January 2005
Volume 46, Issue 1
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Retina  |   January 2005
Differentially Distributed IP3 Receptors and Ca2+ Signaling in Rod Bipolar Cells
Author Affiliations
  • Peter Koulen
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas.
  • Jiao Wei
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas.
  • Christian Madry
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas.
  • Jiyuan Liu
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas.
  • Everett Nixon
    From the Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas.
Investigative Ophthalmology & Visual Science January 2005, Vol.46, 292-298. doi:10.1167/iovs.04-0939
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      Peter Koulen, Jiao Wei, Christian Madry, Jiyuan Liu, Everett Nixon; Differentially Distributed IP3 Receptors and Ca2+ Signaling in Rod Bipolar Cells. Invest. Ophthalmol. Vis. Sci. 2005;46(1):292-298. doi: 10.1167/iovs.04-0939.

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

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Abstract

purpose. Inositol (1,4,5)-trisphosphate receptors (IP3Rs) contribute substantially to cytosolic free calcium ion (Ca2+) concentration transients and thereby modulate neuronal function. The present study was undertaken to determine the contribution of IP3Rs to the function of rod bipolar cells in the retina.

methods. Immunoreactivity for IP3Rs in rod bipolar cells from mouse retinas was detected by immunocytochemical methods. Intracellular Ca2+ concentrations were optically recorded in acutely isolated rod bipolar cells, and biophysical properties of IP3Rs were analyzed with single channel electrophysiology.

results. The distribution of IP3R isoforms was correlated with cytosolic Ca2+ transients induced by activation of group I metabotropic glutamate receptors (mGluRs) and with biophysical properties of differentially expressed IP3Rs.

conclusions. The differential distribution of IP3Rs is used by rod bipolar cells to convey Ca2+ signals that are distinct in their duration, amplitude, and kinetics at the subcellular level, and that serve the functions of individual subcellular compartments. IP3R-mediated Ca2+ signaling indicates a potential mechanism for the adaptation of the ON-pathway of vision and for coincidence and threshold detection in retinal neurons.

