Biology subjects

Wakeham, C. M.

Publications and source records attributed to Wakeham, C. M..

4 recordsLinked to original sources

bioRxiv2024

Dopamine regulates the membrane potential and glycine release of AII amacrine cells via D1-like receptor modulation of gap junction coupling.

Dopamine plays a pivotal role in adjusting the flow of information across the retina as luminance changes from night to day. Here we show, under dim photopic conditions, that both dopamine and a D1-like receptor (D1R) agonist hyperpolarized the resting membrane potential (Vm) of AII amacrine cells (AII-ACs). Surprisingly, in the presence of glutamatergic and GABAergic synaptic blockers that isolate glycinergic synapses, D1R agonists are without effect. However, a D1R antagonist depolarized Vm and reduced the input resistance of AII-ACs in wild type mice, but not in Cx36-/- mice. Accordingly, D1R antagonists enhanced tonic glycinergic transmission to type-2 OFF-cone bipolar cells (OFF-CBCs). D1Rs thus adjust the Vm and excitability of AII-ACs and, thereby, the level of glycine release to OFF-CBCs by regulating gap junction coupling with ON cone bipolar cells. Our findings provide insights into how the retina may use dopamine to adapt crossover inhibitory microcircuits during changes in luminance.

neuroscience↗

bioRxiv2022

Trophoblast glycoprotein is required for efficient synaptic vesicle exocytosis from retinal rod bipolar cells.

Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs. We now examine the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electrophysiological techniques. The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents. Thus, TPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/501380v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@13d5916org.highwire.dtl.DTLVardef@a3c259org.highwire.dtl.DTLVardef@12c74e0org.highwire.dtl.DTLVardef@1ad43a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Identification of PKC -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 (PKC), the targets of PKC phosphorylation in the retina have not been identified. PKC activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKC antibody and antibodies to phosphorylated PKC motifs. PKC activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKC-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKC knockout (PKC-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKC-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 PKC-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.\n\nSignificanceRetinal 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, PKC, though the specific mechanism by which PKC modulates RBC physiology is unknown. This study examined PKC phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKC-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKC-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKC-dependent modulation of RBC physiology.\n\nHighlightsO_LIPKC is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.\nC_LIO_LIProteins showing differential phosphorylation between phorbol ester-treated wild type and PKC-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).\nC_LIO_LIThe PKC-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKC-dependent modulation of RBC physiology.\nC_LI

neuroscience↗

bioRxiv2019

Expression and distribution of Trophoblast Glycoprotein in the mouse retina

We recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKC-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKC knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

neuroscience↗