Uncovering the electrical synapse proteome in retinal neurons via in vivo proximity labeling.

Gap junctions

Adaptation to Background Light Enables Contrast Coding at Rod Bipolar Cell Synapses

We have studied the expression pattern of neuronal connexin36 (Cx36) in the mouse and rat retina. In vertical sections of both retinas, a polyclonal antibody directed against Cx36 produced punctate labeling in the inner plexiform layer (IPL). Intense immunoreactivity was localized to the entire OFF sublamina of the IPL, and much weaker staining could be observed in the ON sublamina. Double-labeling experiments in the rat retina with antibodies directed against parvalbumin indicate that Cx36 is expressed on dendrites of AII amacrine cells. Cx36-like immunoreactivity in sublamina a of the IPL did not overlap with lobular appendages or cell bodies of AII amacrine cells. In a mouse retinal slice preparation, AII amacrine and ON cone bipolar cells were intracellularly injected with Neurobiotin and counterstained with antibody against Cx36. Punctate labeling appeared to be in register with dendritic arborization of AII amacrines and cone bipolar cells in the ON sublamina of the IPL. Whereas AII amacrine cells isolated from the rat retina clearly displayed Cx36-like immunoreactivity, isolated ON cone bipolar cells were negative for Cx36. Axon terminals of rod bipolar cells were decorated with Cx36-positive contacts but did not express Cx36 themselves. These results indicate that Cx36 is expressed by AII amacrine cells in homologous and heterologous gap junctions made with AII amacrines and cone bipolar cells, respectively. The heterologous gap junctions appear to be heterotypic, because ON cone bipolar cells do not express Cx36.

Connexin45 (Cx45) is known to be expressed in the retina, but its functional analysis was problematic because general deletion of Cx45 coding DNA resulted in cardiovascular defects and embryonic lethality at embryonic day 10.5. We generated mice with neuron-directed deletion of Cx45 and concomitant activation of the enhanced green fluorescent protein (EGFP). EGFP labeling was observed in bipolar, amacrine, and ganglion cell populations. Intracellular microinjection of fluorescent dyes in EGFP-labeled somata combined with immunohistological markers revealed Cx45 expression in both ON and OFF cone bipolar cells. The scotopic electroretinogram of mutant mice revealed a normal a-wave but a 40% reduction in the b-wave amplitude, similar to that found in Cx36-deficient animals, suggesting a possible defect in the rod pathway of visual transmission. Indeed, neurotransmitter coupling between AII amacrine cells and Cx45-expressing cone bipolar cells was disrupted in Cx45-deficient mice. These data suggest that both Cx45 and Cx36 participate in the formation of functional heterotypic electrical synapses between these two types of retinal neurons that make up the major rod pathway.

Electrical synapses or gap junctions occur between many retinal neurons. However, in most cases, the gap junctions have not been visualized directly. Instead, their presence has been inferred from tracer spread throughout the network of cells. Thus, tracer coupling is taken as a marker for the presence of gap junctions between coupled cells. AII amacrine cells are critical interneurons in the rod pathway of the mammalian retina. Rod bipolar cell output passes to AII amacrine cells, which in turn make conventional synapses with OFF cone bipolar cells and gap junctions with ON cone bipolar cells. Injections of biotinylated tracers into AII amacrine cells reveals coupling between the AII amacrine cell network and heterologous coupling with a variety of ON cone bipolar cells, including the calbindin-positive cone bipolar cell. To directly visualize gap junctions in this network, we prepared material for electron microscopy that was double labeled with antibodies to calretinin and calbindin to label AII amacrine cells and calbindin-positive cone bipolar cells, respectively. AII amacrine cells were postsynaptic to large vesicle-laden rod bipolar terminals, as previously reported. Gap junctions were identified between AII amacrine cells and calbindin-positive cone bipolar cell terminals identified by the presence of immunostaining and ribbon synapses. This represents direct confirmation of gap junctions between two different yet positively identified cells, which are tracer coupled, and provides additional evidence that tracer coupling with Neurobiotin indicates the presence of gap junctions. These results also definitively establish the presence of gap junctions between AII amacrine cells and calbindin bipolar cells which can therefore carry rod signals to the ON alpha ganglion cell.