In many neurons, transients in the intracellular Ca2+ concentration mediated by release of Ca2+ from intracellular stores functionally depend on the activity of inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ channels/ IP3 receptors (IP3R). Production of IP3 through the stimulation of plasma membrane receptors and activation of the phospholipase C (PLC) pathway leads to the activation of IP3Rs that release Ca2+ from intracellular Ca2+ stores. 2 This second messenger signaling system has effects on global Ca2+ signaling as well as on local, spatially defined Ca2+ signals mediating a variety of physiological processes in many cells and particularly in neurons. 3 4 5 6  
Peng et al. 7 have described the localization in the mammalian retina of type 1 IP3R in synaptic terminals of photoreceptors and bipolar cells, as well as in amacrine cell processes, indicating a possible role in neurotransmission. Wang et al. 8 localize type 1 IP3Rs to outer segments of cone photoreceptors and conclude a possible role of IP3Rs in photoreceptor function. In vertebrate retina, IP3Rs had been identified in photoreceptor cells and plexiform layers, 9 horizontal cells, bipolar cells, and Müller glia 10 and had been implicated predominantly in neuronal and Müller cell function. 11 Indirect evidence from a number of studies indicates that IP3Rs potentially play important roles in regulating neuronal function in the retina by affecting physiological processes governed by transients in the intracellular Ca2+ concentration. 12 13 14 15 16 17 18 19 20  
Bipolar cells are the first interneurons in the glutamatergic vertical pathway of retinal information processing, and integrate signals in the two synaptic layers of the retina. 21 22 Besides evidence for the presence of type 1 IP3R in these cells 7 8 9 10 11 and modulation of neurotransmitter release by transients in the intracellular Ca2+ concentration, 23 24 25 26 27 28 29 30 the function of intracellular Ca2+ channels in bipolar cell physiology, especially in the outer retina, remains elusive. The present study addresses this issue by investigating the distribution of the different types of intracellular Ca2+ channels and by analyzing IP3R-mediated Ca2+ signaling and biophysical properties of individual IP3Rs in an anatomically and physiologically well-characterized type of bipolar cells, rod bipolar cells. These cells express group I metabotropic glutamate receptors, 1 31 a group of G-protein-coupled glutamate receptors, that are coupled to stimulation of PLC and generation of IP3 32 . Rod bipolar cells also have physiologically relevant PLC activity 33 and IP3R-dependent Ca2+ stores. 7 9 10 11  
Materials and Methods
All experiments described in the present study were carried out in accordance with the appropriate National Institutes of Health and University of North Texas Health Science Center Guidelines for the Welfare, Care and Use of Experimental Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Isolation and Immunochemistry of Rod Bipolar Cells
Rod bipolar cells were isolated from retinas of outbred albino Swiss Webster mice (Harlan, Indianapolis, IN) by mechanical and enzymatic dissociation as described previously, and were identified using morphologic and/or immunochemical criteria. 34 35 Freshly isolated rod bipolar cells, which maintain not only their substructural morphology but also important physiological functional properties during enzymatic and mechanical dissociation, 68 69 70 71 72 73 were used in the study. To show preservation of subcellular protein distribution after enzymatic and mechanical dissociation of rod bipolar cells, we used stainings of metabotropic glutamate receptors 1 and 5, two upstream elements of IP3R-mediated signaling in rod bipolar cells, to verify that the dissociation process did not lead to rearrangements of proteins (see Figs. 2G and 2H ). After plating on coverslips coated with poly-d-lysine, cells were processed for either immunocytochemistry or optical imaging of intracellular Ca2+ concentrations. Immunocytochemistry was performed as described previously. 34 35 Briefly, cells were fixed in 4% para-formaldehyde (w/v) in phosphate buffer and incubated in blocking, primary and secondary antibody solutions 2 hours each. Primary antibodies were used at concentrations identified below. Goat anti-mouse or anti-rabbit IgG labeled with Alexa Fluor 488 and Alexa Fluor 594 (diluted 1:500; Molecular Probes, Inc., Eugene, OR) were used to visualize immunoreactivity using standard immunofluorescence microscopy techniques. 34 35  
For Western blot analyses and isolation of endoplasmic reticulum (ER) from bipolar cells, ON-bipolar cells which comprise both ON-cone and rod bipolar cells of which ON-cone bipolar cells make up approximately 36% and rod bipolar cells approximately 64% 36 37 were isolated by immunopanning. After dissociation of retinas, cells were incubated in vials (Corning, Acton, MA) coated with polyclonal antibody to the N-terminal extracellular region of metabotropic glutamate receptor 6 (mGluR6; rabbit polyclonal, 1:500; Novus Biologicals, Littleton, CO). mGluR6-positive ON bipolar cells that adhered to the vials were collected after centrifugation at 500g and used for Western blot analyses (WB) or ER isolation. Immunoreactivity for intracellular Ca2+ channels was assayed with the standard Western blotting technique. 1 Primary antibodies were used at concentrations identified below. 
Antibodies
Intracellular Ca2+ channels were detected with isoform-specific antibodies: type 1 IP3R (EMD Biosciences, La Jolla, CA) rabbit polyclonal, 1:1000 for WB, 1:100 for immunocytochemistry (ICC) 38 ; type 2 IP3R (EMD Biosciences) rabbit polyclonal, 1:1000 for WB, 1:100 for ICC 39 ; type 3 IP3R (BD Biosciences Pharmingen, San Diego, CA) mouse monoclonal, 1:1000 for WB, 1:100 for ICC 40 ; pan RyR (Affinity BioReagents, Golden, CO) mouse monoclonal, 1:1000 for WB, 1:50 for ICC. 41 Additionally, specific antibodies were used to immunolabel mGluR1 (Chemicon, Temecula, CA; rabbit polyclonal, 0.2 μg/mL for ICC) 1 mGluR5 (Chemicon; rabbit polyclonal, 1 μg/mL for ICC) 1 to isolate ON bipolar cells using mGluR6 immunoreactivity (Novus Biologicals; rabbit polyclonal, 1:500 for immunopanning), and to characterize rod bipolar cells using protein kinase Cα immunoreactivity (Seikagaku, Tokyo, Japan; mouse monoclonal, 1:100 for ICC). 35  
Optical Imaging of Intracellular Ca2+ Concentrations
