Rod Bipolar Axon Terminals Research Articles

Multiple Calcium Channel Types with Unique Expression Patterns Mediate Retinal Signaling at Bipolar Cell Ribbon Synapses.

Retinal bipolar cells (BCs) compose the canonical vertical excitatory pathway that conveys photoreceptor output to inner retinal neurons. Although synaptic transmission from BC terminals is thought to rely almost exclusively on Ca2+ influx through voltage-gated Ca2+ (CaV) channels mediating L-type currents, the molecular identity of CaV channels in BCs is uncertain. Therefore, we combined molecular and functional analyses to determine the expression profiles of CaV α1, β, and α2δ subunits in mouse rod bipolar (RB) cells, BCs from which the dynamics of synaptic transmission are relatively well-characterized. We found significant heterogeneity in CaV subunit expression within the RB population from mice of either sex, and significantly, we discovered that transmission from RB synapses was mediated by Ca2+ influx through P/Q-type (CaV2.1) and N-type (CaV2.2) conductances as well as the previously-described L-type (CaV1) and T-type (CaV3) conductances. Furthermore, we found both CaV1.3 and CaV1.4 proteins located near presynaptic ribbon-type active zones in RB axon terminals, indicating that the L-type conductance is mediated by multiple CaV1 subtypes. Similarly, CaV3 α1, β, and α2δ subunits also appear to obey a "multisubtype" rule, i.e., we observed a combination of multiple subtypes, rather than a single subtype as previously thought, for each CaV subunit in individual cells.SIGNIFICANCE STATEMENT Bipolar cells (BCs) transmit photoreceptor output to inner retinal neurons. Although synaptic transmission from BC terminals is thought to rely almost exclusively on Ca2+ influx through L-type voltage-gated Ca2+ (CaV) channels, the molecular identity of CaV channels in BCs is uncertain. Here, we report unexpectedly high molecular diversity of CaV subunits in BCs. Transmission from rod bipolar (RB) cell synapses can be mediated by Ca2+ influx through P/Q-type (CaV2.1) and N-type (CaV2.2) conductances as well as the previously-described L-type (CaV1) and T-type (CaV3) conductances. Furthermore, CaV1, CaV3, β, and α2δ subunits appear to obey a "multisubtype" rule, i.e., a combination of multiple subtypes for each subunit in individual cells, rather than a single subtype as previously thought.

Glutamate Transporters EAAT2 and EAAT5 Differentially Shape Synaptic Transmission from Rod Bipolar Cell Terminals.

Excitatory amino acid transporters (EAATs) control visual signal transmission in the retina by rapidly removing glutamate released from photoreceptors and bipolar cells (BCs). Although it has been reported that EAAT2 and EAAT5 are expressed at presynaptic terminals of photoreceptors and some BCs in mammals, the distinct functions of these two glutamate transporters in retinal synaptic transmission, especially at a single synapse, remain elusive. In this study, we found that EAAT2 was expressed in all BC types while coexisting with EAAT5 in rod bipolar (RB) cells and several types of cone BCs from mice of either sex. Our immunohistochemical study, together with a recently published literature (Gehlen et al., 2021), showed that EAAT2 and EAAT5 were both located in RB axon terminals near release sites. Optogenetic, electrophysiological and pharmacological analyses, however, demonstrated that EAAT2 and EAAT5 regulated neurotransmission at RB→AII amacrine cell synapses in significantly different ways: EAAT5 dramatically affected both the peak amplitude and kinetics of postsynaptic responses in AIIs, whereas EAAT2 had either relatively small or opposite effects. By contrast, blockade of EAAT1/GLAST, which was exclusively expressed in Müller cells, showed no obvious effect on AII responses, indicating that glutamate uptake by Müller cells did not influence synaptic transmission from RB terminals. Furthermore, we found that temporal resolution at RB→AII synapses was reduced substantially by blockade of EAAT5 but not EAAT2. Taken together, our work reveals the distinct functions of EAAT2 and EAAT5 in signal transmission at RB ribbon synapses.

