Direction selectivity in retinal bipolar cell axon terminals.

The ability to encode the direction of image motion is fundamental to our sense of vision. Direction selectivity along the four cardinal directions is thought to originate in direction-selective ganglion cells (DSGCs) because of directionally tuned GABAergic suppression by starburst cells. Here, by utilizing two-photon glutamate imaging to measure synaptic release, we reveal that direction selectivity along all four directions arises earlier than expected at bipolar cell outputs. Individual bipolar cells contained four distinct populations of axon terminal boutons with different preferred directions. We further show that this bouton-specific tuning relies on cholinergic excitation from starburst cells and GABAergic inhibition from wide-field amacrine cells. DSGCs received both tuned directionally aligned inputs and untuned inputs from among heterogeneously tuned glutamatergic bouton populations. Thus, directional tuning in the excitatory visual pathway is incrementally refined at the bipolar cell axon terminals and their recipient DSGC dendrites by two different neurotransmitters co-released from starburst cells.

LRRTM4 is a member of the transsynaptic complex between rod photoreceptors and bipolar cells.

Leucine rich repeat transmembrane (LRRTM) proteins are synaptic adhesion molecules with roles in synapse formation and signaling. LRRTM4 transcripts were previously shown to be enriched in rod bipolar cells (BCs), secondary neurons of the retina that form synapses with rod photoreceptors. Using two different antibodies, LRRTM4 was found to reside primarily at rod BC dendritic tips, where it colocalized with the transduction channel protein, TRPM1. LRRTM4 was not detected at dendritic tips of ON-cone BCs. Following somatic knockout of LRRTM4 in BCs by subretinal injection and electroporation of CRISPR/Cas9, LRRTM4 was abolished or reduced in the dendritic tips of transfected cells. Knockout cells had a normal complement of TRPM1 at their dendritic tips, while GPR179 accumulation was partially reduced. In experiments with heterologously expressed protein, the extracellular domain of LRRTM4 was found to engage in heparan-sulfate dependent binding with pikachurin. These results implicate LRRTM4 in the GPR179-pikachurin-dystroglycan transsynaptic complex at rod synapses.

Identification of PKCα-dependent phosphoproteins in mouse retina.

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906. SIGNIFICANCE: Retinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.

Bipolar cell gap junctions serve major signaling pathways in the human retina.

Connexin36 (Cx36) constituent gap junctions (GJ) throughout the brain connect neurons into functional syncytia. In the retina they underlie the transmission, averaging and correlation of signals prior conveying visual information to the brain. This is the first study that describes retinal bipolar cell (BC) GJs in the human inner retina, whose function is enigmatic even in the examined animal models. Furthermore, a number of unique features (e.g. fovea, trichromacy, midget system) necessitate a reexamination of the animal model results in the human retina. Well-preserved postmortem human samples of this study are allowed to identify Cx36 expressing BCs neurochemically. Results reveal that both rod and cone pathway interneurons display strong Cx36 expression. Rod BC inputs to AII amacrine cells (AC) appear in juxtaposition to AII GJs, thus suggesting a strategic AII cell targeting by rod BCs. Cone BCs serving midget, parasol or koniocellular signaling pathways display a wealth of Cx36 expression to form homologously coupled arrays. In addition, they also establish heterologous GJ contacts to serve an exchange of information between parallel signaling streams. Interestingly, a prominent Cx36 expression was exhibited by midget system BCs that appear to maintain intimate contacts with bistratified BCs serving other pathways. These findings suggest that BC GJs in parallel signaling streams serve both an intra- and inter-pathway exchange of signals in the human retina.

Identification of the minimal promoter for specific expression of the GABAρ1 receptor in retinal bipolar cells.