Calcium imaging was performed as described previously. 34 42 43 Briefly, after the isolation of retinal neurons, cells were transferred to L-15 medium (Leibovitz medium; Sigma-Aldrich, St. Louis, MO) and were incubated in 4 μM cell permeant fluo-3 (fluo-3-acetoxymethylester; Molecular Probes) at 37°C for 30 minutes, washed, and recorded for up to 6 hours after dissociation. Changes in fluorescence intensity of the Ca2+ indicator fluo-3 in loaded cells were measured over time with time-lapse videomicroscopy (Olympus IX70, Olympus, Japan; Hamamatsu ORCA-ER, Hamamatsu, Japan; Lambda DG-4 Ultra High Speed Wavelength Switcher with appropriate filter sets; Sutter Instrument Company, Novato, CA; SimplePCI Imaging Software v. 5.2; Compix Inc., Imaging Systems/Hamamatsu Photonics Management Corporation, Bridgewater, NJ). Cells received fresh L-15 medium constantly using a gravity-fed perfusion system with a flow rate of 1 mL/min. Images were acquired every 0.5 second and Ca2+ transients were calculated as the ratio of the fluorescence intensity during drug application (F) over the average baseline fluorescence intensity 10 s before drug application (F/F 0). Subcellular regions of interest of rod bipolar cells were analyzed independently and their spatiotemporal patterns of Ca2+ transients were analyzed separately. 
During the calcium imaging experiments, the group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) 44 45 (Sigma-Aldrich) was applied to the imaging chamber at pharmacologically relevant concentrations between 0.1 and 250 μM in a 0.5 seconds bolus application. Similarly, thapsigargin (10 μM; EMD Biosciences), an inhibitor of sarcoplasmic and endoplasmic reticulum Ca2+ ATPases, 46 was bath-applied to release Ca2+ from intracellular stores at the end of experiments to test the functionality and Ca2+ levels of intracellular Ca2+ stores. In several experiments, 100 μM dantrolene and/or 1 μM xestospongin C (Sigma-Aldrich) were included in the perfusate to block ryanodine receptors and/or IP3Rs, respectively. To test the dependence of measured Ca2+ transients on the extracellular Ca2+ concentration, Ca2+-free L-15 medium containing 5 mM EGTA to buffer trace amounts of free Ca2+ was used instead of regular L-15 medium. When cells were tested in the Ca2+-free medium environment they were equilibrated in Ca2+-free medium for 2 minutes before the application of mGluR1 agonists. No significant depletion of Ca2+ stores occurred during this time. 
Three or more independent experiments analyzing at least 5 cells each were performed to obtain data for each experimental condition, and statistical analyses used standard one-way or multiple ANOVA for comparisons of parametric populations. 
Single Channel Electrophysiology
Intracellular calcium channels, present in ER vesicles prepared from ON-bipolar cells that had been isolated with mGluR6 immunopanning, were measured as described previously 42 43 after incorporation into planar lipid bilayers. Bilayers had a 250 mM HEPES-Tris solution, pH 7.35 on the cytosolic side and a 250 mM HEPES, 55 mM Ba(OH)2 solution, pH 7.35 on the ER lumen side of the channel. The identity of IP3Rs was verified by activation with its ligand IP3 and inactivation by 50 mg/L heparin or 1 μM xestospongin C. No RyRs were observed in the ER preparations. The activity of IP3Rs was monitored over a range of cytosolic-free Ca2+ and IP3 concentrations. Channel activity was recorded under voltage-clamp conditions, filtered at 3 kHz and digitized using a planar lipid bilayer workstation (Warner Instruments, Inc., Hamden, CT). Data acquisition and analysis were carried out with pClamp version 8.1 (Axon Instruments, Union City, CA) identifying mean dwell times, current amplitudes, and open probability (P o). The identity of IP3R subtypes was determined using known biophysical parameters particularly the channel activity dependence on cytosolic IP3 and free Ca2+ concentrations 52 53 (reviewed in Refs. 2 , 54 , 55 ). Based on these properties the group of IP3Rs with high IP3 sensitivity was identified as IP3R2 and the group of IP3Rs with intermediate IP3 sensitivity as IP3R1. 
The data for each experimental condition were obtained from three or more independent experiments, and statistical analyses used standard one-way or multiple ANOVA for comparisons of parametric populations. 
Results
In the present study, the distribution and function of intracellular calcium channel isoforms in rod bipolar cells of the mouse retina were investigated with specific antibodies, optical imaging of intracellular Ca2+ concentrations, and electrophysiology. We identified a correlation between the isoform-specific differential distribution of intracellular calcium channels and their isoform-specific biophysical and cellular Ca2+ signaling properties in neurons. Our results indicated that the differential distribution of intracellular Ca2+ channel isoforms with different biophysical properties can be used by neurons to produce cellular signaling patterns as described for other cell types. 47 48 49 50 51 Such functional distribution patterns in neurons potentially provide a basis for compartment-specific intracellular signaling mechanisms that convey temporally and spatially distinct signaling patterns. 
In mGluR6-immunopurified ON-bipolar cells (see Materials and Methods), which comprise both ON-cone and rod bipolar cells (approximately 36% and 64%, respectively 36 37 ), Western blot analyses showed the expression of two IP3R isoforms, types 1 and 2 IP3R (Fig. 1) . Both antibodies against type 3 IP3R and RyRs showed no significant signals when compared to mouse control tissues (Fig. 1) . These results from immunoblotting experiments were confirmed by immunocytochemistry. Immunofluorescence staining of acutely isolated rod bipolar cells showed specific label with signals above control levels (Figs. 2A and 2B)only for type 1 and 2 IP3R (Figs. 2C and 2D) . The stainings for type 1 IP3R immunoreactivity indicate that type 1 IP3R can be found throughout the entire rod bipolar cell. Highest levels of immunoreactivity, however, were found in somata and axon terminals (Fig. 1C) . In contrast, immunoreactivity for type 2 IP3R was restricted to the dendrites and only very faint immunofluorescence label for type 2 IP3R immunoreactivity was found in the distal portion of the rod bipolar cell soma (Fig. 1D)