Calmodulin Bidirectionally Regulates Evoked and Spontaneous Neurotransmitter Release at Retinal Ribbon Synapses.

For decades, a role for the Ca2+-binding protein calmodulin (CaM) in Ca2+-dependent presynaptic modulation of synaptic transmission has been recognized. Here, we investigated the influence of CaM on evoked and spontaneous neurotransmission at rod bipolar (RB) cell→AII amacrine cell synapses in the mouse retina. Our work was motivated by the observations that expression of CaM in RB axon terminals is extremely high and that [Ca2+] in RB terminals normally rises sufficiently to saturate endogenous buffers, making tonic CaM activation likely. Taking advantage of a model in which RBs can be stimulated by expressed channelrhodopsin-2 (ChR2) to avoid dialysis of the presynaptic terminal, we found that inhibition of CaM dramatically decreased evoked release by inhibition of presynaptic Ca channels while at the same time potentiating both Ca2+-dependent and Ca2+-independent spontaneous release. Remarkably, inhibition of myosin light chain kinase (MLCK), but not other CaM-dependent targets, mimicked the effects of CaM inhibition on evoked and spontaneous release. Importantly, initial antagonism of CaM occluded the effect of subsequent inhibition of MLCK on spontaneous release. We conclude that CaM, by acting through MLCK, bidirectionally regulates evoked and spontaneous release at retinal ribbon synapses.

Spikes in Mammalian Bipolar Cells Support Temporal Layering of the Inner Retina

In the mammalian retina, 10-12 different cone bipolar cell (BC) types decompose the photoreceptor signal into parallel channels, providing the basis for the functional diversity of retinal ganglion cells (RGCs). BCs differing in their temporal properties appear to project to different strata of the retina's inner synaptic layer, based on somatic recordings of BCs and excitatory synaptic currents measured in RGCs. However, postsynaptic currents in RGCs are influenced by dendritic morphology and receptor types, and the BC signal can be transformed at the axon terminals both through interactions with amacrine cells and through the generation of all-or-nothing spikes. Therefore, the temporal properties of the BC output have not been analyzed systematically across different types of mammalian BCs. We recorded calcium signals directly within axon terminals using two-photon imaging and show that BCs can be divided into ≥eight functional clusters. The temporal properties of the BC output were directly reflected in their anatomical organization within the retina's inner synaptic layer: faster cells stratified closer to the border between ON and OFF sublamina. Moreover, ≥three fastest groups generated clear all-or-nothing spikes. Therefore, the systematic projection pattern of BCs provides distinct temporal "building blocks" for the feature extracting circuits of the inner retina.

Properties of glycine receptors underlying synaptic currents in presynaptic axon terminals of rod bipolar cells in the rat retina

The excitability of presynaptic terminals can be controlled by synaptic input that directly targets the terminals. Retinal rod bipolar axon terminals receive presynaptic input from different types of amacrine cells, some of which are glycinergic. Here, we have performed patch-clamp recordings from rod bipolar axon terminals in rat retinal slices. We used whole-cell recordings to study glycinergic inhibitory postsynaptic currents (IPSCs) under conditions of adequate local voltage clamp and outside-out patch recordings to study biophysical and pharmacological properties of the glycine receptors with ultrafast application. Glycinergic IPSCs, recorded in both intact cells and isolated terminals, were strychnine sensitive and displayed fast kinetics with a double-exponential decay. Ultrafast application of brief (approximately 1 ms) pulses of glycine (3 mM) to patches evoked responses with fast, double-exponential deactivation kinetics, no evidence of desensitization in double-pulse experiments, relatively low apparent affinity (EC(50) approximately 100 microM), and high maximum open probability (0.9). Longer pulses evoked slow, double-exponential desensitization and double-pulse experiments indicated slow, double-exponential recovery from desensitization. Non-stationary noise analysis of IPSCs and patch responses yielded single-channel conductances of approximately 41 pS and approximately 64 pS, respectively. Directly observed single-channel gating occurred at approximately 40-50 pS and approximately 80-90 pS in both types of responses, suggesting a mixture of heteromeric and homomeric receptors. Synaptic release of glycine leads to transient receptor activation, with about eight receptors available to bind transmitter after release of a single vesicle. With a low intracellular chloride concentration, this leads to either hyperpolarizing or shunting inhibition that will counteract passive and regenerative depolarization and depolarization-evoked transmitter release.

