Evanna Gleason

Development of functional calcium channels in cultured avian photoreceptors.

Vertebrate photoreceptors are unusual neurons in that they are capable of continuous calcium-mediated release of neurotransmitter (Trifonov, 1968; Hagins et al., 1970). In this study, we have examined the development and characteristics of calcium currents in chick cone cells placed in culture on embryonic day 8. Cone cells were identified by their lectin-binding properties, rhodopsin-like immunoreactivity, and the presence of an oil droplet. Using the whole-cell patch-clamp method, we have seen calcium currents in these cells after three days in culture, slightly before the appearance of synapses (Gleason & Wilson, 1989). Because cone calcium currents are blocked by cadmium and nifedipine but are enhanced by Bay K 8644, they most closely resemble L-type current (Nowycky et al., 1985). An unexpected feature of these currents is that their gating ranges varied widely between cells so that some cells showed the foot of their activation range at -70 mV and others as positive as -25 mV. Calcium imaging of fura-2 loaded cells was used to confirm the time course of calcium current development and describe the distribution of cytosolic calcium. As expected, depolarization of young cells failed to increase cytosolic calcium but in older cells an increase of threefold to fourfold was usually observed. Both at rest and during depolarization, most cone cells showed regional differences in internal calcium concentration. In the most mature cones, depolarization strongly elevated cytosolic calcium at the terminal end of the cell while producing a lesser change around the oil droplet and the ellipsoid region, suggesting that calcium channels are localized to the terminal.

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)‐dependent Ca2+ elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both Gq and Gs. Selective activation of mGluR5 stimulated Ca2+ elevations that varied in waveform from cell to cell. Experiments using high external K+ revealed that the mGluR5‐dependent Ca2+ elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca2+‐free external solution demonstrated that the variability in the time course of mGluR5‐dependent Ca2+ elevations is largely due to the influx of extracellular Ca2+. The sensitivity of the initial phase of the Ca2+ elevation to thapsigargin indicates that this phase of the response is due to the release of Ca2+ from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5‐mediated Ca2+ elevations are dependent upon the activation of phospholipase C. We rule out a role for L‐type Ca2+ channels and cAMP‐gated channels as pathways for Ca2+ entry, but provide evidence of transient receptor potential (TRP) channel‐like immunoreactivity, suggesting that Ca2+ influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C‐dependent Ca2+ release and Ca2+ influx, possibly through TRP channels.

Immunolocalization of TRPC channel subunits 1 and 4 in the chicken retina

In the vertebrate retina, multiple cell types express G protein-coupled receptors linked to the IP3 signaling pathway. The signaling engendered by activation of this pathway can involve activation of calcium permeable transient receptor potential (TRP) channels. To begin to understand the role of these channels in the retina, we undertake an immunocytochemical localization of two TRP channel subunits. Polyclonal antibodies raised against mammalian TRPC1 and TRPC4 are used to localize the expression of these proteins in sections of the adult chicken retina. Western blot analysis indicates that these antibodies recognize avian TRPC1 and TRPC4. TRPC1 labeling is almost completely confined to the inner plexiform layer (IPL) where it labels a subset of processes that ramify in three broad stripes. Occasionally, cell bodies are labeled. These can be found in the inner nuclear layer (INL) proximal to the IPL, the IPL, and the ganglion cell layer (GCL). Double-labeling experiments using a polyclonal antibody that recognizes brain nitric oxide synthase (bNOS) in the chicken indicate that many of the TRPC1-positive processes and cell bodies also express bNOS. Labeling with the TRPC4 antibody was much more widespread with some degree of labeling found in all layers of the retina. TRPC4 immunoreactivity was found in the photoreceptor layer, in the outer plexiform layer (OPL), in radially oriented cells in the INL, diffusely in the IPL, and in vertically oriented elements below the GCL. Double-labeling experiments with a monoclonal antibody raised against vimentin indicate that the TRPC4-positive structures in the INL and below the GCL are Müller cells. Thus, TRPC1 and TRPC4 subunits have unique expression patterns in the adult chicken retina. The distributions of these two subunits indicate that different retinal cell types express TRP channels containing different subunits.

Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double‐labeling experiments, most metabotropic glutamate receptor 1‐positive cell bodies in the inner nuclear layer also label with anti‐GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca2+ elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca2+ elevations were enhanced by metabotropic glutamate receptor 1‐selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca2+ signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.

Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein‐coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin‐mediated endocytic pathway. Olfactory receptor neurons from the channel catfish (Ictalurus punctatus) express the G protein‐coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin‐dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity‐dependent fluorescent dye FM1‐43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l‐glumate internalized the dye. Odorant‐stimulated neurons showed a consistent pattern of internalized FM1‐43 fluorescence localized in the cell bodies and dendritic knobs. Odorant‐stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor‐mediated internalization pathway exists in olfactory receptor neurons.

