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Axonal stratification patterns and glutamate-gated conductance mechanisms in zebrafish retinal bipolar cells.

1. Whole-cell patch recording and puff pipette techniques were used to identify glutamate receptor mechanisms on bipolar cell (BC) dendrites in the zebrafish retinal slice. Recorded neurons were stained with Lucifer Yellow, to correlate glutamate responses with BC morphology. 2. BC axon terminals (ATs) consisted of swellings or varicosities along the axon, as well as at its end. AT stratification patterns identified three regions in the inner plexiform layer (IPL): a thick sublamina a, with three bands of ATs, a narrow terminal-free zone in the mid-IPL, and a thin sublamina b, with two bands of ATs. BCs occurred with ATs restricted to sublamina a(Group a), sublamina b(Group b) or with ATs in both sublaminae (Group a/b). 3. OFF-BCs belonged to Group a or Group a/b. These cells responded to glutamate or kainate with a CNQX-sensitive conductance increase. Reversal potential (Erev) ranged from -0.6 to +18 mV. Bipolar cells stimulated sequentially with both kainate and glutamate revealed a population of glutamate-insensitive, kainate-sensitive cells in addition to cells sensitive to both agonists. 4. ON-BCs responded to glutamate via one of three mechanisms: (a) a conductance decrease with Erev approximately 0 mV, mimicked by L-(+)-2-amino-4-phosphonobutyric acid (APB) or trans-1-amino-1, 3-cyclopentanedicarboxylic acid (trans-ACPD), (b) a glutamate-gated chloride conductance increase (IGlu-like) characterized by Erev >= ECl (where ECl is the chloride equilibrium potential) and partial blockade by extracellular Li+/Na+ substitution or (c) the activation of both APB and chloride mechanisms simultaneously to produce a response with outward currents at all holding potentials. APB-like responses were found only among BCs in Group b, with a single AT ramifying deep within sublamina b; whereas, cells expressing IGlu-like currents had one or more ATs, and occurred within Groups b or a/b. 5. Multistratified cells (Group a/b) were common and occurred with either ON- or OFF-BC physiology. OFF-BCs typically had one or more ATs in sublamina a and only one AT in sublamina b. In contrast, multistratified ON-BCs had one or more ATs in sublamina b and a single AT ramifying deep in sublamina a. Multistratified ON-BCs expressed the IGlu-like mechanism only. 6. Visual processing in the zebrafish retina involves at least 13 BC types. Some of these BCs have ATs in both the ON- and OFF-sublaminae, suggesting a significant role for ON- and OFF-inputs throughout the IPL.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Role of the low-affinity NGF receptor (p75) in survival of retinal bipolar cells.

We have examined the role of neurotrophins in promoting survival of mammalian rod bipolar cells (RBC) in culture. Retinas taken from 8- to 10-day-old Long-Evans rats were dissociated and cultured in media supplemented with either nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (FGF-2). Survival was measured by the number of cells that were immunoreactive for alpha-, beta-, gamma-PKC, a bipolar cell-specific marker. Compared to untreated cultures, CNTF had no effect on RBC survival, while NGF and NT-3 increased survival only slightly. BDNF, however, increased survival by approximately 300%. Similar results were obtained with FGF-2. Both nerve growth factor (NGF) and an antibody (anti-REX) which interferes with binding to the 75-kD low-affinity neurotrophin receptor (p75NTR) eliminated BDNF-promoted survival, but had no effect on FGF-2-mediated survival. Interestingly, p75NTR was expressed by retinal glia (Müller cells), but not by the bipolar cells themselves, providing for the possibility that BDNF might induce Müller cells to produce a secondary factor, perhaps FGF-2, which directly rescues RBCs. In support of this hypothesis, an antibody that neutralizes FGF-2 attenuated the trophic effects of BDNF, and dramatically reduced survival in cultures with no added growth factors, indicating that there may be an endogenous source of FGF-2 that promotes survival of RBCs in culture. We suggest that BDNF increases production or release of FGF-2 by binding to p75NTR on Müller cells.

Animals↗

Correlation between a bicuculline-resistant response to GABA and GABAA receptor rho 1 subunit expression in single rat retinal bipolar cells.

