against glutamate neurotoxicity. Dopamine receptor antagonists (1 μM) and nicotine (1 μM) were added to the incubation medium for 12 hours until immediately before glutamate exposure and were removed from the culture medium during glutamate exposure followed by 1-hour incubation. SCH23390, a dopamine D1 receptor antagonist, reversed the protective effects of nicotine against glutamate neurotoxicity, whereas domperidone, a dopamine D2 receptor antagonist, did not affect the nicotine-induced protection. Exposing the cells to 1 μM dopamine receptor antagonists for 12 hours did not affect cell viability (**P < 0.01, compared with the glutamate-only group). SCH, SCH23390; DOM, domperidone.
Figure 6.
 
The effects of dopamine receptor antagonists, SCH23390 and domperidone, on nicotine-induced protection against glutamate neurotoxicity. Dopamine receptor antagonists (1 μM) and nicotine (1 μM) were added to the incubation medium for 12 hours until immediately before glutamate exposure and were removed from the culture medium during glutamate exposure followed by 1-hour incubation. SCH23390, a dopamine D1 receptor antagonist, reversed the protective effects of nicotine against glutamate neurotoxicity, whereas domperidone, a dopamine D2 receptor antagonist, did not affect the nicotine-induced protection. Exposing the cells to 1 μM dopamine receptor antagonists for 12 hours did not affect cell viability (**P < 0.01, compared with the glutamate-only group). SCH, SCH23390; DOM, domperidone.
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Figure 1.
 
Photomicrographs showing the effect of ACh on glutamate-induced neurotoxicity in cultured rat retinal neurons. All cultures were photographed after trypan blue staining followed by formalin fixation, by using modulation microscopy. Cells stained with trypan blue dye were regarded as nonviable. (A) Nontreated cells (control). Cells showed almost no stain. (B) Cells treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. Marked cell death occurred. (CE) Cells pretreated with drugs for 12 hours until exposure to glutamate, then treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. (C) Cells pretreated with ACh (1 μM). Cell death was markedly reduced. (D) Cells pretreated with both ACh (1μ M) and mecamylamine (0.5 μM). The number of stained cells increased, and cell viability was markedly reduced. (E) Cells pretreated with both ACh (1 μM) and atropine (0.5 μM). The number of stained cells was reduced, and cell viability was increased. Bar, 50 μm.
Figure 1.
 
Photomicrographs showing the effect of ACh on glutamate-induced neurotoxicity in cultured rat retinal neurons. All cultures were photographed after trypan blue staining followed by formalin fixation, by using modulation microscopy. Cells stained with trypan blue dye were regarded as nonviable. (A) Nontreated cells (control). Cells showed almost no stain. (B) Cells treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. Marked cell death occurred. (CE) Cells pretreated with drugs for 12 hours until exposure to glutamate, then treated with glutamate (500 μM) for 10 minutes, followed by a 1-hour incubation with glutamate-free medium. (C) Cells pretreated with ACh (1 μM). Cell death was markedly reduced. (D) Cells pretreated with both ACh (1μ M) and mecamylamine (0.5 μM). The number of stained cells increased, and cell viability was markedly reduced. (E) Cells pretreated with both ACh (1 μM) and atropine (0.5 μM). The number of stained cells was reduced, and cell viability was increased. Bar, 50 μm.
Figure 2.
 
The quantitative assessment of the effect of ACh and carbachol on glutamate-induced neurotoxicity and effects of cholinergic antagonists on ACh-induced protection against glutamate neurotoxicity. Incubating the cultures with 1 μM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 μM). Simultaneous application of the nicotinic receptor antagonist, 0.5 μM mecamylamine, with ACh for 12 hours before glutamate exposure reversed the protective effects of ACh against glutamate neurotoxicity, whereas the muscarinic receptor antagonist, 0.5 μM atropine, did not affect the ACh-induced protection (**P < 0.01, compared with the glutamate-only group). Error bars in this and the subsequent figure represent the SEM (n = 5). CAR, carbachol; MEC, mecamylamine; AT, atropine.
Figure 2.
 
The quantitative assessment of the effect of ACh and carbachol on glutamate-induced neurotoxicity and effects of cholinergic antagonists on ACh-induced protection against glutamate neurotoxicity. Incubating the cultures with 1 μM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 μM). Simultaneous application of the nicotinic receptor antagonist, 0.5 μM mecamylamine, with ACh for 12 hours before glutamate exposure reversed the protective effects of ACh against glutamate neurotoxicity, whereas the muscarinic receptor antagonist, 0.5 μM atropine, did not affect the ACh-induced protection (**P < 0.01, compared with the glutamate-only group). Error bars in this and the subsequent figure represent the SEM (n = 5). CAR, carbachol; MEC, mecamylamine; AT, atropine.
Figure 3.
 