The absence of the clathrin-dependent endocytosis in rod bipolar cells of the FVB/N mouse retina

The high rate of exocytosis at the ribbon synapses is balanced by following compensatory endocytosis. Unlike conventional synaptic terminals where clathrin-mediated endocytosis (CME) is a predominant mechanism for membrane retrieval, CME is thought to be only a minor mechanism of endocytosis at the retinal ribbon synapses, but CME is present there and it works. We examined the clathrin expression in the FVB/N rd1 mouse, which is an animal model of retinitis pigmentosa. The broadly distributed pattern of clathrin immunoreactivity in the inner plexiform layer was similar in both the control and FVB/N mouse retinas, but the immunoreactive punta within the rod bipolar axon terminals located in the proximal IPL were decreased in number and reduced in size at postnatal days 14 and they came to disappear at postnatal days 21. This preferential decrease of the clathrin expression at ribbon synapses in the rod bipolar cell axon terminals of the FVB/N mouse retina demonstrates another plastic change after photoreceptor degeneration and this suggests that clathrin may be important for normal synaptic function at the rod bipolar ribbon synapses in the mammalian retina.

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Excitatory amino acid transporters (EAATs) control visual signal transmission in the retina by rapidly removing glutamate released from photoreceptors and bipolar cells (BCs). Although it has been reported that EAAT2 and EAAT5 are expressed at presynaptic terminals of photoreceptors and some BCs in mammals, the distinct functions of these two glutamate transporters in retinal synaptic transmission, especially at a single synapse, remain elusive. In this study, we found that EAAT2 was expressed in all BC types while coexisting with EAAT5 in rod bipolar (RB) cells and several types of cone BCs from mice of either sex. Our immunohistochemical study, together with a recently published literature (Gehlen et al., 2021), showed that EAAT2 and EAAT5 were both located in RB axon terminals near release sites. Optogenetic, electrophysiological and pharmacological analyses, however, demonstrated that EAAT2 and EAAT5 regulated neurotransmission at RB→AII amacrine cell synapses in significantly different ways: EAAT5 dramatically affected both the peak amplitude and kinetics of postsynaptic responses in AIIs, whereas EAAT2 had either relatively small or opposite effects. By contrast, blockade of EAAT1/GLAST, which was exclusively expressed in Müller cells, showed no obvious effect on AII responses, indicating that glutamate uptake by Müller cells did not influence synaptic transmission from RB terminals. Furthermore, we found that temporal resolution at RB→AII synapses was reduced substantially by blockade of EAAT5 but not EAAT2. Taken together, our work reveals the distinct functions of EAAT2 and EAAT5 in signal transmission at RB ribbon synapses.

Patch-Clamp Investigations and Compartmental Modeling of Rod Bipolar Axon Terminals in an In Vitro Thin-Slice Preparation of the Mammalian Retina