Using patch-clamp recording in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we show in individual bipolar cells acutely dissociated from the adult rat retina a correlation between the expression of the GABAA receptor rho 1 subunit mRNA and a bicuculline-resistant, diazepam-insensitive component of the GABA-activated whole-cell current response. This "GABAC-like" response, contributing to approximately 42% of the GABA-activated whole-cell current and displaying variable sensitivity to picrotoxin, was found in bipolar cells but not in any of the ganglion cells examined. Expression profiling of GABAA receptor subunit mRNAs in individual electrophysiologically tested retinal neurons revealed that, while both bipolar cells and ganglion cells may express numerous GABAA receptor subunit isoforms, including that of rho 2, the expression of the rho 1 subunit was strictly limited to bipolar cells. We propose a possible link between the presence of a receptor with GABAC-like pharmacological profile and the expression of the retina-specific rho 1 subunit isoform. The results presented in this study constitute the first direct demonstration of such a correlation at the single-cell level.

Animals↗

Light evokes Ca2+ spikes in the axon terminal of a retinal bipolar cell.

Bipolar cells in the vertebrate retina have been characterized as nonspiking interneurons. Using patch-clamp recordings from goldfish retinal slices, we find, however, that the morphologically well-defined Mb1 bipolar cell is capable of generating spikes. Surprisingly, in dark-adapted retina, spikes were reliably evoked by light flashes and had a long (1-2 s) refractory period. In light-adapted retina, most Mb1 cells did not spike. However, an L-type Ca2+ channel agonist could induce periodic spiking in these cells. Spikes were determined to be Ca2+ action potentials triggered at the axon terminal and were abolished by 2-amino-4-phosphonobutyric acid (APB), an agonist that mimics glutamate. Signaling via spikes in a specific class of bipolar cells may serve to accelerate and amplify small photo-receptor signals, thereby securing the synaptic transmission of dim and rapidly changing visual input.

Action Potentials↗

Modulation of Ca(2+)-activated K+ currents and Ca(2+)-dependent action potentials by exocytosis in goldfish bipolar cell terminals.

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na(+)-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca(2+)-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca(2+)-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca2+ current (I(Ca)) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K+ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca2+ influx I(K(Ca). I therefore investigated whether proton-mediated feedback inhibition of I(Ca) affected the activation of I(K(Ca)). A transient inhibition of I(K(Ca)) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I(Ca) but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca(2+)-dependent currents and regenerative potentials in bipolar-cell terminals.

Action Potentials↗

Cannabinoid receptors on goldfish retinal bipolar cells: electron-microscope immunocytochemistry and whole-cell recordings.

Cannabinoid CB1 receptors are distributed throughout the CNS and interact with GABA, glutamate, and dopamine systems. Cannabinoids have effects on the visual system, some of which may have a retinal component, particularly the enhancement of photosensitivity. We used immunocytochemistry and whole-cell recording to study cannabinoids in the goldfish retina. Immunoblots of an antiserum against amino acids (1-14) of the rat CB1 receptor produced a single band in goldfish retina at about 70 kDa. Light microscope immunocytochemistry of CB1 receptor immunoreactivity (CB1R-IR) revealed intense staining of Müller cells and weaker staining of ON bipolar cells (verified with double labeling with PKC-IR) and the outer and inner plexiform layers. Ultrastructural analysis revealed that CB1R-IR was localized intracellularly as well as on the plasma membrane of photoreceptor terminals, bipolar cell terminals and, rarely, amacrine cell boutons. Membrane-associated CB1R-IR was restricted to cone pedicles at sites removed from the synaptic ribbon. Regarding bipolar cells, membrane-associated CB1R-IR was found at 93% of the synaptic terminals in sublamina b (ON-type) and only at 33% of the synaptic terminals in sublamina a (OFF-type). Whole-cell recordings from large ON-type Mb bipolar cells showed that the delayed rectifier (I(K(V))) was rapidly and reversibly inhibited by 1 microM of the cannabinoid agonists CP 54490 and (+)-WIN 55212-2, effects blocked completely by the antagonist SR 141716A (1 microM). Inhibition of I(K(V)) in the Mb bipolar cells by cannabinoids should result in a more tonic ON response to increments of light. As such, cannabinoids may play a role in modulating the temporal aspects of signaling in the retina.

