Noga Vardi

Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca2+ release channels

The inositol trisphosphate (InsP3) receptor (InsP3R) is a ubiquitously expressed intracellular Ca2+ channel that mediates complex cytoplasmic Ca2+ signals, regulating diverse cellular processes, including synaptic plasticity. Activation of the InsP3R channel is normally thought to require binding of InsP3 derived from receptor-mediated activation of phosphatidylinositol lipid hydrolysis. Here we identify a family of neuronal Ca2+-binding proteins as high-affinity protein agonists of the InsP3R, which bind to the channel and activate gating in the absence of InsP3. CaBP/caldendrin, a subfamily of the EF-hand-containing neuronal calcium sensor family of calmodulin-related proteins, bind specifically to the InsP3-binding region of all three InsP3R channel isoforms with high affinity (Ka ≈ 25 nM) in a Ca2+-dependent manner (Ka ≈ 1 μM). Binding activates single-channel gating as efficaciously as InsP3, dependent on functional EF-hands in CaBP. In contrast, calmodulin neither bound with high affinity nor activated channel gating. CaBP1 and the type 1 InsP3R associate in rat whole brain and cerebellum lysates, and colocalize extensively in subcellular regions in cerebellar Purkinje neurons. Thus, InsP3R-mediated Ca2+ signaling in cells is possible even in the absence of InsP3 generation, a process that may be particularly important in responding to and shaping changes in intracellular Ca2+ concentration by InsP3-independent pathways and for localizing InsP3-mediated Ca2+ signals to individual synapses.

Coordinated multivesicular release at a mammalian ribbon synapse

Traditional models of synaptic transmission hold that release sites within an active zone operate independently. Although the release of multiple vesicles (multivesicular release; MVR) from single active zones occurs at some central synapses, MVR is not thought to require coordination among release sites. Ribbon synapses seem to be optimized to release many vesicles over an extended period, but the dynamics of MVR at ribbon synapses is unknown. We examined MVR at a ribbon synapse in a retinal slice preparation using paired recordings from presynaptic rod bipolar and postsynaptic AII amacrine cells. When evoked release was highly desynchronized, discrete postsynaptic events were larger than quantal miniature excitatory postsynaptic currents (mEPSCs) but had the same time course. The amplitude of these multiquantal mEPSCs, which seem to arise from the essentially simultaneous release of multiple vesicles, was reduced by lowering release probability. The release synchrony reflected in these multivesicular events suggests that release within an active zone is coordinated during MVR.

Localization of mGluR6 to Dendrites of ON Bipolar Cells in Primate Retina

We prepared antibodies selective for the C‐terminus of the human mGluR6 receptor and used confocal and electron microscopy to study the patterns of immunostaining in retina of monkey, cat, and rabbit. In all three species punctate stain was restricted to the outer plexiform layer. In monkey, stain was always observed in the central element of the postsynaptic “triad” of rod and cone terminals. In monkey peripheral retina, stain was seen only in central elements, but in the fovea, stain was also observed in some dendrites contacting the base of the cone terminal. S‐cone terminals, identified by staining for S opsin, showed staining of postsynaptic dendrites. These were identified as dendrites of the ON S‐cone bipolar cell by immunostaining for the marker cholecystokinin precursor. The staining pattern suggests that all types of ON bipolar cells, despite their marked differences in function, express a single isoform of mGluR6. Ultrastructurally, mGluR6 was located not on the tip of the central element, near the site of vesicle release, but on its base at the mouth of the invagination, 400–800 nm from the release site. Thus, the mGluR6 receptors of ON bipolar cells lie at about the same distance from sites of vesicle release as the iGluR receptors of OFF bipolar cells at the basal contacts. J. Comp. Neurol. 423:402–412, 2000.

ALPHA SUBUNIT OF GO LOCALIZES IN THE DENDRITIC TIPS OF ON BIPOLAR CELLS

The metabotropic glutamate receptor (mGluR6), expressed by rod bipolar cells and ON cone bipolar cells, activates a trimeric guanine nucleotide‐binding protein (G‐protein) that ultimately closes a cation channel. The G‐protein remains unidentified, but the alpha subunit of Go (Goα) has been suggested as a candidate because it is present in rod bipolar cells. However, the precise subcellular distribution of Go within the rod bipolar cell, and its distribution among cone bipolar cells was not determined. This information is important in assessing the hypothesis that Go couple mGluR6 to its effector. Here I report the distribution of Go (alpha subunit) by immunostaining in several mammalian retinas. The overall distribution is conserved across mammalian species: strongest in the dendrites of ON bipolar cells, moderate in their somas, weak in their axons, and absent from their terminals. Goα is also present in some amacrine somas and processes. In monkey fovea, where rods and rod bipolar cells are absent, Goα is present in about half of the bipolar somas which occupy the upper tiers of the bipolar layer, and are therefore identified as ON cone bipolar cells. Ultrastructurally, in monkey and cat, Goα is present in the dendritic tips of rod bipolar cells and ON cone bipolar cells, which are identified by their invaginating contacts. It is absent from OFF cone bipolar dendrites, which are identified by their flat contacts. It is also absent from axons entering the inner plexiform layer, and their terminals. In the primary dendrites, stain for Goα mainly associates with the plasma membrane, but in the dendritic tips it is also present in the cytosol. Apparently, Goα is expressed by the same bipolar cells that also express mGluR6, and is concentrated at the same subcellular location. Thus, Goα could serve to couple mGluR6 to later stages of its signaling cascade. J. Comp. Neurol. 395:43–52, 1998.

Axons and dendrites originate from neuroepithelial-like processes of retinal bipolar cells.

