Insulin-mediated regulation of neuronal maturation.

Exposure to insulin increased stimulus-evoked transmission at synapses formed in culture by cholinergic retinal neurons derived from fetal rats. This effect occurred at physiological concentrations and was long lasting. The findings support the hypothesis that insulin may serve as a developmental signal to regulate the emergence of effective neurotransmission across nascent synapses.

Dopamine regulates synaptic transmission mediated by cholinergic neurons of the rat retina.

The purpose of this study was to investigate the effect of dopamine on the function of synapses formed by cholinergic neurons derived from the rat retina. We used an experimental culture system in which rat striated muscle cells served as postsynaptic targets for cholinergic neurons of the retina. This culture system permitted the physiological monitoring of acetylcholine release at synapses formed by retinal neurons. We found that dopamine could facilitate evoked transmission at retina-muscle synapses. This facilitation by dopamine was reversible and could be blocked by haloperidol, a dopamine receptor antagonist. The adenosine 3':5'-phosphate analogue, 8-bromoadenosine 3':5'-phosphate, mimicked the facilitating effect of dopamine. In addition, dopamine elevated markedly the levels of adenosine 3':5'-phosphate in cultures of rat retinal cells. The results suggest that dopamine can regulate transmission through retinal neurons. Our findings support the hypothesis that a dopamine-induced facilitation of stimulus-evoked transmission involves the activation of dopamine receptors and the intracellular accumulation of adenosine 3':5'-phosphate.

Epinephrine regulates cholinergic transmission mediated by rat retinal neurons in culture.

The purpose of this study was to investigate the effects of epinephrine on neurotransmission mediated by cholinergic neurons derived from the rat retina. We used a culture system in which striated muscle cells served as postsynaptic targets for cholinergic neurons of the embryonic retina. This culture system permitted the physiological monitoring of acetylcholine released by retinal neurons. Here, we report that epinephrine facilitates evoked transmission across retina-muscle synapses. This facilitation of cholinergic transmission by epinephrine is reversible, can be mimicked by isoproterenol (a beta adrenoceptor agonist) and blocked by propranolol (a beta adrenoceptor antagonist). Neither the alpha-2 adrenoceptor blocker, yohimbine, nor the dopamine receptor antagonist, haloperidol, blocked this effect of epinephrine. Since epinephrine was found not to influence the membrane potential of muscle cells nor the responses of myotubes to acetylcholine, epinephrine appeared to have mediated its facilitatory effect on cholinergic transmission by affecting retinal cells. Because previous findings indicated that adenosine 3',5'-cyclic monophosphate may be involved in the modulation of transmission at retina-muscle synapses, the effect of epinephrine on adenosine 3',5'-cyclic monophosphate levels was investigated. Our biochemical studies demonstrated that epinephrine could increase adenosine 3',5'-cyclic monophosphate levels markedly in cultured retinal cells. The accumulation of adenosine 3',5'-cyclic monophosphate induced by epinephrine could be blocked by propranolol, but not by yohimbine nor haloperidol. Taken together, the results indicate that the facilitatory effect of epinephrine is mediated via a beta adrenoceptor and may involve an increase in adenosine 3',5'-cyclic monophosphate levels. Our findings are in agreement with the hypothesis that epinephrine may be a modulatory neurotransmitter in the rat retina.

Use of a culture system to identify possible causes of abnormal retinal development.

The aim of this study was to apply a recently developed cell culture system to the problem of identifying possible etiologies of altered retinal maturation. Using techniques of electrophysiology and neuropharmacology, it was possible to monitor the release of acetylcholine at synapses formed by cultured retinal neurons derived from the fetal rat. The major finding of this study was that the functional development of cholinergic retinal neurons of the fetus could be altered if the mother rat had been injected with a synthetic glucocorticoid hormone or stressed by cold exposure during a critical period in pregnancy. Thus, the maturation of at least certain fetal retinal neurons appears to be influenced by factors affecting the mother. The culture system described here provides a potentially useful approach to the identification and investigation of possible causes of abnormalities in the functional development of the mammalian retina.