To extend the usefulness of rod bipolar cells for studies of chemical synaptic transmission, we have performed electrophysiological recordings from rod bipolar axon terminals in an in vitro slice preparation of the rat retina. Whole cell recordings from axon terminals and cell bodies were used to investigate the passive membrane properties of rod bipolar cells and analyzed with a two-compartment equivalent electrical circuit model developed by Mennerick et al. For both terminal- and soma-end recordings, capacitive current decays were well fitted by biexponential functions. Computer simulations of simplified models of rod bipolar cells demonstrated that estimates of the capacitance of the axon terminal compartment can depend critically on the recording location, with terminal-end recordings giving the best estimates. Computer simulations and whole cell recordings demonstrated that terminal-end recordings can yield more accurate estimates of the peak amplitude and kinetic properties of postsynaptic currents generated at the axon terminals due to increased electrotonic filtering of these currents when recorded at the soma. Finally, we present whole cell and outside-out patch recordings from axon terminals with responses evoked by GABA and glycine, spontaneous inhibitory postsynaptic currents, voltage-gated Ca(2+) currents, and depolarization-evoked reciprocal synaptic responses, verifying that the recorded axon terminals are involved in normal pre- and postsynaptic relationships. These results demonstrate that axon terminals of rod bipolar cells are directly accessible to whole cell and outside-out patch recordings, extending the usefulness of this preparation for detailed studies of pre- and postsynaptic mechanisms of synaptic transmission in the CNS.

Diurnal and circadian variation of protein kinase C immunoreactivity in the rat retina.

We studied the dependence of the expression of protein kinase C immunoreactivity (PKC-IR) in the rat retina on the light:dark (LD) cycle and on circadian rhythmicity in complete darkness (DD). Two anti-PKC alpha antibodies were employed: One, which we call PKCalphabeta recognized the hinge region; the other, here termed PKCalpha, recognized the regulatory region of the molecule. Western blots showed that both anti-PKC antibodies stained an identical single band at approximately 80 kD. The retinal neurons showing PKC-IR were rod bipolar cells and a variety of amacrine neurons. After 3 weeks on an LD cycle, PKCalphabeta-IR in both rod bipolar and certain amacrine cells manifested a clear rhythm with a peak at zeitgeber time (ZT) of 06-10 hours and a minimum at ZT 18. No rhythm in total PKC-IR was observed when using the PKCalpha antibody, but, at ZT 06-10 hours, rod bipolar axon terminals showed increased immunostaining. After 48 hours in DD, with either antibody, rod bipolar cells showed increased PKC-IR. The PKCalpha antibody alone revealed that, after 48 hours, AII amacrine neurons, which lacked PKC-IR in an LD cycle, manifested marked PKC-IR, which became stronger after 72 hours. Light administered early in the dark period greatly increased PKCalphabeta-IR in rod bipolar and some amacrine neurons. Our data indicate that light and darkness exert a strong regulatory influence on PKC synthesis, activation, and transport in retinal neurons.

The synaptic organization of the dopaminergic amacrine cell in the cat retina

The dopaminergic amacrine cells of the cat retina have been stained by immunocytochemistry using an antibody to tyrosine hydroxylase (Toh). The complete population of Toh+ cells has been studied by light microscopy of retinal wholemounts to evaluate morphological details of dendritic structure and branching patterns. Selected Toh+ amacrine cells have been studied by serial-section electron microscopy to analyse synaptic input and output relationships. The majority of Toh+ amacrine cells occur in the amacrine cell layer of the retina and have their dendrites ramifying and forming the characteristic rings in stratum 1 of the inner plexiform layer. A minority of Toh+ cells have cell bodies displaced to the ganglion cell layer but their dendrites also stratify in stratum 1. All Toh+ cells have some dendritic branches running in stratum 2 as well as in stratum 1, and frequently they have long 'axon-like' processes (500-1000 microns long) dipping down to run in stratum 5 before passing up to rejoin the major dendritic arbors in stratum 1. In addition Toh+ stained processes follow blood vessels in the inner plexiform layer and in the ganglion cell layer. A population of Toh+ cells found in the inferior retina appears to give rise to stained processes that pass to the outer plexiform layer and therein to run for as far as one millimeter. Electron microscopy reveals that Toh+ amacrine cells are postsynaptic to amacrine cells and a few bipolar cell terminals in stratum 1 of the inner plexiform layer and are primarily presynaptic to AII amacrine cell bodies and lobular appendages, and to another type of amacrine cell body and amacrine dendrites hypothesized to be the A17 amacrine cell. The Toh+ dendrites in stratum 2 are presynaptic to AII lobular appendages primarily. Stained 'axon-like' processes running in stratum 5 prove to be presynaptic to AII amacrine dendrites as they approach the rod bipolar axon terminals and they may also be presynaptic to the rod bipolar terminal itself. The Toh+ stained dendrites that have been followed in the outer plexiform layer run along the top of the B-type horizontal cell somata and may have small synapses upon them. The only clear synapses seen in the outer plexiform layer are from the Toh+ profiles upon vesicle filled amacrine-like profiles that are in turn presynaptic to bipolar cell dendrites in the outer plexiform layer. We presume the cells postsynaptic to the Toh+ dendrites in the outer plexiform layer are interplexiform cells.(ABSTRACT TRUNCATED AT 400 WORDS)