Animals↗

Bullfrog retinal bipolar cells may express heterogeneous glycine receptors at dendrites and axon terminals.

Subcellular localization and properties of glycine receptors on bipolar cells (BCs) were studied using whole-cell recordings and non-stationary noise analysis (NSNA) in bullfrog retinal slices. The currents elicited by focally applied glycine were of comparable amplitudes at the dendrites and axon terminals of both OFF and ON BCs. Moreover, glycine receptors were also expressed at the axons of some BCs. NSNA revealed that the weighted mean single-channel conductance of the glycine receptors at the dendrites (18.2 pS) was significantly larger than that of those at the axon terminals (8.1 pS), thus implying that the glycine receptors on bullfrog retinal BCs may be heterogeneous at these two sites.

Algorithms↗

Temporal filtering in retinal bipolar cells. Elements of an optimal computation?

Recent experiments indicate that the dark-adapted vertebrate visual system can count photons with a reliability limited by dark noise in the rod photoreceptors themselves. This suggests that subsequent layers of the retina, responsible for signal processing, add little if any excess noise and extract all the available information. Given the signal and noise characteristics of the photoreceptors, what is the structure of such an optimal processor? We show that optimal estimates of time-varying light intensity can be accomplished by a two-stage filter, and we suggest that the first stage should be identified with the filtering which occurs at the first anatomical stage in retinal signal processing, signal transfer from the rod photoreceptor to the bipolar cell. This leads to parameter-free predictions of the bipolar cell response, which are in excellent agreement with experiments comparing rod and bipolar cell dynamics in the same retina. As far as we know this is the first case in which the computationally significant dynamics of a neuron could be predicted rather than modeled.

Animals↗

Molecular identity, synaptic localization, and physiology of calcium channels in retinal bipolar cells.

Bipolar cells convey information through the retina via graded changes in their membrane potential and modulate transmitter release through the influx of calcium via L-type calcium channels. However, the molecular identity of the alpha(1) subunit has not been confirmed. We report the presence of the newly cloned alpha(1F) subunit in mouse bipolar cell synaptic terminals. The alpha(1F) subunits are localized to hot spots, possibly corresponding to active zones. We also report the physiological properties of two calcium currents present in mouse bipolar cells, a low-voltage-activated L-type current and a low-voltage-activated T-type calcium current. The physiological properties of the T-type current suggest that it is completely inactivated under physiological conditions. The L-type current may be mediated by the alpha(1F) subunit, and influx of calcium through the alpha(1F) channel may control neurotransmitter release from the bipolar cell terminal.

Animals↗

L-glutamate-induced responses and cGMP-activated channels in three subtypes of retinal bipolar cells dissociated from the cat.

Effects of L-glutamate (Glu), the neurotransmitter released by photoreceptors, on isolated cat bipolar cells were examined. Membrane currents of bipolar cells were recorded by the patch-clamp technique in a conventional whole-cell recording configuration using pipettes containing 1 mM cGMP, which has been known to activate a cationic current sensitive to Glu in ON-type bipolar cells. ON-type bipolar cells (depolarized by light in in situ) and OFF-type bipolar cells (hyperpolarized by light) were identified by their response polarity to Glu. When the whole-cell configuration was established, ON-type bipolar cells showed a steady inward current which was suppressed by Glu, consistent with the response polarity observed in in situ recordings. In contrast, OFF-type cells did not show a steady current during the recordings. However, they responded to Glu with an increase in cationic conductance. Among recorded cells, rod-driven bipolar cells were identified by their immunoreactivity to anti-protein kinase C (PKC-IR) antibody. Examination of PKC-IR revealed that ON-type bipolar cells included both rod- and cone-driven bipolar cells, while OFF-type cells were all cone-driven bipolar cells. The cGMP-activated current observed in ON-type cells was accompanied by a change in the current fluctuation due to the opening and closing of underlying channels. Fluctuation analysis gave a unitary conductance value of 13 pS. In half of the cells examined, maximum open probability reached almost 100%. The cGMP-activated channel in bipolar cells seems novel, fundamentally different from those found in photoreceptor cells or olfactory receptor cells.

Animals↗

Ethanol modulation of GABA-activated current responses in acutely dissociated retinal bipolar cells and ganglion cells.