Phagocytosis by human retinal glial cells in culture.

Under a variety of pathologic conditions, glial cells of the retina are capable of phagocytosis. Although phagocytosis may play a role in retinal pathobiology, the regulation of the phagocytic activity of retinal glial cells is poorly understood. We used a culture system to study phagocytosis by human retinal glial cells. The cultured cells were obtained from adult postmortem eyes and were immunoreactive to antibodies for glial fibrillary acidic protein and Muller cells. Electron microscopy demonstrated that the glial cells in culture were capable of phagocytosing fragments of retinal cells as well as latex beads. To rapidly quantitate phagocytosis, flow cytometry was used to detect glial cells that had internalized fluorescein-labeled microspheres. We found that reducing the extracellular calcium concentration decreased the phagocytic activity of the retinal glia. An inhibitory effect by nifedipine, a calcium channel blocker, on phagocytosis suggests a role for these ion channels in mediating a phagocytic response by retinal glial cells. A possible modulatory action of cyclic AMP was indicated by a decrease in phagocytic activity with exposure to 8-bromo-cyclic AMP. In addition to identifying conditions that reduce phagocytosis, we found that vitamin D3 can stimulate the phagocytic activity of retinal glia. Our experiments establish that phagocytosis by human retinal glial cells can be studied in a culture system and demonstrate that certain molecules can regulate the phagocytic activity of these cells.

Mitogenic and chemotactic effects of platelet-derived growth factor on human retinal glial cells.

Glial cell migration and proliferation appear to play a role in many of the proliferative retinopathies. Knowledge concerning the regulation of the migratory and proliferative responses of glial cells to pathophysiologic conditions in the human retina is limited. Here, we report that platelet-derived growth factor (PDGF) has both mitogenic and chemotactic effects on human retinal glial cells in culture. These effects of PDGF support the idea that this growth factor may be one of the molecules influencing glial cell activities in the proliferative retinopathies. Both the mitogenic and chemotactic responses of retinal glial cells to PDGF could be inhibited by the calcium-channel blocker, nifedipine. Although this finding suggests that nifedipine-sensitive calcium channels may help mediate these responses to PDGF, an electrophysiologic effect of PDGF on voltage-gated calcium channels was not detected. Also, the concentration of nifedipine required to inhibit proliferation was higher than the dose needed to block calcium channels. It seems likely that nifedipine inhibits the mitogenic and chemotactic responses of human retinal glial cells to PDGF by affecting cellular processes in addition to calcium channels.

PDGF-induced coupling of function with metabolism in microvascular pericytes of the retina.

PURPOSE: The aim of this study was to test the hypothesis that platelet-derived growth factor (PDGF)-BB regulates the physiology of retinal pericytes, which are contractile cells located on the abluminal surface of capillaries. The expression of PDGF-BB and its cognate receptor in retinal vessels suggests a vasoactive function. However, although endothelium-derived PDGF-BB appears vital for the development of pericyte-containing microvessels, its role in the mature vasculature remains uncertain. METHODS: Based on the premise that ion channels mediate the responses of pericytes to vasoactive signals, the perforated-patch configuration of the patch-clamp technique was used to determine the effect of PDGF-BB on the ionic currents and membrane potential of pericytes located on microvessels freshly isolated from the adult rat retina. Changes in pericyte calcium levels were monitored with the calcium indicator fluo-4. Differential interference contrast optics and image analysis software aided in assessing the effects of PDGF-BB on the lumens of isolated pericyte-containing microvessels. In some experiments, blockers of adenosine triphosphate (ATP) synthesis created chemical ischemia. RESULTS: Electrophysiological recordings from pericytes showed that PDGF-BB can activate nonspecific cation channels, chloride channels, and ATP-sensitive potassium channels. The metabolic status of an isolated capillary determined which of these ion channels were activated by PDGF-BB and thereby whether the membrane potential decreased or increased, the cell calcium rose or fell, and the vessel lumen constricted or dilated. CONCLUSIONS: The ability of PDGF-BB to be a vasoconstrictor when energy supplies are ample and to be a vasodilator under ischemic conditions may provide an efficient mechanism to link capillary function to local metabolic needs.