Diabetes leads to dysfunction of the neural retina before and independent of classical microvascular diabetic retinopathy, but previous studies have failed to demonstrate which neurons and circuits are affected at the earliest stages. Here, using patch-clamp recording and two-photon Ca(2+) imaging in rat retinal slices, we investigated diabetes-evoked changes in a microcircuit consisting of rod bipolar cells and their dyad postsynaptic targets, AII and A17 amacrine cells, which play an essential role in processing scotopic visual signals. AII amacrines forward their signals to ON- and OFF-cone bipolar cells and A17 amacrines provide GABAergic feedback inhibition to rod bipolar cells. Whereas Ca(2+)-permeable AMPA receptors mediate input from rod bipolar cells to both AII and A17 amacrines, diabetes changes the synaptic receptors on A17, but not AII amacrine cells. This was expressed as a change in pharmacological properties and single-channel conductance of the synaptic receptors, consistent with an upregulation of the AMPA receptor GluA2 subunit and reduced Ca(2+) permeability. In addition, two-photon imaging revealed reduced agonist-evoked influx of Ca(2+) in dendritic varicosities of A17 amacrine cells from diabetic compared with normal animals. Because Ca(2+)-permeable receptors in A17 amacrine cells mediate synaptic release of GABA, the reduced Ca(2+) permeability of these receptors in diabetic animals leads to reduced release of GABA, followed by disinhibition and increased release of glutamate from rod bipolar cells. This perturbation of neuron and microcircuit dynamics can explain the decreased dynamic range and sensitivity of scotopic vision that has been observed in diabetes.

Gap junction channels constitute specialized intercellular contacts that can serve as electrical synapses. In the rod pathway of the retina, electrical synapses between AII amacrine cells express connexin 36 (Cx36) and electrical synapses between AII amacrines and on-cone bipolar cells express Cx36 on the amacrine side and Cx36 or Cx45 on the bipolar side. For physiological investigations of the properties and functions of these electrical synapses, it is highly desirable to have access to potent pharmacological blockers with selective and reversible action. Here we use dual whole cell voltage-clamp recordings of pairs of AII amacrine cells and pairs of AII amacrine and on-cone bipolar cells in rat retinal slices to directly measure the junctional conductance (G(j)) between electrically coupled cells and to study the effect of the drug meclofenamic acid (MFA) on G(j). Consistent with previous tracer coupling studies, we found that MFA reversibly blocked the electrical synapse currents in a concentration-dependent manner, with complete block at 100 muM. Whereas MFA evoked a detectable decrease in G(j) within minutes of application, the time to complete block of G(j) was considerably longer, typically 20-40 min. After washout, G(j) recovered to 20-90% of the control level, but the time to maximum recovery was typically >1 h. These results suggest that MFA can be a useful drug to investigate the physiological functions of electrical synapses in the rod pathway, but that the slow kinetics of block and reversal might compromise interpretation of the results and that explicit monitoring of G(j) is desirable.

The synaptic connections of the narrow-field, bistratified rod amacrine cell (AII) in the inner plexiform layer (IPL) of the rabbit retina were reconstructed from electron micrographs of continuous series of thin sections. The AII amacrine cell receives a large synaptic input from the axonal endings of rod bipolar cells in the most vitreal region of the IPL (sublamina b, S5) and a smaller input from axonal endings of cone bipolar cells in the scleral region of the IPL (sublamina a, S1-S2). Amacrine input, localized at multiple levels in the IPL, equals the total number of synapses received from bipolar cells. The axonal endings of cone bipolar cells represent the major target for the chemical output of the AII amacrine cell: these synapses are established by the lobular appendages in sublamina a (S1-S2). Ganglion cell dendrites represent only 4% of the output of the AII amacrine and most of them are also postsynaptic to the cone bipolars which receive AII input. The AII amacrine is not presynaptic to other amacrine cells. Finally, the AII amacrine makes gap junctions with the axonal arborizations of cone bipolars that stratify in sublamina b (S3-S4) as well as with other AII amacrine cells in S5. Therefore, in the rabbit retina 1) the rod pathway consists of five neurons arranged in series: rod-->rod bipolar-->AII amacrine-->cone bipolar-->ganglion cell; 2) it seems unlikely that a class of ganglion cells exists that is exclusively devoted to scotopic functions. In ventral, midperipheral retina, about nine rod bipolar cells converge onto a single AII amacrine, but one of them establishes a much higher proportion of synaptic contacts than the rest. Conversely, each rod bipolar cell diverges onto four AII amacrine cells, but one of them receives the largest fraction of synapses. Thus, within the pattern of convergence and divergence suggested by population studies, preferential synaptic pathways are established.

