Terminals Of Retinal Bipolar Cells Research Articles

Transient expression of a GABA receptor subunit during early development is critical for inhibitory synapse maturation and function

Developing neural circuits, including GABAergic circuits, switch receptor types. But the role of early GABA receptor expression for establishment of functional inhibitory circuits remains unclear. Tracking the development of GABAergic synapses across axon terminals of retinal bipolar cells (BCs), we uncovered a crucial role of early GABAA receptor expression for the formation and function of presynaptic inhibitory synapses. Specifically, early α3-subunit-containing GABAA (GABAAα3) receptors are a key developmental organizer. Before eye opening, GABAAα3 gives way to GABAAα1 at individual BC presynaptic inhibitory synapses. The developmental downregulation of GABAAα3 is independent of GABAAα1 expression. Importantly, lack of early GABAAα3 impairs clustering of GABAAα1 and formation of functional GABAA synapses across mature BC terminals. This impacts the sensitivity of visual responses transmitted through the circuit. Lack of early GABAAα3 also perturbs aggregation of LRRTM4, the organizing protein at GABAergic synapses of rod BC terminals, and their arrangement of output ribbon synapses.

Ascorbic acid antagonizes the sedative effect of diazepam possibly through inhibition of GABA(Aρ₁) and GABA(B1) receptors

Gamma-aminobutyric acid (GABA) receptors belong to a ligand-gated ion channels family and are markedly expressed at the axon terminals of retinal bipolar cells. Ascorbic acid (AA), a known and vital antioxidant in the brain can modulate GABA receptors. We postulate that AA would antagonize benzodiazepines' effect via GABA receptor(s) interacting pathway. Here, we evaluated the modulatory sedative effect of AA on diazepam (DZP)'s anxiolytic effects in Swiss albino mice. The anxiolytic study was accomplished by using open-field, hole-board, and by swing and light-dark tests taking DZP as a standard anxiolytic drug. To understand the possible modulatory effects of AA, animals were co-administered with AA and DZP and/or its antagonist flumazenil (FLU). Additionally, an in-silico study was undertaken against GABA(A1), GABA(B1), and GABA(Aρ₁) receptors. Data suggest that AA at 25 mg/kg (i.p.) increased (p<0.05) the number of field cross, rearing, number of hole cross, and swing and residence, while decreased grooming and dark residence parameters as compared to the control and DZP groups. In addition, AA and/or FLU combined with DZP (2 mg/kg, i.p.) reversed DZP-mediated sedative effects in mice. Results from in silico study suggest that AA has good interactions with GABA(Aρ₁) and GABA(B1) receptors. In conclusion, DZP is a GABA receptor agonist and AA may reverse DZP-mediated sedative effects in a non-competitive binding fashion in mice through inhibition of GABA(Aρ₁) and GABA(B1) receptors.

Paired-Pulse Plasticity in the Strength and Latency of Light-Evoked Lateral Inhibition to Retinal Bipolar Cell Terminals

Synapses in the inner plexiform layer of the retina undergo short-term plasticity that may mediate different forms of adaptation to regularities in light stimuli. Using patch-clamp recordings from axotomized goldfish Mb bipolar cell (BC) terminals with paired-pulse light stimulation, we isolated and quantified the short-term plasticity of GABAergic lateral IPSCs (L-IPSCs). Bright light stimulation evoked ON and OFF L-IPSCs in axotomized BCs, which had distinct onset latencies (∼50-80 and ∼70-150 ms, respectively) that depended on background light adaptation. We observed plasticity in both the synaptic strength and latency of the L-IPSCs. With paired light stimulation, latencies of ON L-IPSCs increased at paired-pulse intervals (PPIs) of 50 and 300 ms, whereas OFF L-IPSC latencies decreased at the 300 ms PPI. ON L-IPSCs showed paired-pulse depression at intervals <1 s, whereas OFF L-IPSCs showed depression at intervals ≤1 s and amplitude facilitation at longer intervals (1-2 s). This biphasic form of L-IPSC plasticity may underlie adaptation and sensitization to surround temporal contrast over multiple timescales. Block of retinal signaling at GABA(A)Rs and AMPARs differentially affected ON and OFF L-IPSCs, confirming that these two types of feedback inhibition are mediated by distinct and convergent retinal pathways with different mechanisms of plasticity. We propose that these plastic changes in the strength and timing of L-IPSCs help to dynamically shape the time course of glutamate release from ON-type BC terminals. Short-term plasticity of L-IPSCs may thus influence the strength, timing, and spatial extent of amacrine and ganglion cell inhibitory surrounds.