Retinal glial cell proliferation and ion channels: a possible link.

Under a variety of pathological conditions, glial cells of the retina proliferate. Although glial cell proliferation may play a role in a number of important retinal disorders, the regulation of this proliferative response is understood poorly. We examined the possibility that the function of certain ion channels in retinal glial cells may be linked with the induction of proliferation. Experiments were performed on glial cells derived from the adult rat retina and maintained in culture. The cells stained positively by immunocytochemistry with antibodies specific for glial fibrillary acidic protein and for Muller cells. Patch clamp studies of these retinal glial cells demonstrated the presence of a large conductance, calcium-activated potassium channel that is sensitive to tetraethylammonium, a classical blocker of potassium channels. The activity of this type of potassium channel was monitored by patch clamp recordings in the cell-attached configuration before and after exposure of the glial cells to a mitogenic conditioned medium. Exposure to this conditioned medium was associated with a marked increase in the activity of the ion channel. A possible link between the activity of potassium channels and the action of mitogens was suggested further by the finding that tetraethylammonium significantly blocked the proliferative response of retinal glial cells to the conditioned medium. Our findings support the hypothesis that there is a link between the biophysical processes involved in ion channel activity and the proliferation of retinal glial cells.

Diabetes-induced disruption of gap junction pathways within the retinal microvasculature.

PURPOSE: Microvascular damage caused by diabetes is a leading cause of visual loss. Identifying events early in the course of diabetic retinopathy may help in understanding and, perhaps, preventing this disorder. The hypothesis that cell-to-cell communication within the retinal microvasculature may be affected soon after the onset of diabetes was tested. METHODS: Streptozotocin was used to induce diabetes in rats. To assess cell-to-cell coupling the gap junction-permeant tracer, Neurobiotin, was delivered via patch pipettes into pericytes located on microvessels freshly isolated from the retinas of diabetic and control animals. Subsequently, immunohistochemical methods revealed the extent of the intercellular spread of the tracer. Electrophysiological methods were also used to detect intercellular communication. RESULTS: In retinal microvessels of control rats, Neurobiotin spread hundreds of micrometers from the tracer-loaded pericytes. However, within days after the onset of diabetes, this cell-to-cell coupling was dramatically reduced. In contrast, microvessels of insulin-treated diabetic rats showed no significant loss of intercellular communication. Consistent with protein kinase C (PKC) playing a role in the diabetes-induced inhibition of gap junction pathways, exposure of microvessels to a PKC activator (phorbol myristate acetate) markedly reduced tracer coupling. CONCLUSIONS: Within retinal microvessels there is extensive cell-to-cell coupling, which is markedly reduced soon after the onset of streptozotocin-induced diabetes. The closure of gap junction pathways disrupts the multicellular organization of retinal microvessels and may contribute to vascular dysfunction.

Energy metabolism in human retinal Müller cells.