Inhibitory interneurons represent a diverse population of cell types in the central nervous system, whose general role is to suppress activity of target neurons. The timing of spikes in principal neurons has millisecond precision, and I asked what are the roles of inhibition in shaping the temporal codes that emerge from different parallel local neural circuits. First I investigated the local circuitry of melanopsin-containing ganglion cells in the mouse retina, which are intrinsically photosensitive and responsible for circadian photoentrainment. Using transsynaptic viral tracing, I identified three types of melanopsin-containing ganglion cell, and found that inhibitory (GABAergic) dopaminergic amacrine cells are presynaptic to one of these types. These results provided a direct circuitry link between the medium time scale process of light-dark adaptation, which involves dopamine, and the longer time scale of the circadian rhythm. Next I characterised a subpopulation of genetically-identified neurons in the mouse retina, in order to compare the precise timing of inhibition in different circuits at a high temporal resolution. I identified eight physiologically and morphologically distinct ganglion cell types and found that each circuit could be described by a 'motif' that represented the inhibitory-excitatory interactions that lead to cell-type-specific firing patterns. The cell would fire only when the change in excitation was faster than the change in inhibition. Therefore the role of inhibition is to detect 'irrelevance' in the visual scene, only allowing the ganglion cell to fire at specific time points relating to functions that are both parallel and complementary to the other cell types. Finally, I looked deeper within the neural circuitry of one of the genetically-identified cell types, to study the mechanism of 'fast inhibition' in detecting approaching objects. Through two-photon targeted paired recordings of postsynaptic ganglion cells and presynaptic amacrine cells, I found evidence that the AII amacrine cell - a well-characterised glycinergic inhibitory interneuron known to be involved in night vision circuits - conveys fast inhibitory information to the ganglion cell via an electrical synapse with an excitatory neuron of day vision circuitry only during non-approach motion. Therefore, it appears that the role of inhibition is to dynamically interact with direct excitatory neural pathways during 'irrelevant' stimulation, suppressing or completely blocking activity, resulting in precisely timed spikes that occur in the brief moments when excitation changes faster than inhibition.

In the rod pathway of the mammalian retina, axon terminals of glutamatergic rod bipolar cells are presynaptic to AII and A17 amacrine cells in the inner plexiform layer. Recent evidence suggests that both amacrines express NMDA receptors, raising questions concerning molecular composition, localization, activation, and function of these receptors. Using dual patch-clamp recording from synaptically connected rod bipolar and AII or A17 amacrine cells in retinal slices from female rats, we found no evidence that NMDA receptors contribute to postsynaptic currents evoked in either amacrine. Instead, NMDA receptors on both amacrine cells were activated by ambient glutamate, and blocking glutamate uptake increased their level of activation. NMDA receptor activation also increased the frequency of GABAergic postsynaptic currents in rod bipolar cells, suggesting that NMDA receptors can drive release of GABA from A17 amacrines. A striking dichotomy was revealed by pharmacological and immunolabeling experiments, which found GluN2B-containing NMDA receptors on AII amacrines and GluN2A-containing NMDA receptors on A17 amacrines. Immunolabeling also revealed a clustered organization of NMDA receptors on both amacrines and a close spatial association between GluN2B subunits and connexin 36 on AII amacrines, suggesting that NMDA receptor modulation of gap junction coupling between these cells involves the GluN2B subunit. Using multiphoton Ca2+ imaging, we verified that activation of NMDA receptors evoked an increase of intracellular Ca2+ in dendrites of both amacrines. Our results suggest that AII and A17 amacrines express clustered, extrasynaptic NMDA receptors, with different and complementary subunits that are likely to contribute differentially to signal processing and plasticity.SIGNIFICANCE STATEMENT Glutamate is the most important excitatory neurotransmitter in the CNS, but not all glutamate receptors transmit fast excitatory signals at synapses. NMDA-type glutamate receptors act as voltage- and ligand-gated ion channels, with functional properties determined by their specific subunit composition. These receptors can be found at both synaptic and extrasynaptic sites on neurons, but the role of extrasynaptic NMDA receptors is unclear. Here, we demonstrate that retinal AII and A17 amacrine cells, postsynaptic partners at rod bipolar dyad synapses, express extrasynaptic (but not synaptic) NMDA receptors, with different and complementary GluN2 subunits. The localization of GluN2A-containing receptors to A17s and GluN2B-containing receptors to AIIs suggests a mechanism for differential modulation of excitability and signaling in this retinal microcircuit.