Effects of simultaneous application of ACh with glutamate on glutamate-induced neurotoxicity. Simultaneous application of a selective NMDA channel blocker, MK-801 (1 μM), with glutamate markedly reduced the neurotoxic effect of glutamate. By contrast, simultaneous application of ACh (1 μM) with glutamate did not inhibit glutamate-induced neurotoxicity (**P < 0.01, compared with the glutamate-only group).
Figure 3.
 
Effects of simultaneous application of ACh with glutamate on glutamate-induced neurotoxicity. Simultaneous application of a selective NMDA channel blocker, MK-801 (1 μM), with glutamate markedly reduced the neurotoxic effect of glutamate. By contrast, simultaneous application of ACh (1 μM) with glutamate did not inhibit glutamate-induced neurotoxicity (**P < 0.01, compared with the glutamate-only group).
Figure 4.
 
The effects of the cholinergic agonists, nicotine and muscarine, on glutamate-induced neurotoxicity. Incubating the cultures with 1 μM nicotine for 12 hours before glutamate exposure reduced glutamate-induced neurotoxicity. However, 1 μM muscarine did not inhibit glutamate neurotoxicity. Cell viability was not affected by a 12-hour exposure of the cells to nicotine or muscarine (**P < 0.01, compared with the glutamate-only group).
Figure 4.
 
The effects of the cholinergic agonists, nicotine and muscarine, on glutamate-induced neurotoxicity. Incubating the cultures with 1 μM nicotine for 12 hours before glutamate exposure reduced glutamate-induced neurotoxicity. However, 1 μM muscarine did not inhibit glutamate neurotoxicity. Cell viability was not affected by a 12-hour exposure of the cells to nicotine or muscarine (**P < 0.01, compared with the glutamate-only group).
Figure 5.
 
(A) Time dependence of the protective effect of nicotine, an nAChR agonist, against glutamate neurotoxicity. Incubating the cultures with 1 μM nicotine for 1 to 12 hours before glutamate exposure reduced the glutamate-induced neurotoxicity in a time-dependent manner. The cultures pretreated with nicotine (1 μM) for more than 2 hours significantly inhibited the cell death induced by glutamate (500 μM). Maximal protection was observed in the culture pretreated with nicotine for 12 hours before glutamate exposure (**P < 0.01, compared with the glutamate-only group). (B) The dose–response relationship of the protective action of nicotine against glutamate neurotoxicity. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 μM for 12 hours before glutamate exposure demonstrated dose-dependent protection against glutamate-induced neurotoxicity. Maximal protection was observed in the culture pretreated with nicotine at concentrations of 0.1 and 1 μM (*P < 0.05, **P < 0.01, compared with the glutamate-only group).
Figure 5.
 
(A) Time dependence of the protective effect of nicotine, an nAChR agonist, against glutamate neurotoxicity. Incubating the cultures with 1 μM nicotine for 1 to 12 hours before glutamate exposure reduced the glutamate-induced neurotoxicity in a time-dependent manner. The cultures pretreated with nicotine (1 μM) for more than 2 hours significantly inhibited the cell death induced by glutamate (500 μM). Maximal protection was observed in the culture pretreated with nicotine for 12 hours before glutamate exposure (**P < 0.01, compared with the glutamate-only group). (B) The dose–response relationship of the protective action of nicotine against glutamate neurotoxicity. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 μM for 12 hours before glutamate exposure demonstrated dose-dependent protection against glutamate-induced neurotoxicity. Maximal protection was observed in the culture pretreated with nicotine at concentrations of 0.1 and 1 μM (*P < 0.05, **P < 0.01, compared with the glutamate-only group).
Figure 6.
 
The effects of dopamine receptor antagonists, SCH23390 and domperidone, on nicotine-induced protection against glutamate neurotoxicity. Dopamine receptor antagonists (1 μM) and nicotine (1 μM) were added to the incubation medium for 12 hours until immediately before glutamate exposure and were removed from the culture medium during glutamate exposure followed by 1-hour incubation. SCH23390, a dopamine D1 receptor antagonist, reversed the protective effects of nicotine against glutamate neurotoxicity, whereas domperidone, a dopamine D2 receptor antagonist, did not affect the nicotine-induced protection. Exposing the cells to 1 μM dopamine receptor antagonists for 12 hours did not affect cell viability (**P < 0.01, compared with the glutamate-only group). SCH, SCH23390; DOM, domperidone.
Figure 6.
 
The effects of dopamine receptor antagonists, SCH23390 and domperidone, on nicotine-induced protection against glutamate neurotoxicity. Dopamine receptor antagonists (1 μM) and nicotine (1 μM) were added to the incubation medium for 12 hours until immediately before glutamate exposure and were removed from the culture medium during glutamate exposure followed by 1-hour incubation. SCH23390, a dopamine D1 receptor antagonist, reversed the protective effects of nicotine against glutamate neurotoxicity, whereas domperidone, a dopamine D2 receptor antagonist, did not affect the nicotine-induced protection. Exposing the cells to 1 μM dopamine receptor antagonists for 12 hours did not affect cell viability (**P < 0.01, compared with the glutamate-only group). SCH, SCH23390; DOM, domperidone.
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