When rod bipolar cells were stained with antibodies against type 3 IP3R and RyRs that had been used for immunocytochemistry successfully in previous publications, 40 41 no specific immunofluorescence signals were detected (Figs. 2E and 2F) . However, with immunolabeling of acutely isolated rod bipolar cells we could corroborate findings from a previous study 1 that had identified group I mGluRs in rod bipolar cells postsynaptically to rod photoreceptor cells using vertical cryosections of the retina and ultrastructural immunolocalization. Immunoreactivity for mGluR1 and mGluR5, two upstream elements of IP3R-mediated signaling in rod bipolar cells, was found in the dendrites and distal one-quarter to one-third of rod bipolar cell somata (Figs. 2G and 2H) , consistent with synaptic release sites for the ligand of group I mGluRs, glutamate, by rod photoreceptor cells in the outer plexiform layer 1 and, at the same time, indicating the major sites of IP3 generation in the distal portion of rod bipolar cells. 
Based on these findings of the expression of specific isoforms of intracellular Ca2+ channels by rod bipolar cells (Fig. 1)and of the differential subcellular distribution of these isoforms (Fig. 2) , we hypothesized that stimulation of IP3Rs would produce functionally different Ca2+ signaling patterns dependent on the amount of IP3 generated by group I mGluR activation. 
Intracellular Ca2+ concentrations were recorded optically in rod bipolar cells after ester loading with fluo-3 in the absence of extracellular Ca2+. Changes in fluo-3 emission wavelength maximum as an indicator of changes in intracellular calcium concentrations were monitored. Fluorescence intensity of the Ca2+ indicator dye fluo-3 was normalized to the baseline intensity and was used as a measure of relative changes in the intracellular Ca2+ concentration and displayed as intensity change over time. Freshly isolated rod bipolar cells were stimulated with bolus applications of a specific agonist of group I mGluR, S-DHPG, to induce IP3 generation in the distal portion of rod bipolar cells (Fig. 3) . Figure 3Ashows an image montage of a representative rod bipolar cell response to S-DHPG stimulation. Low concentrations of the IP3-generating agonist (0.1–10 μM S-DHPG) produced a temporally and spatially well-defined increase in the cytosolic Ca2+ concentration that was restricted to the dendrites (Fig. 3B , black trace). The mean maximum amplitude at 10 μM S-DHPG was 9 ± 3% with an average signal duration of 7 ± 3 seconds (mean ± SEM; n = 17). No signals were observed in the soma at low agonist concentrations. At higher agonist concentrations (50–250 μM S-DHPG), Ca2+ transients with distinct spatial and temporal characteristics could be observed in dendrites and soma (Figs. 3A and 3B) . Whereas Ca2+ transients in the dendrites immediately followed the stimulus (Fig. 3B , gray trace), somata showed a delayed onset response of 22 ± 4 seconds (mean ± SEM; n = 17; Fig. 3B , gray trace). Ca2+ concentrations in both compartments returned to baseline levels during constant perfusion of the cell with medium. The mean maximum amplitude at 100 μM S-DHPG in the dendrites was 11 ± 4% with an average signal duration of 82 ± 7 seconds (mean ± SEM; n = 16). In the soma, mean maximum amplitude at 100 μM S-DHPG was 15 ± 3% with an average signal duration of 104 ± 9 seconds (mean ± SEM; n = 16). All measurements were obtained in Ca2+-free medium excluding a contribution of ligand- or store-operated Ca2+ channels located on the plasma membrane to the Ca2+ transients. The absence of the contribution of extracellular Ca2+ to the cytosolic Ca2+ transients through Ca2+ channels of the plasma membrane and the receptor-specific stimulation of group I mGluRs enabled us to focus on the spatio-temporal patterns of IP3-induced intracellular Ca2+ release via mGluR1 in rod bipolar cells. 
Based on our findings from the immunochemistry experiments, we hypothesized that the observed Ca2+ transients induced by group I mGluR stimulation are mediated by IP3R and not RyR. Preincubation and perfusion of rod bipolar cells for 5 minutes before and during group I mGluR stimulation with 100 μM dantrolene to inactivate RyRs 2 did not alter signal amplitude or duration, whereas addition of 1 μM xestospongin C 2 to the perfusion medium for 5 minutes before and during S-DHPG application completely abolished agonist-induced Ca2+ transients. Based on the same findings from the immunochemistry experiments, we further hypothesized the existence of IP3Rs with distinct biophysical properties in rod bipolar cells that can be attributed to the differential expression of both types 1 and 2 IP3R. To test this hypothesis, we analyzed single channel electrophysiological properties of intracellular calcium channels isolated from mGluR6-immunopurified ON-bipolar cells. Intracellular calcium channels with RyR characteristics (activation by Ca2+, cyclic ADP ribose, caffeine, and block by ryanodine, ruthenium red 2 ) were not observed, whereas two populations of IP3R distinct in their activity dependence on IP3 concentrations were identified (Fig. 4) . A range of physiologically relevant concentrations of IP3 on the cytoplasmic side of the channel was tested to evaluate activation of the channels by their ligand. Figures 4A and 4Bshow representative recordings from IP3Rs with high and intermediate IP3 sensitivity, respectively. Dose–response analyses showed that IP3Rs with high IP3 sensitivity had an EC50 of 63 ± 4 nM and IP3Rs with an intermediate IP3 sensitivity had an EC50 of 196 ± 7 nM. Based on previously published data on the IP3 dependence of IP3Rs, 52 53 reviewed in Refs. 54 , 55 , we tentatively identified the group of IP3Rs with high IP3 sensitivity as IP3R2 and the group of IP3Rs with intermediate IP3 sensitivity as IP3R1 (Figs. 4C and 4D)
Discussion
In neurons, intracellular Ca2+ signaling is critically determined by IP3R-mediated release of Ca2+ from intracellular Ca2+ stores. 4 Especially for retinal neurons, the second messenger substance IP3 has been implicated indirectly in several studies as an important physiological component of intracellular and neuronal signaling. 12 13 14 15 16 17 18 19 20 In bipolar cells that integrate signals in the two synaptic layers of the retina, 21 22 as the first interneurons in the glutamatergic vertical pathway of retinal information processing changes in the intracellular Ca2+ concentration have been shown to control neurotransmitter release. 23 24 25 26 27 28 29 30 Specifically, rod bipolar cells express group I metabotropic glutamate receptors, 1 31 and have physiologically relevant PLC activity 33 and IP3R dependent Ca2+ stores. 7 9 10 11 Therefore, these findings of IP3-mediated Ca2+ signaling via group I mGluRs in rod bipolar cells set the stage for experimental analyses presented in the present study. Our findings indicated that, depending on the strength of glutamatergic input, more specifically group I mGluR activation, Ca2+ transients with differential varying temporal and spatial properties ensue. Using immunoblotting, immunocytochemistry, optical imaging of Ca2+ concentrations, and single channel electrophysiology, we identified the presence and differential distribution of types 1 and 2 IP3R and potentially excluded the functional expression of other isoforms of intracellular Ca2+ channels in rod bipolar cells. 