This study examined the effect of acute ethanol exposure on GABA-activated whole-cell current responses elicited in bipolar cells and ganglion cells of the rat retina. Acute exposure to ethanol potentiated GABA responses in 86% of the bipolar cells and in 52% of the ganglion cells tested. As determined in bipolar cells, ethanol was maximally effective at a concentration of 50 mM. In bipolar cells, a GABAC receptor-mediated component of the whole-cell response to GABA could be uncovered which was also potentiated by ethanol. However, ethanol was ineffective in enhancing bipolar cell responses to glycine. GABA-activated current responses monitored in ganglion cells that were insensitive to modulation by ethanol were sensitive to potentiation by diazepam. At higher concentrations (100-175 mM), ethanol by itself occasionally induced a chloride-mediated current but this occurred independent of an ethanol-induced potentiation of GABA responses. These data establish that ethanol can modulate the sensitivity of retinal neurons to GABA. Overall, the results presented in this study set the stage for future studies to examine the cellular and molecular bases for a differential neuronal sensitivity to an ethanol-induced modulation of GABA responses.

Animals↗

Modulation of voltage-dependent K+ currents (IK(V)) in retinal bipolar cells by ascorbate is mediated by dopamine D1 receptors.

Ascorbic acid (AA), a neuromodulator in the vertebrate CNS, is released from glutamatergic neurons in exchange with glutamate uptake and, in turn, modulates the release of both glutamate and dopamine. We have reported that voltage-gated K+ currents (IK(V)) in ON-mixed rod/cone bipolar cells (Mb) were suppressed 60% by 100-200 microM AA when added to an ascorbate-free solution. However, as the in vivo [AA]o in retina is about 200 microM, we studied the effects of changes in [AA]o on IK(V) when [AA]o was varied around a baseline concentration of 200 microM. Whole-cell currents were recorded with patch-clamp methods from goldfish Mb cells in retinal slices, bathed in a solution containing 200 microM AA. We found that (1) IK(V) was enhanced (180+/-36%, n = 9) by increases of [AA]o less than 40 microM with an average latency of 8 min. (2) However, IK(V) was suppressed without an appreciable latent period by two conditions: increases more than 40 microM [AA]o and decreases by any amount greater than 10 microM. (3) Effects of delta[AA]o on IK(V) were blocked by a D1 dopamine receptor antagonist, SCH 23390, but not by a D2 receptor antagonist, spiperone. Increased concentrations of a D1 agonist (SKF 38390) and dopamine had similar concentration-dependent effects on IK(V) as did AA, even in the presence of 200 microM ascorbate. Ascorbate has complicated concentration-dependent effects on IK(V) of Mb cells in vitro that were mediated by D1 dopamine receptors, suggesting that dopamine and ascorbate may be involved reciprocally in modulating IK(V), with consequences on the transmission of rod signals to the inner retina.

Animals↗

Retinal bipolar cell input mechanisms in giant danio. I. Electroretinographic analysis.

UNLABELLED: Electroretinograms (ERGs) were recorded from the giant danio (Danio aequipinnatus) to study glutamatergic input mechanisms onto bipolar cells. Glutamate analogs were applied to determine which receptor types mediate synaptic transmission from rods and cones to on and off bipolar cells. Picrotoxin, strychnine, and tetrodotoxin were used to isolate the effects of the glutamate analogs to the photoreceptor-bipolar cell synapse. Under photopic conditions, the group III metabotropic glutamate receptor (mGluR) antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) only slightly reduced the b-wave, whereas the excitatory amino acid transporter (EAAT) blocker dl-threo-beta-benzyl-oxyaspartate (TBOA) removed most of it. Complete elimination of the b-wave required both antagonists. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) blocked the d-wave. Under scotopic conditions, rod and cone inputs onto on bipolar cells were studied by comparing the sensitivities of the b-wave to photopically matched green and red stimuli. The b-wave was >1 log unit more sensitive to the green than to the red stimulus under control conditions. In CPPG or l-AP4 (l-(+)-2-amino-4-phosphonobutyric acid, a group III mGluR agonist), the sensitivity of the b-wave to the green stimulus was dramatically reduced and the b-waves elicited by the 2 stimuli became nearly matched. The d-wave elicited by dim green stimuli, which presumably could be detected only by the rods, was eliminated by NBQX. IN CONCLUSION: 1) cone signals onto on bipolar cells involve mainly EAATs but also mGluRs (presumably mGluR6) to a lesser extent; 2) rods signal onto on bipolars by mainly mGluR6; 3) off bipolar cells receive signals from both photoreceptor types by AMPA/kainate receptors.