Pharmacological Analysis of the Activation and Receptor Properties of the Tonic GABACR Current in Retinal Bipolar Cell Terminals

GABAergic inhibition in the central nervous system (CNS) can occur via rapid, transient postsynaptic currents and via a tonic increase in membrane conductance, mediated by synaptic and extrasynaptic GABAA receptors (GABAARs) respectively. Retinal bipolar cells (BCs) exhibit a tonic current mediated by GABACRs in their axon terminal, in addition to synaptic GABAAR and GABACR currents, which strongly regulate BC output. The tonic GABACR current in BC terminals (BCTs) is not dependent on vesicular GABA release, but properties such as the alternative source of GABA and the identity of the GABACRs remain unknown. Following a recent report that tonic GABA release from cerebellar glial cells is mediated by Bestrophin 1 anion channels, we have investigated their role in non-vesicular GABA release in the retina. Using patch-clamp recordings from BCTs in goldfish retinal slices, we find that the tonic GABACR current is not reduced by the anion channel inhibitors NPPB or flufenamic acid but is reduced by DIDS, which decreases the tonic current without directly affecting GABACRs. All three drugs also exhibit non-specific effects including inhibition of GABA transporters. GABACR ρ subunits can form homomeric and heteromeric receptors that differ in their properties, but BC GABACRs are thought to be ρ1-ρ2 heteromers. To investigate whether GABACRs mediating tonic and synaptic currents may differ in their subunit composition, as is the case for GABAARs, we have examined the effects of two antagonists that show partial ρ subunit selectivity: picrotoxin and cyclothiazide. Tonic and synaptic GABACR currents were differentially affected by both drugs, suggesting that a population of homomeric ρ1 receptors contributes to the tonic current. These results extend our understanding of the multiple forms of GABAergic inhibition that exist in the CNS and contribute to visual signal processing in the retina.

Activation of the Tonic GABACReceptor Current in Retinal Bipolar Cell Terminals by Nonvesicular GABA Release

Within the second synaptic layer of the retina, bipolar cell (BC) output to ganglion cells is regulated by inhibitory input to BC axon terminals. GABA(A) receptors (GABA(A)Rs) mediate rapid synaptic currents in BC terminals, whereas GABA(C) receptors (GABA(C)Rs) mediate slow evoked currents and a tonic current, which is strongly regulated by GAT-1 GABA transporters. We have used voltage-clamp recordings from BC terminals in goldfish retinal slices to determine the source of GABA for activation of these currents. Inhibition of vesicular release with concanamycin A or tetanus toxin significantly inhibited GABA(A)R inhibitory postsynaptic currents and glutamate-evoked GABA(A)R and GABA(C)R currents but did not reduce the tonic GABA(C)R current, which was also not dependent on extracellular Ca(2+). The tonic current was strongly potentiated by inhibition of GABA transaminase, under both normal and Ca(2+)-free conditions, and was activated by exogenous taurine; however inhibition of taurine transport had little effect. The tonic current was unaffected by GAT-2/3 inhibition and was potentiated by GAT-1 inhibition even in the absence of vesicular release, indicating that it is unlikely to be evoked by reversal of GABA transporters or by ambient GABA. In addition, GABA release does not appear to occur via hemichannels or P2X(7) receptors. BC terminals therefore exhibit two forms of GABA(C)R-mediated inhibition, activated by vesicular and by nonvesicular GABA release, which are likely to have distinct functions in visual signal processing. The tonic GABA(C)R current in BC terminals exhibits similar properties to tonic GABA(A)R and glutamate receptor currents in the brain.

