Scott Nawy

A Transient Receptor Potential-Like Channel Mediates Synaptic Transmission in Rod Bipolar Cells

On bipolar cells are connected to photoreceptors via a sign-inverting synapse. At this synapse, glutamate binds to a metabotropic receptor which couples to the closure of a cation-selective transduction channel. The molecular identity of both the receptor and the G protein are known, but the identity of the transduction channel has remained elusive. Here, we show that the transduction channel in mouse rod bipolar cells, a subtype of On bipolar cell, is likely to be a member of the TRP family of channels. To evoke a transduction current, the metabotropic receptor antagonist LY341495 was applied to the dendrites of cells that were bathed in a solution containing the mGluR6 agonists l-AP4 or glutamate. The transduction current was suppressed by ruthenium red and the TRPV1 antagonists capsazepine and SB-366791. Furthermore, focal application of the TRPV1 agonists capsaicin and anandamide evoked a transduction-like current. The capsaicin-evoked and endogenous transduction current displayed prominent outward rectification, a property of the TRPV1 channel. To test the possibility that the transduction channel is TRPV1, we measured rod bipolar cell function in the TRPV1−/− mouse. The ERG b-wave, a measure of On bipolar cell function, as well as the transduction current and the response to TRPV1 agonists were normal, arguing against a role for TRPV1. However, ERG measurements from mice lacking TRPM1 receptors, another TRP channel implicated in retinal function, revealed the absence of a b-wave. Our results suggest that a TRP-like channel, possibly TRPM1, is essential for synaptic function in On bipolar cells.

Regulation of the On Bipolar Cell mGluR6 Pathway by Ca2

Glutamate produces a hyperpolarizing synaptic potential in On bipolar cells by binding to the metabotropic glutamate receptor mGluR6, leading to closure of a cation channel. Here it is demonstrated that this cation channel is regulated by intracellular Ca2+. Glutamate-evoked currents were recorded from On bipolar cells in light-adapted salamander retinal slices in the presence of 2 mm external Ca2+. When glutamate was applied almost continuously, interrupted only briefly to measure the size of the response, the glutamate response remained robust. However, currents elicited by intermittent and brief applications of glutamate exhibited time-dependent run down. Run down of the glutamate response was also voltage dependent, because it was accelerated by membrane hyperpolarization. Run down was triggered, at least in part, by a rise in intracellular Ca2+; measured as a function of time or voltage, it was attenuated by intracellular buffering of Ca2+ with BAPTA or by omitting Ca2+ from the bathing solution. Current–voltage measurements demonstrated that Ca2+induced run down of the glutamate response by downregulating cation channel function, rather than by preventing closure of the channel by glutamate and mGluR6. A major source of the Ca2+that mediated this inhibition is the cation channel itself, which was found to be permeable to Ca2+, accounting for the use dependence of the run down. These results suggest that Ca2+ influx through the cation channel during background illumination could provide a signal to close the cation channel and repolarize the membrane toward its dark potential, an adaptive mechanism for coping with changes in ambient light.

cGMP-Dependent Kinase Regulates Response Sensitivity of the Mouse On Bipolar Cell

The visual system can adjust its sensitivity over a wide range of light intensities. Photoreceptors account for some of this adjustment, but there is evidence that postreceptoral processes also exist. To investigate the latter, we pharmacologically mimicked the effects of light stimulation on mouse On bipolar cells, thus avoiding confounding effects of receptoral mechanisms. Here, we report that cGMP selectively enhances responses to dim, but not bright, stimuli through a purely postsynaptic mechanism. This action of cGMP was completely blocked by inhibitors of cGMP-dependent kinase. We propose that cGMP-dependent kinase decreases coupling of the On bipolar cell glutamate receptor to the downstream cascade, thus amplifying small decreases in photoreceptor transmitter levels that would otherwise go undetected by the visual system.

A Role for Nyctalopin, a Small Leucine-Rich Repeat Protein, in Localizing the TRP Melastatin 1 Channel to Retinal Depolarizing Bipolar Cell Dendrites

Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.

Regulation of ON bipolar cell activity.

Synaptic transmission from photoreceptors to all types of ON bipolar cells is primarily mediated by the mGluR6 receptor. This receptor, which is apparently expressed uniquely in the nervous system by ON bipolar cells, couples negatively to a nonselective cation channel. This arrangement results in a sign reversal at photoreceptor/ON bipolar cell synapse, which is necessary in order to establish parallel ON and OFF pathways in the retina. The synapse is an important target for second messenger molecules that are known to modulate synaptic transmission elsewhere in the nervous system, second messengers that act on a time scale ranging from milliseconds to minutes. This review focuses on two of these molecules, Ca2+ and cGMP, summarizing our current knowledge of how they modulate gain at the photoreceptor/ON bipolar cell synapse, as well as their proposed sites of action within the mGluR6 cascade. The implications of plasticity at this synapse for retinal function will also be examined.