Freshly isolated rod bipolar cells maintain not only their substructural morphology but also important physiological functional properties during enzymatic and mechanical dissociation. 68 69 70 71 72 73 Therefore, they were used as model systems in the present study to allow a detailed analysis of IP3R localization and function in mammalian rod bipolar cells. Control experiments using the immunolocalization of metabotropic glutamate receptors 1 and 5 (Figs. 2G and 2H)indicate that the native subcellular protein distribution 1 69 was preserved after enzymatic and mechanical dissociation of rod bipolar cells and did not lead to rearrangements of proteins. 
These findings corroborated data related to the immunolocalization of type 1 IP3R 7 9 10 11 and expanded these reports to include the localization of type 2 IP3R. But more importantly, they incorporate functional properties as well as mechanisms of action of IP3R-mediated Ca2+ signaling in rod bipolar cells. Our results also supported the notion that the differential localization of functionally distinct isoforms of intracellular Ca2+ channels determines cellular signaling patterns and functions. 47 48 49 50 51 The results also identify IP3R-mediated signaling initiated by glutamatergic neurotransmission as a potential mechanism of action in rod bipolar cells. We determined that the differential distribution of IP3R isoforms influences group I mGluR and IP3-mediated Ca2+ signaling in mouse rod bipolar cells and might play a critical role in the modulation of signaling in these first interneurons in the glutamatergic vertical pathway of retinal information processing. 
Low agonist and therefore low cytosolic IP3 concentrations would preferentially activate IP3R isoforms with a high affinity for IP3 52 54 55 in the dendrites (Figs. 2D 3B 4A 4C 4D) . In contrast, higher agonist and therefore higher cytosolic IP3 concentrations would also be able to activate IP3R isoforms with a lower affinity for IP3, 52 54 55 that in rod bipolar cells are found throughout the cell, but predominantly in the soma and the axon terminal (Figs. 2C 3 4B 4C 4D ). These correlations between localization of receptor isoforms and their functional and biophysical properties resulting in specific Ca2+ signaling patterns are further supported by the diffusion properties of IP3 56 and spatial constraints of the group I mGluR 1 and IP3R signaling system (Figs. 2C 2D 2G 2H) . Allbritton and colleagues 56 identified IP3 as a global intracellular messenger measured in cytosolic extracts with a fast diffusion coefficient of 283 μm2 and a long, effective range of 24 μm taking diffusion coefficient and lifetime into account. Therefore, even if one assumes that IP3 is exclusively being generated at the tips of rod bipolar cell dendrites, which is not supported by the group I mGluR immunolocalization data 1 (Figs. 2G 2H)showing extrasynaptic expression of group I mGluRs, IP3Rs in the soma would still be activated if they had a similar affinity to IP3 as IP3Rs in the dendrite, (i.e., the same IP3R isoforms in both soma and dendrites). However, the existence of IP3Rs with different affinities for IP3 in the soma and the dendrites (Figs. 2C 2D 4)allows rod bipolar cells to discriminate the strength of incoming glutamate/IP3 signals (Fig. 3) . Recent studies show that mGluR 1 function in addition to binding of the receptor to l-glutamate also depends on the extracellular calcium concentration. 74 75 76 The present study used native group I mGluRs as an indirect means of stimulating IP3 production in rod bipolar cells, allowing not only the control of the amount but more importantly the location of the IP3 release better than other current pharmacological or cell biological techniques, and at the same time, limiting the observed effects to a ligand-specific interaction and subsequent second-messenger mobilization. Even though high S-DHPG doses used in the present study produced maximal receptor activity, future studies investigating the involvement of changes in extracellular calcium concentrations need to address modulating effects of extracellular calcium concentrations on the results of the present study. 
In ON-bipolar cells, glutamate released by photoreceptor cells produces hyperpolarization by binding to mGluR6, which closes a cGMP-gated cation channel on the plasma membrane. 57 As reported previously 58 59 60 (reviewed in Ref. 61 ), the cytosolic Ca2+ concentration is critical for the regulation of this cation channel and thereby the mGluR6-mediated adaptation of the ON-pathway of visual neurotransmission to changing light levels. Results of the present study potentially indicate that the group I mGluR-mediated effects of glutamate could modulate the mGluR6 pathway through regulation of the cytosolic Ca2+ concentration 58 59 60 (reviewed in Ref. 61 ), and potentially adapt the sensitivity of rod bipolar cells to changing light levels. 
In addition to this potential function of the group I mGluR/IP3R pathway, other reports also support the notion of group I mGluR activation resulting in hyperpolarization via activation of Ca2+-activated K+ channels as a result of IP3R mediated Ca2+ signaling 62 63 (reviewed in Ref. 32 ), which is relevant in light of reported Ca2+-activated K+ channel activity in bipolar cells. 64 However, it should be noted that depending on the system and physiological environment, other functions of group I mGluR including neuronal depolarization are also being discussed. 32  
Similar to processes observed in other portions of the CNS, 65 66 67 the expression of a low affinity IP3R in the soma of rod bipolar cells could also serve as a coincidence and threshold detector for elevated levels of IP3 in the cytosol, providing signal integration in the soma. In contrast, the expression of a high affinity IP3R in the dendrites of rod bipolar cells would help to maintain regulation of synaptic events as discussed above for a potential involvement in mGluR6-mediated signaling. 58 59 60 61 In summary, rod bipolar cells can use the differential distribution of IP3R isoforms to produce spatio-temporally distinct cytosolic Ca2+ signals that contribute to Ca2+-dependent functions of subcellular compartments. 
 