γ-aminobutyric acid (GABA)ρ receptors regulate rapid synaptic ion currents in the axon end of retinal ON bipolar neurons, acting as a point of control along the visual pathway. In the GABAρ1 subunit knock out mouse, inhibition mediated by this receptor is totally eliminated, showing its role in neural transmission in retina. GABAρ1 mRNA is expressed in mouse retina after post-natal day 7, but little is known about its transcriptional regulation. To identify the GABAρ1 promoter, in silico analyses were performed and indicated that a 0.290-kb fragment, flanking the 5'-end of the GABAρ1 gene, includes putative transcription factor-binding sites, two Inr elements, and lacks a TATA-box. A rapid amplification of cDNA ends (RACE) assay showed three transcription start sites (TSS) clustered in the first exon. Luciferase reporter assays indicated that a 0.232-kb fragment upstream from the ATG is the minimal promoter in transfected cell lines and in vitro electroporated retinae. The second Inr and AP1 site are important to activate transcription in secretin tumor cells (STC-1) and retina. Finally, the 0.232-kb fragment drives green fluorescent protein (GFP) expression to the inner nuclear layer, where bipolar cells are present. This first work paves the way for further studies of molecular elements that control GABAρ1 transcription and regulate its expression during retinal development.

Optimization of a GCaMP calcium indicator for neural activity imaging.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

PKCζ-interacting protein ZIP3 is generated by intronic polyadenylation, and is expressed in the brain and retina of the rat.

Scaffold proteins contain multiple protein-protein interaction modules that physically assemble functionally related proteins into larger complexes. ZIPs [PKC (protein kinase C) ζ-interacting proteins] link the enzymatic activity of the atypical PKC isoforms PKCλ/ι or PKCζ to target proteins and are associated with neurodegenerative disorders. In the rat, alternative splicing generates three ZIP variants. Previously, we identified the ZIP3 transcript, containing 13 C-terminal amino acids encoded by intron 4, in the rat CNS (central nervous system). In the present study, we identified intronic polyadenylation signals in rat and human ZIP genes [known as SQSTM1 (sequestosome-1) in humans] and detected the corresponding ZIP3-like transcripts. In addition, we generated ZIP3-specific immune sera and observed expression of the protein in the brain and retina of the adult rat. In the retina, ZIP3 is present in nuclear layers where it co-localizes with PKCζ. An immune serum recognizing all three ZIP isoforms labelled the same cells as the newly generated ZIP3-specific antibodies and, in addition, stained both synaptic layers of the retina. There, ZIPs are localized in axon terminals of rod bipolar cells that also contain ZIP-interacting PKCζ and GABA(C) (γ-aminobutyric acid type C) receptors. In summary, we detected ZIP3-like transcripts in rat- and human-derived samples and describe the expression of ZIP3 in the rat CNS.

Role of afferents in the differentiation of bipolar cells in the mouse retina.

To establish dendritic arbors that integrate properly into a neural circuit, neurons must rely on cues from the local environment. The neurons presynaptic to these arbors, the afferents, are one potential source of these cues, but the particular dendritic features they regulate remain unclear. Retinal bipolar cells can be classified by the type of photoreceptor, cone or rod, forming synaptic contacts with their dendrites, suggesting a potential role of these afferents in shaping the bipolar cell dendritic arbor. In the present investigation, the role of photoreceptors in directing the differentiation of bipolar cells has been studied using two genetically modified "coneless" and "conefull" mice. Single cone (Type 7/CB4a) and rod bipolar cells were labeled with DiI to reveal the entire dendritic arbor and subsequently analyzed for several morphological features. For both cone and rod bipolar cells, the dendritic field area, number of dendritic terminals, and stratification of terminals in the outer plexiform layer were comparable among coneless, conefull, and wild-type retinas, and the overall morphological appearance of each type of cell was essentially conserved, indicating an independence from afferent specification. The presence of normal afferents was, however, found to be critical for the proper spatial distribution of dendritic terminals, exhibiting a clustered distribution for the cone bipolar cells and a dispersed distribution for the rod bipolar cells. These results demonstrate a selectivity in the afferent dependency of bipolar cell differentiation, their basic morphogenetic plan commanded cell intrinsically, and their fine terminal connectivity directed by the afferents themselves.

Immunocytochemical analysis of photoreceptors in the tiger salamander retina.