Aminobutyrates↗

HCN channels are expressed differentially in retinal bipolar cells and concentrated at synaptic terminals.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels codetermine the integrative behaviour of neurons and shape their response to synaptic stimulation. We used immunohistochemistry and patch-clamp recording to study the composition and distribution of HCN channels in the rat retina. All four HCN channel isoforms (HCN1-4) are expressed differentially in the retina. In particular, different classes of bipolar cells have a different inventory of HCN channels. We found no evidence for the formation of heterooligomeric HCN channels. HCN channels are densely clustered at synaptic terminals of bipolar cells and photoreceptors. This suggests that HCN channels are involved in the control of transmitter release.

Animals↗

Differential distribution of Shaker-like and Shab-like K+-channel subunits in goldfish retina and retinal bipolar cells.

The distributions of Shaker subfamily Kv1.1 and Kv1.2 and Shab subfamily Kv2.1 subunits of voltage-gated K+ channels were determined in the retina and ON bipolar cells of goldfish by using double-label light and electron microscopic immunocytochemistry. All labeling to be described was blocked by preabsorption of the primary antibodies with antigen. The retina was labeled throughout with all three antibodies. However, labeling was densest in the inner plexiform layer for Kv1.1, more concentrated in the outer nuclear layer for Kv2.1, and uniform throughout for Kv1.2. All ON mixed rod/cone (mb) and cone (cb) bipolar somata and the proximal portions of their axons and dendrites were labeled for anti-Kv1.1, Kv1.2, and Kv2.1. Labeling of axons rarely extended over the mb axon terminal. Only Kv1.2 antibodies labeled mb bipolar cell dendrites in the outer plexiform layer. No evidence for Kv1.1, 1.2, or 2.1 antibody labeling of OFF bipolar cells was found. Ultrastructurally, Kv1.2-immunoreactivity was associated with the plasma membrane of bipolar cell bodies and with dendrites that make narrow-cleft junctions with cone terminals (ON-type). Kv immunoreactivity was not found associated with presynaptic membranes in the inner plexiform layer and was found only rarely with membranes, postsynaptic to an amacrine cell process. Although both Shaker and Shab subfamilies include delayed rectifiers, their activation properties differ, suggesting differential modulation of K+ conductances in bipolar cells based not only on the presence or absence of rod photoreceptor input but also whether the bipolar cells are of the ON or OFF type.

Animals↗

Glycine-receptor immunoreactivity in retinal bipolar cells is postsynaptic to glycinergic and GABAergic amacrine cell synapses.

Glycinergic innervation of the synaptic terminals of mixed rod-cone bipolar cells in the goldfish retina was investigated by electron microscopical immunocytochemistry with presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and polyclonal antisera against a glycine/BSA conjugate. Conventional "glycinergic" synaptic contacts, made by amacrine cell processes, accounted for 7-10% of the input to the bipolar cell terminals, whether determined by glycine receptor immunoreactivity (GlyR-IR) or glycine-IR. In addition to the conventional synapses, the large bipolar cell terminals in the proximal inner plexiform layer (type Mb) gave rise to spinules (spine-like protrusions) that invaginated into presynaptic amacrine cell processes. Although 85% of the spinules were GlyR-IR, no spinules were postsynaptic to glycine-IR processes; yet 86% of the spinules were postsynaptic to GAD-IR processes, suggesting that the GlyR-IR spinules were postsynaptic to GABAergic terminals. Furthermore, a single amacrine cell process could make two synapses with an Mb terminal: a GlyR-IR contact onto a spinule and a conventional synapse that was not GlyR-IR. We suggest that glycinergic innervation of bipolar cell terminals involves conventional glycinergic synapses as well as an unconventional situation in which GABA and glycine may interact in as yet undetermined manner, perhaps by potentiation.

Animals↗