Figure 2.
 
Localization of IP3R and RyR immunoreactivity in mouse rod bipolar cells. Acutely isolated rod bipolar cells were fixed and stained for type 1 (C), type 2 (D), and type 3 (E) IP3R immunoreactivity, as well as for RyR immunoreactivity (F). Controls include secondary antibody controls for both types of secondary antibodies used [anti-mouse (A); anti-rabbit (B)], as well as labeling of mGluR1 (G) and mGluR5 (H) immunoreactivity. For each panel, differential interference contrast images show the typical rod bipolar cell morphology with dendritic tree, soma axon, and axon terminal system.
Figure 2.
 
Localization of IP3R and RyR immunoreactivity in mouse rod bipolar cells. Acutely isolated rod bipolar cells were fixed and stained for type 1 (C), type 2 (D), and type 3 (E) IP3R immunoreactivity, as well as for RyR immunoreactivity (F). Controls include secondary antibody controls for both types of secondary antibodies used [anti-mouse (A); anti-rabbit (B)], as well as labeling of mGluR1 (G) and mGluR5 (H) immunoreactivity. For each panel, differential interference contrast images show the typical rod bipolar cell morphology with dendritic tree, soma axon, and axon terminal system.
Figure 1.
 
Western blot showing the expression of IP3Rs and RyRs in ON-bipolar cells (ONBC) and controls (C). Homogenates of enzymatically isolated mouse rod bipolar cells were analyzed. Mouse cerebellum, liver, pancreas, and brain homogenates were loaded as controls for IP3R type 1, IP3R type 2, IP3R type 3, and RyRs, respectively. Twenty micrograms total protein per lane were loaded on 4%–12% gradient gels to detect individual intracellular calcium channels. IP3R isoform specific antibodies detected approximately 250 kDa bands, the pan-RyR antibody detected a high molecular weight band of approximately 550 kDa (numbers and arrows, position and size of protein standards in kDa, respectively). Only IP3Rs type 1 and 2 could be detected in RBCs.
Figure 1.
 
Western blot showing the expression of IP3Rs and RyRs in ON-bipolar cells (ONBC) and controls (C). Homogenates of enzymatically isolated mouse rod bipolar cells were analyzed. Mouse cerebellum, liver, pancreas, and brain homogenates were loaded as controls for IP3R type 1, IP3R type 2, IP3R type 3, and RyRs, respectively. Twenty micrograms total protein per lane were loaded on 4%–12% gradient gels to detect individual intracellular calcium channels. IP3R isoform specific antibodies detected approximately 250 kDa bands, the pan-RyR antibody detected a high molecular weight band of approximately 550 kDa (numbers and arrows, position and size of protein standards in kDa, respectively). Only IP3Rs type 1 and 2 could be detected in RBCs.
Figure 3.
 
Group I mGluR agonists induce spatiotemporally differential Ca2+ transients in isolated mouse rod bipolar cells. (A) Montage of a representative imaging experiment visualizing the cytosolic Ca2+ concentration at 0 seconds, during 10 and 50 seconds, and after 200 seconds, application of 100 μM S-DHPG. A differential interference contrast image to the right shows the main subcellular regions of the rod bipolar cell, dendritic tree (D), soma (S), axon (A), and axon terminal system (AT). Although a Ca2+ transient in the dendrites was observed immediately after stimulation (10 seconds), a Ca2+ transient with larger amplitude was seen in the soma after a delay and after the peak of the dendritic Ca2+ transient (50 seconds). (B) Changes in the intracellular Ca2+ concentration in two regions of a representative rod bipolar cell, dendrites (black) and soma (gray). After a single 0.5 second bolus application of a low dose of an mGluR1 agonist (10 μM S-DHPG, indicated by arrow to the left), only the dendrites responded with a Ca2+ transient, whereas a higher concentration of mGluR1 agonist (100 μM S-DHPG, 0.5 second bolus application indicated by the arrow to the right) induced Ca2+ transients in both dendrites and soma.
Figure 3.
 