In the tiger salamander retina, visual signals are transmitted to the inner retina via six morphologically distinct types of photoreceptors: large/small rods, large/small single cones, and double cones composed of principal and accessory members. The objective of this study was to determine the morphology of these photoreceptors and their synaptic interconnection with bipolar cells and horizontal cells in the outer plexiform layer (OPL). Here we showed that glutamate antibodies labeled all photoreceptors and recovering antibodies strongly labeled all cones and weakly labeled all rods. Antibodies against calbindin selectively stained accessory members of double cones. Antibodies against S-cone opsin stained small rods, a subpopulation of small single cones, and the outer segments of accessory double cones and a subtype of unidentified single cones. On average, large rods and small S-cone opsin positive rods accounted for 98.6% and 1.4% of all rods, respectively. Large/small cones, principle/accessory double cones, S-cone opsin positive small single cones, and S-cone opsin positive unidentified single cones accounted for about 66.9%, 23%, 4.5%, and 5.6% of the total cones, respectively. Moreover, the differential connection between rods/cones and bipolar/horizontal cells and the wide distribution of AMPA receptor subunits GluR2/3 and GluR4 at the rod/cone synapses were observed. These results provide anatomical evidence for the physiological findings that bipolar/horizontal cells in the salamander retina are driven by rod/cone inputs of different weights, and that AMPA receptors play an important role in glutamatergic neurotransmission at the first visual synapses. The different photoreceptors selectively contacting bipolar and horizontal cells support the idea that visual signals may be conveyed to the inner retina by different functional pathways in the outer retina.

Novel processes invaginate the pre-synaptic terminal of retinal bipolar cells.

Mixed-rod cone bipolar (Mb) cells of goldfish retina have large synaptic terminals (10 microm in diameter) that make 60-90 ribbon synapses mostly onto amacrine cells and rarely onto ganglion cells and, in return, receive 300-400 synapses from gamma-aminobutyric acid (GABA)-ergic amacrine cells. Tissue viewed by electron microscopy revealed the presence of double-membrane-bound processes deep within Mb terminals. No membrane specializations were apparent on these invaginating processes, although rare vesicular fusion was observed. These invaginating dendrites were termed "InDents". Mb bipolar cells were identified by their immunoreactivity for protein kinase C. Double-label immunofluorescence with other cell-type-specific labels eliminated Müller cells, efferent fibers, other Mb bipolar cells, dopaminergic interplexiform cells, and somatostatin amacrine cells as a source of the InDents. Confocal analysis of double-labeled tissue clearly showed dendrites of GABA amacrine cells, backfilled ganglion cells, and dendrites containing PanNa immunoreactivity extending into and passing through Mb terminals. Nearly all Mb terminals showed evidence for the presence of InDents, indicating their common presence in goldfish retina. No PanNa immunoreactivity was found on GABA or ganglion cell InDents, suggesting that a subtype of glycine amacrine cell contained voltage-gated Na channels. Thus, potassium and calcium voltage-gated channels might be present on the InDents and on the Mb terminal membrane opposed to the InDents. In addition to synaptic signaling at ribbon and conventional synapses, Mb bipolar cells may exchange information with InDents by an alternative signaling mechanism.

Distribution of phosphorylated protein kinase C alpha in goldfish retinal bipolar synaptic terminals: control by state of adaptation and pharmacological treatment.

Protein kinase C (PKC) is a signalling enzyme critically involved in many aspects of synaptic plasticity. In cyprinid retinae, the PKC alpha isoform is localized in a subpopulation of depolarizing bipolar cells that show adaptation-related morphological changes of their axon terminals. We have studied the subcellular localization of phosphorylated PKC alpha (pPKC alpha) in retinae under various conditions by immunohistochemistry with a phosphospecific antibody. In dark-adapted retinae, pPKC alpha immunoreactivity is weak in the cytoplasm of synaptic terminals, labelling being predominantly associated with the membrane compartment. In light-adapted cells, immunoreactivity is diffusely distributed throughout the terminal. Western blot analysis has revealed a reduction of pPKC alpha immunoreactivity in cytosolic fractions of homogenized dark-adapted retinae compared with light-adapted retinae. Pharmacological experiments with the isoform-specific PKC blocker Goe6976 have shown that inhibition of the enzyme influences immunolabelling for pPKC alpha, mimicking the effects of light on the subcellular distribution of immunoreactivity. Our findings suggest that the state of adaptation modifies the subcellular localization of a signalling molecule (PKC alpha) at the ribbon-type synaptic complex. We propose that changes in the subcellular distribution of PKC alpha immunoreactivity might be one component regulating the strength of the signal transfer of the bipolar cell terminal.