PURPOSE: To measure selected parameters of energy metabolism and adenosine triphosphate (ATP) production in passaged monolayer cultures of human retinal glial (Müller) cells to assess the effects of varying substrate and oxygen availability on the biochemistry and histologic integrity of these cells. METHODS: Confluent Müller cell cultures were incubated for up to 4 hours at 37 degrees C in a modified minimal essential medium (no serum) under aerobic or mitochondrial-inhibited conditions in the presence and absence of 5 mM glucose or in the presence of lactate, pyruvate, glutamate, or glutamine. Cellular ATP levels, lactic acid production, and (14)CO(2) production from labeled glucose or glutamate were measured along with an examination of cellular morphology. Immunohistochemistry with antibodies to glial cell-specific proteins was also performed. Cells were positive for vimentin, but negative for glial fibrillary acidic protein and glutamine synthetase. RESULTS: Human Müller cells maintained ATP content aerobically at the same level for 4 hours in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Müller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate. CONCLUSIONS: These results suggest that, in the presence of glucose and oxygen, cultured Müller cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Müller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Müller cells are less susceptible than neurons to ischemia or hypoglycemia.

Replication of human cytomegalovirus in human retinal glial cells.

PURPOSE: The purpose of these studies was to characterize the replication cycle of human cytomegalovirus (HCMV) in human retinal glial cells in vitro. METHODS: Cultured human retinal glial cells were exposed to HCMV strain AD169 or low-passage clinical isolates for a 2-hour adsorption period and then incubated in the appropriate growth medium at 37 degrees C. Cultures were examined by microscopy for cytopathic effect and by immunofluorescence staining using monoclonal antibodies directed against immediate-early, early, and late HCMV proteins. Viral DNA was analyzed by field inversion gel electrophoresis and detected using Southern blot analysis or the polymerase chain reaction. RESULTS: Immunocytochemical staining revealed that the glial cells expressed all three classes of HCMV proteins and that infectious virus could be transferred from the medium of the infected cultures to susceptible MRC-5 cell monolayers. Less than 1% of the glial cells expressed the S-phase enzyme, thymidine kinase, at the time of infection compared to MRC-5 fibroblasts, of which 81% expressed it. Progeny virus was found to be highly cell associated in glial cells (80%) at peak virus titer compared to MRC-5 cells (39% cell associated at peak titer). Four low-passage clinical isolates of HCMV from patients with acquired immune deficiency virus also productively infected cultures of human retinal glial cells. Field inversion gel electrophoresis of HCMV-infected glial cell lysates was performed to identify the replicative forms of DNA. Southern blots probed with HCMV-specific probes showed that HCMV DNA replication proceeds through high molecular weight intermediates before forming the 230-kb unit length genome. CONCLUSIONS: The full permissive replication of HCMV in human retinal glial cells indicates that glial cells are a likely site of HCMV replication in the retina and thus may play an important role in the pathogenesis of HCMV retinitis.

Characterization of an L-type calcium channel expressed by human retinal Müller (glial) cells.

The traditional notion that glial cells are permeable only to potassium has been revised. For example, glia from various parts of the nervous system have calcium-permeable ion channels. Since characterization of the calcium channels in glia is limited, the purpose of this study was to determine the molecular identity and examine the functional properties of a voltage-gated calcium channel expressed by Müller cells, the predominant glia of the retina. Whole-cell and perforated-patch recordings of human Müller cells in culture revealed a high threshold voltage-activated calcium current that is blocked by dihydropyridines, but not by omega-conotoxin GVIA or omega-conotoxin MVIIC. RT-PCR of cultured human Müller cells using primers specific for the calcium channel subunits demonstrated the expression of an L-type channel composed of the alpha 1D, alpha 2 and beta 3 subunits. The alpha 2 subunit of the Müller cell calcium channel is a splice variant which is distinct from either the skeletal muscle alpha 2s or the brain alpha 2b. Our electrophysiological experiments indicate that the alpha 1D/alpha 2/beta 3 calcium channel is functionally linked with the activation of a potassium channel that may serve as one of the pathways for the redistribution by Müller cells of excess retinal potassium.

Barbiturates protect retinal cells from hypoxia in cell culture.