Group I mGluR agonists induce spatiotemporally differential Ca2+ transients in isolated mouse rod bipolar cells. (A) Montage of a representative imaging experiment visualizing the cytosolic Ca2+ concentration at 0 seconds, during 10 and 50 seconds, and after 200 seconds, application of 100 μM S-DHPG. A differential interference contrast image to the right shows the main subcellular regions of the rod bipolar cell, dendritic tree (D), soma (S), axon (A), and axon terminal system (AT). Although a Ca2+ transient in the dendrites was observed immediately after stimulation (10 seconds), a Ca2+ transient with larger amplitude was seen in the soma after a delay and after the peak of the dendritic Ca2+ transient (50 seconds). (B) Changes in the intracellular Ca2+ concentration in two regions of a representative rod bipolar cell, dendrites (black) and soma (gray). After a single 0.5 second bolus application of a low dose of an mGluR1 agonist (10 μM S-DHPG, indicated by arrow to the left), only the dendrites responded with a Ca2+ transient, whereas a higher concentration of mGluR1 agonist (100 μM S-DHPG, 0.5 second bolus application indicated by the arrow to the right) induced Ca2+ transients in both dendrites and soma.
Figure 4.
 
Two types of IP3R with respect to their IP3 sensitivity can be isolated from the ER of mouse rod bipolar cells. Activity of individual IP3Rs was recorded with 0.5 μM free Ca2+, 500 μM ATP as IP3R co-agonists, 10 μM ruthenium red to block RyRs present on the cytoplasmic side of the channel and Ba2+ as the current carrier on the luminal side of the channel. Bars to the right of each trace indicate zero current levels of channel activity and downward deflections indicate channel openings. (A) and (B) Electrophysiological recordings of two representative experiments with three traces at 0.01, 0.1, and 1 μM IP3, exemplifying the two types of IP3Rs that could be observed in mouse rod bipolar cells. (C) and (D) summarize the IP3 dependence of the absolute and normalized open probability (P o), respectively, for the two types of IP3Rs isolated from mouse rod bipolar cells. Based on the biophysical properties, the group of IP3Rs with high IP3 sensitivity is identified as IP3R2 (type 2 IP3R; n = 9) and the IP3R with intermediate IP3 sensitivity as IP3R1 (type 1 IP3R; n = 11).
Figure 4.
 
Two types of IP3R with respect to their IP3 sensitivity can be isolated from the ER of mouse rod bipolar cells. Activity of individual IP3Rs was recorded with 0.5 μM free Ca2+, 500 μM ATP as IP3R co-agonists, 10 μM ruthenium red to block RyRs present on the cytoplasmic side of the channel and Ba2+ as the current carrier on the luminal side of the channel. Bars to the right of each trace indicate zero current levels of channel activity and downward deflections indicate channel openings. (A) and (B) Electrophysiological recordings of two representative experiments with three traces at 0.01, 0.1, and 1 μM IP3, exemplifying the two types of IP3Rs that could be observed in mouse rod bipolar cells. (C) and (D) summarize the IP3 dependence of the absolute and normalized open probability (P o), respectively, for the two types of IP3Rs isolated from mouse rod bipolar cells. Based on the biophysical properties, the group of IP3Rs with high IP3 sensitivity is identified as IP3R2 (type 2 IP3R; n = 9) and the IP3R with intermediate IP3 sensitivity as IP3R1 (type 1 IP3R; n = 11).
The authors thank Margaret, Richard, and Sara Koulen for generous support and encouragement. 
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Figure 2.
 
Localization of IP3R and RyR immunoreactivity in mouse rod bipolar cells. Acutely isolated rod bipolar cells were fixed and stained for type 1 (C), type 2 (D), and type 3 (E) IP3R immunoreactivity, as well as for RyR immunoreactivity (F). Controls include secondary antibody controls for both types of secondary antibodies used [anti-mouse (A); anti-rabbit (B)], as well as labeling of mGluR1 (G) and mGluR5 (H) immunoreactivity. For each panel, differential interference contrast images show the typical rod bipolar cell morphology with dendritic tree, soma axon, and axon terminal system.
Figure 2.
 
Localization of IP3R and RyR immunoreactivity in mouse rod bipolar cells. Acutely isolated rod bipolar cells were fixed and stained for type 1 (C), type 2 (D), and type 3 (E) IP3R immunoreactivity, as well as for RyR immunoreactivity (F). Controls include secondary antibody controls for both types of secondary antibodies used [anti-mouse (A); anti-rabbit (B)], as well as labeling of mGluR1 (G) and mGluR5 (H) immunoreactivity. For each panel, differential interference contrast images show the typical rod bipolar cell morphology with dendritic tree, soma axon, and axon terminal system.
Figure 1.
 
Western blot showing the expression of IP3Rs and RyRs in ON-bipolar cells (ONBC) and controls (C). Homogenates of enzymatically isolated mouse rod bipolar cells were analyzed. Mouse cerebellum, liver, pancreas, and brain homogenates were loaded as controls for IP3R type 1, IP3R type 2, IP3R type 3, and RyRs, respectively. Twenty micrograms total protein per lane were loaded on 4%–12% gradient gels to detect individual intracellular calcium channels. IP3R isoform specific antibodies detected approximately 250 kDa bands, the pan-RyR antibody detected a high molecular weight band of approximately 550 kDa (numbers and arrows, position and size of protein standards in kDa, respectively). Only IP3Rs type 1 and 2 could be detected in RBCs.
Figure 1.
 