A17: a broad-field amacrine cell in the rod system of the cat retina.

A17 amacrine cells of the cat retina have been penetrated with horseradish peroxidase (HRP)-filled microelectrodes and their light responses recorded. These cells depolarize in sustained fashion to steps of light. Viewed in retinal wholemounts, HRP-injected cells have a spokelike radiating splay of very fine dendrites (0.1 micron diam) passing diffusely through all strata of the inner plexiform layer (IPL) to run primarily in strata 4 and 5. There are as many as 1,000 large, regularly spaced beads borne on the 500- to 1,200-micron diameter dendritic field. Cell body sizes range from 9 to 13 micron. In the electron microscope, the dendritic beads in sublamina b of the IPL are seen to synapse reciprocally with rod bipolar axon terminals. Dendritic beads in sublamina a rarely make synapses, but between the beads in this layer, input from at least three distinctive amacrine profiles occurs. Though diffuse at the light microscopic level, A17 thus appears to be structurally bistratified, with amacrine input in sublamina a and bipolar input in sublamina b. It is likely that A17 can be identified with AI. A17 signals are driven almost exclusively by rods. The spectral sensitivity peaks at 507 nm, identical with that of pigment epithelial cells. Light adaptation abolishes all but a small hyperpolarizing component of the signal. The overall intensity-response range is similar to that of AII amacrine cells. When receptive fields of A17 cells are mapped with slit stimuli, a broad, single-component curve is measured approximately covering the dendritic field. The receptive field is well described by a linear electrical model with a mean space constant of 259 +/- 97 micron (SD). On the other hand, responses to centered slit stimuli of varying width yielded space constants of only 38 +/- 29 micron. A17 amacrines are thus broad-field components of the cat's rod system but with very little capacity for spatial integration. Receptive-field measurements are not supportive of the notion of isolated dendritic regions.

The inner plexiform layer in the retina of the cat: electron microscopic observations.

Neural connections of cells ramifying in the inner plexiform layer of the cat retina have been studied by serial section electron microscopy. Flat cone bipolars and invaginating cone bipolars segregate their axon terminals to different sublaminae of the IPL (sublamina a and sublamina b, respectively) where they relate to different subtypes of the same class of ganglion cell (a and b types respectively). Rod bipolar axon terminals end solely in sublamina b and synapse with amacrine cells (AI and AII). AI provides reciprocal synapses to clusters of rod bipolar axon terminals. The AII amacrine provides rod input to a type ganglion cells by means of chemical synapses and to b type ganglion cells through gap junctions with invaginating cone bipolar terminals. Amacrine cells exist which interconnect rod and cone bipolars, but some amacrines appear to be related specifically to neurons branching in particular sublaminae. Both large- and small-bodied ganglion cells have amacrine-dominated input while the medium-bodied ganglion cells with small dendritic trees have cone bipolar-dominated input.