A culture system was used to screen for drugs that can protect mammalian retinal cells from damage induced by hypoxia. Using a special incubator, cultures could be made hypoxic for defined periods. Phase contrast photomicroscopy facilitated comparison of retinal cells before hypoxia and 1 to 2 days after hypoxia. Using 2- to 3-week-old cultures, certain glutamate antagonists, anesthetics, calcium blockers, and thiopental sodium were screened for their effect in protecting cells from hypoxia. The most remarkable effect was noted with thiopental. Quantitative measurements showed a significant increase in the percent of cells surviving after exposure to hypoxia in the presence of 100 mumol/L of thiopental sodium compared with control hypoxic cultures--82% vs 59% at 48 hours. A dose-response curve demonstrated maximal effect at 50 mumol/L of thiopental sodium, with toxic effects noted at 200 mumol/L of thiopental sodium. Our results show that thiopental reduces hypoxia-induced damage to retinal cells in culture.

Maintenance of replicative intermediates in ganciclovir-treated human cytomegalovirus-infected retinal glia.

OBJECTIVES: To characterize the molecular structure of the human cytomegalovirus (HCMV) DNA maintained in cultures of human retinal glia following ganciclovir treatment and to determine the biological activity of the DNA. METHODS: Cultures of human retinal glia were established, infected with HCMV, treated with ganciclovir, and embedded in agarose, and the viral DNA was analyzed by field inversion gel electrophoresis. RESULTS: The HCMV DNA was found to persist in cultures of infected, ganciclovir-treated retinal glial cells in the form of replicative intermediates. After removal of ganciclovir, processed forms of DNA in the 500-to 1000-kilobase range were found as well as 230-kb unit length genome. Infectious virus was recovered after termination of ganciclovir treatment. CONCLUSION: The data are consistent with the concept that ganciclovir's virostatic nature permits maintenance of HCMV DNA in retinal glia in a biologically active form that is capable of replication after removal of the drug.

Quantification of synapse turnover in cell culture.

The on and off rates of synaptogenesis can be quantitated using a cell culture system in which embryonic retinal cells form functional synapses with striated muscle cells. Quantification showed that synapse formation and termination can occur simultaneously in culture. Quantification also revealed that some retina-muscle synapses are transient, terminating within 8 h, while other synapses are much more stable. Relatively stable and transient synaptic pairs could be identified prospectively based on the strength of evoked transmission across the retina-muscle synapse.

A calcium-activated, calcium-permeable ion channel in human retinal glial cells: modulation by basic fibroblast growth factor.

A calcium-permeable, voltage-insensitive non-specific cation channel that is activated by cytoplasmic calcium was found in approximately 50% of the cell-attached patches in cultured human retinal glial cells sampled by the patch clamp technique. Spontaneous openings of this channel were infrequent, but increased markedly when glial cells were exposed to basic fibroblast growth factor. Although the role of these cation channels is uncertain, they provide a mechanism to perpetuate a transient rise in cytosolic calcium induced by the release of calcium from intracellular stores.

Glucocorticoid regulation of synaptic development.

The effect of glucocorticoid hormones on the developmental step in which a presynaptic neuron acquires the ability to transmit excitatory information across a synapse was explored using a retina muscle cell culture system. Cholinergic neurons dissociated from the perinatal rat retina form functional synapses in culture with rat striated muscle cells. Early in the functional maturation of these retina muscle synapses, there is a period in which release of acetylcholine occurs spontaneously, but cannot be evoked. This stage is followed by the emergence of neurotransmitter release that is stimulus-evoked and dependent on extracellular calcium. Here, it is reported that glucocorticoid hormones accelerate this developmental sequence. Experimental findings indicate that this hormonal effect occurs at physiological concentrations, involves glucocorticoid receptors, acts at the transcriptional level and requires protein synthesis. A hypothesis is that glucocorticoids regulate the development of mechanisms which couple neuronal depolarization with release of neurotransmitter. The acceleration of the functional maturation of cholinergic retinal neurons also can occur in utero if pregnant rats are injected with a synthetic glucocorticoid or stressed by cold exposure. Thus, alterations in the time-course of synaptic maturation are not restricted to manipulation of culture conditions. The results presented here indicate that glucocorticoid hormones can regulate the timing of the developmental step in which cholinergic neurons of the rat retina become capable of releasing acetylcholine in response to excitatory stimulation.