Mobility and Turnover of Vesicles at the Synaptic Ribbon

Ribbon synapses release neurotransmitter continuously at high rates, and the ribbons tether a large pool of synaptic vesicles. To determine whether the tethered vesicles are actually released, we tracked vesicles labeled with styryl dye in mouse retinal bipolar cell terminals whose ribbons had been labeled with a fluorescent peptide. We photobleached vesicles in regions with ribbons and without them and then followed recovery of fluorescence as bleached regions were repopulated by labeled vesicles. In the resting terminal, fluorescence recovered by approximately 50% in non-ribbon regions but by only approximately 20% at ribbons. Thus, at rest, vesicles associated with ribbons cannot exchange freely with cytoplasmic vesicles. Depolarization stimulated vesicle turnover at ribbons as bleached, immobile vesicles were released by exocytosis and were then replaced by fluorescent vesicles from the cytoplasm, producing an additional increase in fluorescence specifically at the ribbon location. We conclude that vesicles immobilized at synaptic ribbons participate in the readily releasable pool that is tapped rapidly during depolarization.

Different roles of ribbon-associated and ribbon-free active zones in retinal bipolar cells

Synaptic ribbons with a halo of synaptic vesicles are seen at the active zones of sensory neurons that release transmitter tonically. Thus, ribbons are assumed to be a prerequisite for sustained exocytosis. By applying total internal reflection fluorescence microscopy to goldfish retinal bipolar cell terminals, we visualized Ca2+ entry sites, ribbons, and vesicle fusion events. Here we show that the main Ca2+ entry sites were located at ribbons, and that activation of the Ca2+ current induced immediate and delayed vesicle fusion events at ribbon-associated and ribbon-free 'hot spots', respectively. The activation of protein kinase C (PKC) specifically potentiated vesicle fusion at ribbon-free sites. Electron microscopy showed that PKC activation selectively increased the number of docked vesicles at ribbon-free sites, which faced neuronal processes with the postsynaptic density. Retinal bipolar cells have both ribbon-associated and ribbon-free active zones in their terminals and might send functionally distinct signals through ribbon-associated and ribbon-free synapses to postsynaptic neurons.

Functional significance of dual vesicle fusion sites in retinal bipolar cell terminals

Functional significance of dual vesicle fusion sites in retinal bipolar cell terminals

Streamlined Synaptic Vesicle Cycle in Cone Photoreceptor Terminals

Cone photoreceptors tonically release neurotransmitter in the dark through a continuous cycle of exocytosis and endocytosis. Here, using the synaptic vesicle marker FM1-43, we elucidate specialized features of the vesicle cycle. Unlike retinal bipolar cell terminals, where stimulation triggers bulk membrane retrieval, cone terminals appear to exclusively endocytose small vesicles. These retain their integrity until exocytosis, without pooling their membranes in endosomes. Endocytosed vesicles rapidly disperse through the terminal and are reused with no apparent delay. Unlike other synapses where most vesicles are immobilized and held in reserve, only a small fraction (<15%) becomes immobilized in cones. Photobleaching experiments suggest that vesicles move by diffusion and not by molecular motors on the cytoskeleton and that vesicle movement is not rate limiting for release. The huge reservoir of vesicles that move rapidly throughout cone terminals and the lack of a reserve pool are unique features, providing cones with a steady supply for continuous release.

Timing of Quantal Release from the Retinal Bipolar Terminal Is Regulated by a Feedback Circuit

In isolation, a presynaptic terminal generally releases quanta according to Poisson statistics, but in a circuit its release statistics might be shaped by synaptic interactions. We monitored quantal glutamate release from retinal bipolar cell terminals (which receive GABA-ergic feedback from amacrine cells) by recording spontaneous EPSCs (sEPSCs) in their postsynaptic amacrine and ganglion cells. In about one-third of these cells, sEPSCs were temporally correlated, arriving in brief bursts (10–55 ms) more often than expected from a Poisson process. Correlations were suppressed by antagonizing the GABA C receptor (expressed on bipolar terminals), and correlations were induced by raising extracellular calcium or osmolarity. Simulations of the feedback circuit produced “bursty” release when the bipolar cell escaped intermittently from inhibition. Correlations of similar duration were present in the light-evoked sEPSCs and spike trains of sluggish-type ganglion cells. These correlations were suppressed by antagonizing GABA C receptors, indicating that glutamate bursts from bipolar terminals induce spike bursts in ganglion cells.