DSCAM and DSCAML1 Function in Self-Avoidance in Multiple Cell Types in the Developing Mouse Retina

In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell.

AII amacrine cells, which are the third-order neurons in the rod pathway, can be differentially labelled in rabbit retina by injecting Nuclear Yellow into the posterior chamber. Under ultraviolet excitation, the labelled retina appears strongly metachromatic, with the AII nuclei fluorescing silvery-yellow and the nuclei of other amacrine cells fluorescing blue. Labelled AII cells were injected with Lucifer Yellow under direct microscopic control in a superfused retinal preparation, and the dye was later photoconverted to an opaque reaction product. Rabbit AII amacrines, which number about 525,000 cells, reach a maximum density of 2,500-3,000 cells/mm2 on the peak visual streak, dropping to 400-500 cells/mm2 at the superior margin. These narrow-field amacrines have a bistratified dendritic morphology, with distinctive "lobular appendages" in sublamina a of the inner plexiform layer and wider ranging "arboreal dendrites" in sublamina b. Although the lobular field area increases 10-fold from the visual streak to the far periphery, the lobular field coverage is almost uniform across the retina, averaging 1.0 in inferior retina and 0.8 in superior retina. The dendritic field area of the arboreal dendrites also increases with eccentricity from the visual streak, but there are pronounced differences between inferior and superior retina. The arboreal fields are 2 to 3 times larger than the lobular fields throughout the inferior retina but up to 15 times larger in the superior retina. The arboreal field overlap is only 1.8 at the peak visual streak, increasing slightly to about 2.4 over most of the inferior retina; the overlap increases sharply in the superior retina, however, reaching values of 10 or more in the far periphery. Both the lobular and arboreal fields of AII cells are spaced more regularly than the somata, thus covering apparent gaps in the somatic array. An analysis of the potential convergence and divergence between rod bipolar cells and AII amacrine cells in the rabbit retina indicates that the neuronal architecture of the rod circuit is not organized in a uniform module that is simply scaled-up from central to peripheral retina. Moreover, peripheral fields in the superior and inferior retina that have equivalent densities of interneurons show markedly different rod bipolar----AII amacrine convergence ratios, with the result that many more rod photoreceptors converge on an AII amacrine cell in the superior retina than in the inferior retina.(ABSTRACT TRUNCATED AT 400 WORDS)

AII (Rod) Amacrine Cells Form a Network of Electrically Coupled Interneurons in the Mammalian Retina

In the retina, dopaminergic amacrine (interplexiform) cells establish multiple synapses on the perikarya of AII amacrines, the neurons that distribute rod signals to on- and off-cone bipolars. We used triple-label immunocytochemistry and confocal microscopy to identify the receptors contained within the postsynaptic active zone of these synapses in both mouse and rat retinas. We found that at the interface between the dendrites of the dopaminergic neurons and the AII amacrine cell perikarya clusters of postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors are situated in register with aggregates of presynaptic organelles immunoreactive for GABA, the GABA vesicular transporter, and the vesicular monoamine transporter-2. D1 and D23 dopamine receptors, on the other hand, do not form clusters on the surface of the perikarya of AII amacrine cells. We suggest that the synapses between retinal dopaminergic neurons and AII amacrine cells are GABAergic and that both GABA and dopamine are released by the presynaptic endings. GABA acts on the ionotropic receptors clustered at the postsynaptic active zone, whereas dopamine diffuses to more distant, slower-acting metabotropic receptors.