G-protein-mediated inhibition of the Trp channel TRPM1 requires the Gβγ dimer

ON bipolar cells are critical for the function of the ON pathway in the visual system. They express a metabotropic glutamate receptor (mGluR6) that, when activated, couples to the Go class of G protein. The channel that is primarily responsible for the synaptic response has been recently identified as the transient receptor potential cation channel subfamily M member 1 (TRPM1); TRPM1 is negatively coupled to the mGluR6/Go cascade such that activation of the cascade results in closure of the channel. Light indirectly opens TRPM1 by reducing transmitter release from presynaptic photoreceptors, resulting in a decrease in mGluR6 activation. Conversely, in the dark, binding of synaptic glutamate to mGluR6 inhibits TRPM1 current. Closure of TRPM1 by G-protein activation in the dark is a critical step in the process of ON bipolar cell signal transduction, but the precise pathway linking these two events is not understood. To address this question, we measured TRPM1 activity in retinal bipolar cells, in human ependymal melanocytes (HEMs) that endogenously express TRPM1, and in HEK293 cells transfected with TRPM1. Dialysis of the Gβγ subunit dimer, but not Gαo, closed TRPM1 channels in every cell type that we tested. In addition, activation of an endogenous G-protein–coupled receptor pathway in HEK293 cells that releases Gβγ without activating Go protein also closed TRPM1 channels. These results suggest a model in which the Gβγ dimer that is released as a result of the dissociation from Gαo upon activation of mGluR6 closes the TRPM1 channel, perhaps via a direct interaction.

Role of the low-affinity NGF receptor (p75) in survival of retinal bipolar cells

We have examined the role of neurotrophins in promoting survival of mammalian rod bipolar cells (RBC) in culture. Retinas taken from 8- to 10-day-old Long-Evans rats were dissociated and cultured in media supplemented with either nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (FGF-2). Survival was measured by the number of cells that were immunoreactive for alpha-, beta-, gamma-PKC, a bipolar cell-specific marker. Compared to untreated cultures, CNTF had no effect on RBC survival, while NGF and NT-3 increased survival only slightly. BDNF, however, increased survival by approximately 300%. Similar results were obtained with FGF-2. Both nerve growth factor (NGF) and an antibody (anti-REX) which interferes with binding to the 75-kD low-affinity neurotrophin receptor (p75NTR) eliminated BDNF-promoted survival, but had no effect on FGF-2-mediated survival. Interestingly, p75NTR was expressed by retinal glia (Müller cells), but not by the bipolar cells themselves, providing for the possibility that BDNF might induce Müller cells to produce a secondary factor, perhaps FGF-2, which directly rescues RBCs. In support of this hypothesis, an antibody that neutralizes FGF-2 attenuated the trophic effects of BDNF, and dramatically reduced survival in cultures with no added growth factors, indicating that there may be an endogenous source of FGF-2 that promotes survival of RBCs in culture. We suggest that BDNF increases production or release of FGF-2 by binding to p75NTR on Müller cells.

Desensitization of the mGluR6 transduction current in tiger salamander On bipolar cells.

Light depolarizes retinal On bipolar cells, opening the cation‐selective channels that are responsible for producing the synaptic current. In this study, the basic features of light‐induced signals were mimicked by bathing slices of salamander retina with an agonist for the mGluR6 receptor that is expressed on the dendrites of On cells, and then displacing the agonist with the mGluR6 antagonist (RS)‐a‐cyclopropyl‐4‐phosphonophenylglycine (CPPG). The transduction current that is activated by this protocol rapidly shuts off, or desensitizes. Desensitization was highly correlated with the concentration and the type of Ca2+ buffer that was dialysed into the cell: When Ca2+ buffering was minimized by dialysing cells with 0.5 mm EGTA, the steady‐state response was reduced to approximately 40% of the peak response. Buffering with 10 mm EGTA reduced desensitization, while BAPTA completely eliminated it. Removing external Ca2+ also prevented desensitization, suggesting that entry of  Ca2+ through the transduction channel provides the trigger. The time course of desensitization was measured by using a voltage jump protocol to rapidly increase Ca2+ influx, and could be fitted with a single time constant on the order of 1 s, in good agreement with previously published rates of desensitization to steps of light in this species. It is proposed that Ca2+‐dependent shut‐off of the On bipolar cell transduction current may contribute to the conversion of sustained to transient light responses that predominate in the inner retina.

Depolarizing bipolar cell dysfunction due to a Trpm1 point mutation

Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.

Switching between transient and sustained signalling at the rod bipolar-AII amacrine cell synapse of the mouse retina.

At conventional synapses, invasion of an action potential into the presynaptic terminal produces a rapid Ca2+ influx and ultimately the release of synaptic vesicles. However, retinal rod bipolar cells (RBCs) generally do not produce action potentials, and the rate of depolarization of the axon terminal is instead governed by the rate of rise of the light response, which can be quite slow. Using paired whole‐cell recordings, we measured the behaviour of the RBC‐AII amacrine cell synapse while simulating light‐induced depolarizations either by slowly ramping the RBC voltage or by depolarizing the RBC with the mGluR6 receptor antagonist (R,S)‐α‐cyclopropyl‐4‐phosphonophenylglycine (CPPG). Both voltage ramps and CPPG evoked slow activation of presynaptic Ca2+ currents and severely attenuated the early, transient component of the AII EPSC compared with voltage steps. We also found that the duration of the transient component was limited in time, and this limitation could not be explained by vesicle depletion, inhibitory feedback, or proton inhibition. Limiting the duration of the fast transient insures the availability of readily releasable vesicles to support a second, sustained component of release. The mGluR6 pathway modulator cGMP sped the rate of RBC depolarization in response to puffs of CPPG and consequently potentiated the transient component of the EPSC at the expense of the sustained component. We conclude that the rod bipolar cell is capable of both transient and sustained signalling, and modulation of the mGluR6 pathway by cGMP allows the RBC to switch between these two time courses of transmitter release.