Western blot showing the expression of IP3Rs and RyRs in ON-bipolar cells (ONBC) and controls (C). Homogenates of enzymatically isolated mouse rod bipolar cells were analyzed. Mouse cerebellum, liver, pancreas, and brain homogenates were loaded as controls for IP3R type 1, IP3R type 2, IP3R type 3, and RyRs, respectively. Twenty micrograms total protein per lane were loaded on 4%–12% gradient gels to detect individual intracellular calcium channels. IP3R isoform specific antibodies detected approximately 250 kDa bands, the pan-RyR antibody detected a high molecular weight band of approximately 550 kDa (numbers and arrows, position and size of protein standards in kDa, respectively). Only IP3Rs type 1 and 2 could be detected in RBCs.
Figure 3.
 
Group I mGluR agonists induce spatiotemporally differential Ca2+ transients in isolated mouse rod bipolar cells. (A) Montage of a representative imaging experiment visualizing the cytosolic Ca2+ concentration at 0 seconds, during 10 and 50 seconds, and after 200 seconds, application of 100 μM S-DHPG. A differential interference contrast image to the right shows the main subcellular regions of the rod bipolar cell, dendritic tree (D), soma (S), axon (A), and axon terminal system (AT). Although a Ca2+ transient in the dendrites was observed immediately after stimulation (10 seconds), a Ca2+ transient with larger amplitude was seen in the soma after a delay and after the peak of the dendritic Ca2+ transient (50 seconds). (B) Changes in the intracellular Ca2+ concentration in two regions of a representative rod bipolar cell, dendrites (black) and soma (gray). After a single 0.5 second bolus application of a low dose of an mGluR1 agonist (10 μM S-DHPG, indicated by arrow to the left), only the dendrites responded with a Ca2+ transient, whereas a higher concentration of mGluR1 agonist (100 μM S-DHPG, 0.5 second bolus application indicated by the arrow to the right) induced Ca2+ transients in both dendrites and soma.
Figure 3.
 
Group I mGluR agonists induce spatiotemporally differential Ca2+ transients in isolated mouse rod bipolar cells. (A) Montage of a representative imaging experiment visualizing the cytosolic Ca2+ concentration at 0 seconds, during 10 and 50 seconds, and after 200 seconds, application of 100 μM S-DHPG. A differential interference contrast image to the right shows the main subcellular regions of the rod bipolar cell, dendritic tree (D), soma (S), axon (A), and axon terminal system (AT). Although a Ca2+ transient in the dendrites was observed immediately after stimulation (10 seconds), a Ca2+ transient with larger amplitude was seen in the soma after a delay and after the peak of the dendritic Ca2+ transient (50 seconds). (B) Changes in the intracellular Ca2+ concentration in two regions of a representative rod bipolar cell, dendrites (black) and soma (gray). After a single 0.5 second bolus application of a low dose of an mGluR1 agonist (10 μM S-DHPG, indicated by arrow to the left), only the dendrites responded with a Ca2+ transient, whereas a higher concentration of mGluR1 agonist (100 μM S-DHPG, 0.5 second bolus application indicated by the arrow to the right) induced Ca2+ transients in both dendrites and soma.
Figure 4.
 
Two types of IP3R with respect to their IP3 sensitivity can be isolated from the ER of mouse rod bipolar cells. Activity of individual IP3Rs was recorded with 0.5 μM free Ca2+, 500 μM ATP as IP3R co-agonists, 10 μM ruthenium red to block RyRs present on the cytoplasmic side of the channel and Ba2+ as the current carrier on the luminal side of the channel. Bars to the right of each trace indicate zero current levels of channel activity and downward deflections indicate channel openings. (A) and (B) Electrophysiological recordings of two representative experiments with three traces at 0.01, 0.1, and 1 μM IP3, exemplifying the two types of IP3Rs that could be observed in mouse rod bipolar cells. (C) and (D) summarize the IP3 dependence of the absolute and normalized open probability (P o), respectively, for the two types of IP3Rs isolated from mouse rod bipolar cells. Based on the biophysical properties, the group of IP3Rs with high IP3 sensitivity is identified as IP3R2 (type 2 IP3R; n = 9) and the IP3R with intermediate IP3 sensitivity as IP3R1 (type 1 IP3R; n = 11).
Figure 4.
 
Two types of IP3R with respect to their IP3 sensitivity can be isolated from the ER of mouse rod bipolar cells. Activity of individual IP3Rs was recorded with 0.5 μM free Ca2+, 500 μM ATP as IP3R co-agonists, 10 μM ruthenium red to block RyRs present on the cytoplasmic side of the channel and Ba2+ as the current carrier on the luminal side of the channel. Bars to the right of each trace indicate zero current levels of channel activity and downward deflections indicate channel openings. (A) and (B) Electrophysiological recordings of two representative experiments with three traces at 0.01, 0.1, and 1 μM IP3, exemplifying the two types of IP3Rs that could be observed in mouse rod bipolar cells. (C) and (D) summarize the IP3 dependence of the absolute and normalized open probability (P o), respectively, for the two types of IP3Rs isolated from mouse rod bipolar cells. Based on the biophysical properties, the group of IP3Rs with high IP3 sensitivity is identified as IP3R2 (type 2 IP3R; n = 9) and the IP3R with intermediate IP3 sensitivity as IP3R1 (type 1 IP3R; n = 11).
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