Submillisecond Kinetics of Glutamate Release from a Sensory Synapse

Exocytosis-mediated glutamate release from ribbon-type synaptic terminals of retinal bipolar cells was studied using AMPA receptors and simultaneous membrane capacitance measurements. Release onset (delay <0.8 ms) and offset were closely tied to Ca2+ channel opening and closing. Asynchronous release was not copious and we estimate that there are ∼5 Ca2+ channels per docked synaptic vesicle. Depending on Ca2+ current amplitude, release occurred in a single fast bout or in two successive bouts with fast and slow onset kinetics. The second, slower bout may reflect a mobilization rate of reserve vesicles toward fusion sites that is accelerated by increasing Ca2+ influx. Bipolar cell synaptic ribbons thus are remarkably versatile signal transducers, capable of transmitting rapidly changing sensory input, as well as sustained stimuli, due to their large pool of releasable vesicles.

Ca 2+-activated K + current at presynaptic terminals of goldfish retinal bipolar cells

Properties of the Ca 2+-activated K + current ( I K(Ca)) were investigated in bipolar cells isolated from the goldfish retina. Pharmacological experiments and single channel current recordings demonstrated that I K(Ca) represented currents through BK channels, which were confined mostly to the presynaptic terminal. The ensemble noise analysis of I K(Ca), which was evoked following the activation of presynaptic Ca 2+ current, revealed that the single channel conductance and open probability ( P o) were approximately 50 pS ([K +] o=2.6 mM, [K +] i=140 mM) and 0.6 at 0 mV, respectively. To estimate [Ca 2+] i at the cytosolic side of BK channels, activation of I K(Ca) was examined in Ca 2+-loaded bipolar cells bathed in Co 2+ solution. [Ca 2+] i was monitored using furaptra fluorimetry. It was found that [Ca 2+] i ranged between 10 and 20 μM when P o was 0.6. A high concentration of BAPTA (>20 mM) was required to suppress I K(Ca). Under this condition, channel number was reduced without changing P o. Therefore, it is likely that some BK channels are co-localized with Ca 2+ channels in presynaptic terminals of retinal bipolar cells.

Multiple GABA receptor subtypes mediate inhibition of calcium influx at rat retinal bipolar cell terminals

Inhibitory effects of GABA on K(+)-evoked Ca2+ influx into rat retinal bipolar cell terminals were studied using calcium imaging methods. Application of high K+ evokes a sustained, reversible increase in [Ca2+]i at bipolar cell terminals, which occurs mainly via dihydropyridine-sensitive (L-type) Ca2+ channels. There are at least two GABA receptor subtypes coexisting at bipolar cell terminals: a conventional GABAA receptor and a bicuculline/baclofen-insensitive GABA receptor. Activation of either GABA receptor inhibited the K(+)-evoked Ca2+ response. However, these two GABA receptor subtypes have distinct properties. GABAA receptors suppress the Ca2+ response only at relatively high concentrations of agonist, and with fas kinetics and a narrow dynamic range. In contrast, the bicuculline/baclofen-insensitive GABA receptors produce inhibition on the Ca2+ response at a much lower concentration of agonist, and with slow onset and a wider dynamic range. The pharmacologic profile of the bicuculline/baclofen-insensitive GABA receptor at bipolar cell terminals is most similar to the GABAC receptor reported by Feigenspan et al. (1993). Unlike the GABAC receptors described in other species, it is extremely insensitive to picrotoxin. Therefore, it may be appropriate to refer to this receptor as a picrotoxin-insensitive GABAc receptor. 3-Aminopropyl(methyl)phosphinic acid (3-APMPA) and 3-aminopropylphosphonic (3-APA), two phosphate analogs of GABA, selectively antagonize the picrotoxin-insensitive GABAc receptors but not the GABAA receptors in this system. These results imply a functional role for multiple GABA receptors in regulating synaptic transmission at bipolar cell terminals.