The cellular mechanisms underlying axogenesis and dendritogenesis are not completely understood. The axons and dendrites of retinal bipolar cells, which contact their synaptic partners within specific laminae in the inner and outer retina, provide a good system for exploring these issues. Using transgenic mice expressing enhanced green fluorescent protein (GFP) in a subset of bipolar cells, we determined that axonal and dendritic arbors of these interneurons develop directly from apical and basal processes attached to the outer and inner limiting membranes, respectively. Selective stabilization of processes contributed to stratification of axonal and dendritic arbors within the appropriate synaptic layer. This unusual mode of axogenesis and dendritogenesis from neuroepithelial-like processes may act to preserve neighbor-neighbor relationships in synaptic wiring between the outer and inner retina.

ON cone bipolar cells in rat express the metabotropic receptor mGluR6.

The rod bipolar cell and about five types of ON cone bipolar cells depolarize to light by employing a sign-reversing metabotropic glutamate receptor. Glutamate responses are similar in both rod bipolar and cone bipolar cells, but the receptor mediating this response (mGluR6) was so far demonstrated only in rod bipolar cells. To test if ON cone bipolar cells also express mGluR6, we immunoreacted rat retina with an antibody specific for mGluR6, and studied the staining from serial ultrathin sections. We demonstrate that mGluR6 is indeed expressed in the dendritic tips of cone bipolar cells, the majority of which receive a ribbon synapse, and thus probably are ON cone bipolar cells. We further show that half of the dendritic tips contacting the cones stain for mGluR6, thus implying that all ON cone bipolar cell types express mGluR6.

Differential expression of ionotropic glutamate receptor subunits in the outer retina

Ionotropic glutamate receptors (iGluRs) are extremely diverse in their subunit compositions. To understand the functional consequences of this diversity, it is necessary to know the subunits that are expressed by known cell types. By using immunocytochemistry with light and electron microscopy, we localized several subunits (GluR2/3, GluR4, and GluR6/7) in cat retinal neurons, postsynaptic to photoreceptors. Type A horizontal cells express all three subunits strongly, whereas type B horizontal cells express GluR2/3 strongly, GluR6/7 weakly, and do not express GluR4. When they are present, the subunits are expressed strongly throughout the cytoplasm of the somata and primary dendrites; however, in the terminals, they are concentrated at the postsynaptic region, just opposite the presumed site of photoreceptor glutamate release. Surprisingly, all bipolar cell classes (OFF cone bipolar cells, ON cone bipolar cells, and rod bipolar cells) express at least one iGluR subunit at their dendritic tips. Cone bipolar cells forming basal contacts with the cones (presumably OFF cells) express all three subunits in association with the electron‐dense postsynaptic membrane. Invaginating dendrites of cone bipolar cells (presumably ON cells) express GluR2/3 and GluR4. Rod bipolar cells (ON cells) express GluR2/3 in their invaginating dendrites. The function of iGluRs in horizontal cells and OFF bipolar cells clearly is to mediate their light responses. GluR6/7 subunit in the receptor of these cells may be responsible for the dopamine‐mediated enhancement of glutamate responses that have been observed previously in these cells. The function of iGluRs in ON bipolar cells remains an enigma. J. Comp. Neurol. 405:173–184, 1999.

Subcellular localization of GABAA receptor on bipolar cells in macaque and human retina

The subcellular distribution of GABAA receptor in the macaque and human retina was studied by immunocytochemistry with monoclonal antibodies for the alpha and beta subunits with a particular focus on bipolar cells. Immunoreactivity to GABAA receptor was present on dendritic tips of all bipolar cells. The stain was strongest on bipolar membranes in apposition to horizontal cell processes. Stain was concentrated on the tips of flat and invaginating cone bipolar cells at the base of the cone pedicle and on the invaginating tips of rod bipolar cells. Stain on the cone pedicle membrane was restricted to sites of apposition to stained bipolar dendrites; pedicle membrane in apposition to horizontal cell processes was unstained. Stain was also present on bipolar axon terminals in both on and off strata of the inner plexiform layer. All bipolar cell somas stained faintly; horizontal and Müller cell somas were unstained. The alpha and beta subunits distributed similarly in monkey and human retina. Presence of GABAA receptor on the bipolar dendritic tips suggests that horizontal cells directly affect bipolar cells. Thus, GABAA receptor might mediate the receptive field surround of both off and on bipolar cells. Presence of GABAA receptor on bipolar axon terminals suggests that much of the inhibition feeding back from GABAergic amacrine to bipolar cells is GABAA-mediated.

Horizontal cells in cat and monkey retina express different isoforms of glutamic acid decarboxylase

The neurotransmitter used by horizontal cells in mammals has not been identified. GABA has been the leading candidate, but doubt has remained because of failure to clearly demonstrate the GABA synthetic enzyme, glutamic acid decarboxylase (GAD) in these cells. Because GAD was recently shown to exist as two isoforms, 65 kDa and 67 kDa, we considered whether there might be a mismatch between the forms of GAD expressed in horizontal cells and the probes used to detect it. Accordingly, we stained sections of mammalian retina with antibodies specific for each isoform. Cat horizontal cells of both types (A and B) were immunoreactive for GAD67 but negative for GAD65; monkey horizontal cells of both types (H(I) and HII) were positive for GAD65 and negative for GAD67. The findings reconcile previous, apparently conflicting, observations and strengthen considerably the hypothesis that mammalian horizontal cells are GABAergic.

Identification of a G-protein in depolarizing rod bipolar cells

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G(o), reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). G(o) antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G(o) is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.