Sphingosine-1-Phosphate Elicits Receptor-Dependent Calcium Signaling in Retinal Amacrine Cells

Evidence is emerging indicating that sphingosine-1-phosphate (S1P) participates in signaling in the retina. To determine whether S1P might be involved in signaling in the inner retina specifically, we examine the effects of this sphingolipid on cultured retinal amacrine cells. Whole cell voltage-clamp recordings reveal that S1P activates a cation current that is dependent on signaling through G(i) and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-protein-coupled receptors in the production of the current. Immunocytochemistry and PCR amplification provide evidence for the expression of S1P1R and S1P3R in amacrine cells. The receptor-mediated channel activity is shown to be highly sensitive to blockade by lanthanides consistent with the behavior of transient receptor potential canonical (TRPC) channels. PCR products amplified from amacrine cells reveal that TRPCs 1 and 3-7 channel subunits have the potential to be expressed. Because TRPC channels provide a Ca(2+) entry pathway, we asked whether S1P caused cytosolic Ca(2+) elevations in amacrine cells. We show that S1P-dependent Ca(2+) elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca(2+) elevations are partially due to release from internal stores, but the largest contribution is from influx across the plasma membrane. The effect of inhibition of sphingosine kinase suggests that the production of cytosolic S1P underlies the sustained nature of the Ca(2+) elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function.

IL-1β reciprocally regulates chemokine and insulin secretion in pancreatic β-cells via NF-κB.

Proinflammatory cytokines impact islet β-cell mass and function by altering the transcriptional activity within pancreatic β-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1β, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-κB controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1β. Nitric oxide production, which is markedly elevated in pancreatic β-cells exposed to IL-1β, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1β-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1β were dependent on NF-κB transcriptional activity. We conclude that IL-1β-induced transcriptional reprogramming via NF-κB reciprocally regulates chemokine and insulin secretion while also negatively regulating β-cell proliferation. These findings are consistent with NF-κB as a major regulatory node controlling inflammation-associated alterations in islet β-cell function and mass.

Local influence of mitochondrial calcium transport in retinal amacrine cells

Ca2+-dependent synaptic transmission from retinal amacrine cells is thought to be initiated locally at dendritic processes. Hence, understanding the spatial and temporal impact of Ca2+ transport is fundamental to understanding how amacrine cells operate. Here, we provide the first examination of the local effects of mitochondrial Ca2+ transport in neuronal processes. By combining mitochondrial localization with measurements of cytosolic Ca2+, the local impacts of mitochondrial Ca2+ transport for two types of Ca2+ signals were investigated. Disruption of mitochondrial Ca2+ uptake with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) produces cytosolic Ca2+ elevations. The amplitudes of these elevations decline with distance from mitochondria suggesting that they are related to mitochondrial Ca2+ transport. The time course of the FCCP-dependent Ca2+ elevations depend on the availability of ER Ca2+ and we provide evidence that Ca2+ is released primarily via nearby ryanodine receptors. These results indicate that interactions between the ER and mitochondria influence cytosolic Ca2+ in amacrine cell processes and cell bodies. We also demonstrate that the durations of glutamate-dependent Ca2+ elevations are dependent on their proximity to mitochondria in amacrine cell processes. Consistent with this observation, disruption of mitochondrial Ca2+ transport alters the duration of glutamate-dependent Ca2+ elevations near mitochondria but not at sites more than 10 microm away. These results indicate that mitochondria influence local Ca2+-dependent signaling in amacrine cell processes.

Immunolocalization of metabotropic glutamate receptors 1 and 5 in the synaptic layers of the chicken retina

We have examined the distribution of metabotropic glutamate receptors (mGluRs) 1 and 5 in the adult chicken retina using preembedding immuno-electronmicroscopy. Immunoreactivity for mGluRs 1 and 5 was found in both the outer plexiform layer (OPL) and the inner plexiform layer (IPL). For mGluR1, OPL labeling was observed at cone pedicles and horizontal and bipolar cell processes. In the IPL, mGluR1 labeling could be found on bipolar cell terminals, as well as postsynaptic processes, including amacrine cell processes. Neither presynaptic nor postsynaptic elements were labeled at rod synapses. For mGluR5, OPL labeling was associated with cone pedicles as well as bipolar and horizontal cell processes. As for mGluR1, rod synapses were unlabeled. In the IPL, labeling for mGluR5 was found on bipolar cell terminals and amacrine cell processes. The presynaptic expression of these receptors in the OPL was confirmed at the light level by double-labeling experiments with SV2. The distributions of mGluRs 1 and 5 indicate that they have the potential to regulate function in both synaptic layers. Furthermore, the similar expression patterns for these two receptors indicate that they might be co-expressed and thus have the potential to interact functionally.

The influences of metabotropic receptor activation on cellular signaling and synaptic function in amacrine cells

Amacrine cells receive glutamatergic input from bipolar cells and GABAergic, glycinergic, cholinergic, and dopaminergic input from other amacrine cells. Glutamate, GABA, glycine, and acetylcholine (ACh) interact with ionotropic receptors and it is these interactions that form much of the functional circuitry in the inner retina. However, glutamate, GABA, ACh, and dopamine also activate metabotropic receptors linked to second messenger pathways that have the potential to modify the function of individual cells as well as retinal circuitry. Here, the physiological effects of activating dopamine receptors, metabotropic glutamate receptors, GABAB receptors, and muscarinic ACh receptors on amacrine cells will be discussed. The retina also expresses metabotropic receptors and the biochemical machinery associated with the synthesis and degradation of endocannabinoids and sphingosine-1-phosphate (S1P). The effects of activating cannabinoid receptors and S1P receptors on amacrine cell function will also be addressed.