Cholinergic transmission by embryonic retinal neurons in culture: inhibition by dopamine.

The function of neurotransmitters in ontogeny remains unclear, although it is well known that both pre- and postsynaptic components of certain neurotransmitter systems are present from early in morphogenesis. The objective of this study was to establish a culture system that would permit an analysis of the physiological effects of dopamine on immature neurons. Specifically, dopamine-mediated effects on synaptic transmission by cholinergic neurons of the embryonic chick retina were explored. To do this, a retina-muscle culture system was used. In previous physiological studies, striated muscle cells in culture have proved useful as postsynaptic targets for cholinergic neurons of the immature retina. It is reported here that dopamine can inhibit synaptic responses of cultured muscle cells that are innervated by neurons of the embryonic chick retina. This inhibitory effect is blocked reversibly by the dopamine antagonists, haloperidol and fluphenazine. With the culture system used in this developmental study, dopamine-mediated inhibition can be examined with either explants of retina or with dissociated retinal neurons. When a low density of dissociated cells is plated, it is possible to examine relatively isolated, visually identified, presynaptic, cholinergic neurons. The results show that an inhibitory response to dopamine is expressed by neurons derived from retinas which are at an early stage of ontogeny. The finding that inhibition by dopamine could be demonstrated to develop at 90% of the retina-muscle synapses indicates that the cholinergic neurons studied in this experimental system are a relatively homogenous population with respect to their responsiveness to dopamine.

Adenosine activates ATP-sensitive K(+) currents in pericytes of rat retinal microvessels: role of A1 and A2a receptors.

In the CNS, contractile pericytes are positioned on the endothelial walls of microvessels where they are thought to play a role in adjusting blood flow to meet local metabolic needs. This function may be particularly important in the retina where pericytes are more numerous than at any other site. Despite the putative importance of pericytes, knowledge of the mechanisms by which vasoactive molecules, such as adenosine, regulate their function is limited. Using the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on microvessels freshly isolated from the adult rat retina, we found that adenosine reversibly activated a hyperpolarizing current in 98% of the sampled pericytes. This adenosine-induced current is likely to be due to the opening of ATP-sensitive potassium (K(ATP)) channels since it had a reversal potential near the equilibrium potential for K(+), was inhibited by the K(ATP) channel blocker, glibenclamide, and was mimicked by pinacidil, which is a K(ATP) channel opener. Experiments with specific agonists and antagonists indicated that both the high affinity A1 and the lower affinity A2a adenosine receptors provided effective pathways for activating K(ATP) currents in pericytes recorded under normal metabolic conditions. However, during chemical ischemia, the A1 receptor pathway rapidly became ineffective. In contrast, activation of A2a adenosine receptors continued to open K(ATP) channels in ischemic pericytes. These results suggest that the regulation of K(ATP) channels via A1 and A2a receptors allows adenosine to serve over a broad range of metabolic conditions as a vasoactive signal in the retinal microvasculature.

Glutamate as a neuron-to-glial signal for mitogenesis: role of glial N-methyl-D-aspartate receptors.

The presence on glial cells of receptors for neurotransmitters suggests the capability of neuron-to-glial signalling. Here, we asked whether the retinal transmitter, glutamate, may serve as a mitogenic signal for human retinal glial cells. Using cultured glial from the adult postmortem retina, we found that glutamate stimulated the proliferation of these glial cells. Pharmacological experiments indicated that this proliferative effect involved activation of N-methyl-D-aspartate (NMDA) receptor channels. Activation of NMDA receptors on retinal glial cells may mediate a proliferative response of these cells to pathophysiologic conditions causing a sustained elevation of glutamate.