Jun Zhang

Retinal Parallel Processors: More than 100 Independent Microcircuits Operate within a Single Interneuron

Most neurons are highly polarized cells with branched dendrites that receive and integrate synaptic inputs and extensive axons that deliver action potential output to distant targets. By contrast, amacrine cells, a diverse class of inhibitory interneurons in the inner retina, collect input and distribute output within the same neuritic network. The extent to which most amacrine cells integrate synaptic information and distribute their output is poorly understood. Here, we show that single A17 amacrine cells provide reciprocal feedback inhibition to presynaptic bipolar cells via hundreds of independent microcircuits operating in parallel. The A17 uses specialized morphological features, biophysical properties, and synaptic mechanisms to isolate feedback microcircuits and maximize its capacity to handle many independent processes. This example of a neuron employing distributed parallel processing rather than spatial integration provides insights into how unconventional neuronal morphology and physiology can maximize network function while minimizing wiring cost.

Subunit- and pathway-specific localization of NMDA receptors and scaffolding proteins at ganglion cell synapses in rat retina

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from ON and OFF bipolar cells in distinct sublaminae of the inner plexiform layer (IPL). AMPA and NMDA receptors (AMPARs and NMDARs) mediate excitatory inputs in both synaptic layers, but specific roles for NMDARs at RGC synapses remain unclear. NMDARs comprise NR1 and NR2 subunits and are anchored by membrane-associated guanylate kinases (MAGUKs), but it is unknown whether particular NR2 subunits associate preferentially with particular NR1 splice variants and MAGUKs. Here, we used postembedding immunogold electron microscopy techniques to examine the subsynaptic localization of NMDAR subunits and MAGUKs at ON and OFF synapses onto rat RGCs. We found that the NR2A subunit, the NR1C2′ splice variant, and MAGUKs PSD-95 and PSD-93 are localized to the postsynaptic density (PSD), preferentially at OFF synapses, whereas the NR2B subunit, the NR1C2 splice variant, and the MAGUK SAP102 are localized perisynaptically, with NR2B exhibiting a preference for ON synapses. Consistent with these anatomical data, spontaneous EPSCs (sEPSCs) recorded from OFF cells exhibited an NMDAR component that was insensitive to the NR2B antagonist Ro 25-6981. In ON cells, sEPSCs expressed an NMDAR component, partially sensitive to Ro 25-6981, only when glutamate transport was inhibited, indicating perisynaptic expression of NR2B NMDARs. These results provide the first evidence for preferential association of particular NR1 splice variants, NR2 subunits, and MAGUKs at central synapses and suggest that different NMDAR subtypes may play specific roles at functionally distinct synapses in the retinal circuitry.

Distinct perisynaptic and synaptic localization of NMDA and AMPA receptors on ganglion cells in rat retina.

At most excitatory synapses, AMPA and NMDA receptors (AMPARs and NMDARs) occupy the postsynaptic density (PSD) and contribute to miniature excitatory postsynaptic currents (mEPSCs) elicited by single transmitter quanta. Juxtaposition of AMPARs and NMDARs may be crucial for certain types of synaptic plasticity, although extrasynaptic NMDARs may also contribute. AMPARs and NMDARs also contribute to evoked EPSCs in retinal ganglion cells (RGCs), but mEPSCs are mediated solely by AMPARs. Previous work indicates that an NMDAR component emerges in mEPSCs when glutamate uptake is reduced, suggesting that NMDARs are located near the release site but perhaps not directly beneath in the PSD. Consistent with this idea, NMDARs on RGCs encounter a lower glutamate concentration during synaptic transmission than do AMPARs. To understand better the roles of NMDARs in RGC function, we used immunohistochemical and electron microscopic techniques to determine the precise subsynaptic localization of NMDARs in RGC dendrites. RGC dendrites were labeled retrogradely with cholera toxin B subunit (CTB) injected into the superior colliculus (SC) and identified using postembedding immunogold methods. Colabeling with antibodies directed toward AMPARs and/or NMDARs, we found that nearly all AMPARs are located within the PSD, while most NMDARs are located perisynaptically, 100–300 nm from the PSD. This morphological evidence for exclusively perisynaptic NMDARs localizations suggests a distinct role for NMDARs in RGC function. J. Comp. Neurol. 498:810–820, 2006. Published 2006 Wiley‐Liss, Inc.

Coagonist Release Modulates NMDA Receptor Subtype Contributions at Synaptic Inputs to Retinal Ganglion Cells

NMDA receptors (NMDARs) are tetrameric protein complexes usually comprising two NR1 and two NR2 subunits. Different combinations of four potential NR2 subunits (NR2A-D) confer diversity in developmental expression, subsynaptic localization, and functional characteristics, including affinity for neurotransmitter. NR2B-containing NMDARs, for example, exhibit relatively high affinity both for glutamate and the coagonist glycine. Although multiple NMDAR subtypes can colocalize at individual synapses, particular subtypes often mediate inputs from distinct functional pathways. In retinal ganglion cells (RGCs), NMDARs contribute to synaptic responses elicited by light stimulus onset (“ON”) and offset (“OFF”), but roles for particular NMDAR subtypes, and potential segregation between the ON and OFF pathways, have not been explored. Moreover, elements in the retinal circuitry release two different NMDAR coagonists, glycine and d-serine, but the effects of endogenous coagonist release on the relative contribution of different NMDAR subtypes are unclear. Here, we show that coagonist release within the retina modulates the relative contribution of different NMDARs in the ON pathway of the rat retina. By pharmacologically stimulating functional pathways independently in acute slices and recording synaptic responses in RGCs, we show that ON inputs, but not OFF inputs, are mediated in part by NMDARs exhibiting NR2B-like pharmacology. Furthermore, suppressing release of NMDAR coagonist reduces NMDAR activation at ON synapses and increases the relative contribution of these putative NR2B-containing receptors. These results demonstrate direct evidence for evoked coagonist release onto NMDARs and indicate that modulating coagonist release may regulate the relative activation of different NMDAR subtypes in the ON pathway.

Requirement for Microglia for the Maintenance of Synaptic Function and Integrity in the Mature Retina

Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.

Passive diffusion as a mechanism underlying ribbon synapse vesicle release and resupply.

Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, “analog” sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbons surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-μm-thick sections of retinal tissue at an isotropic spatial resolution of ∼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon–vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates.

Complex inhibitory microcircuitry regulates retinal signaling near visual threshold

Neuronal microcircuits, small, localized signaling motifs involving two or more neurons, underlie signal processing and computation in the brain. Compartmentalized signaling within a neuron may enable it to participate in multiple, independent microcircuits. Each A17 amacrine cell in the mammalian retina contains within its dendrites hundreds of synaptic feedback microcircuits that operate independently to modulate feedforward signaling in the inner retina. Each of these microcircuits comprises a small (<1 μm) synaptic varicosity that typically receives one excitatory synapse from a presynaptic rod bipolar cell (RBC) and returns two reciprocal inhibitory synapses back onto the same RBC terminal. Feedback inhibition from the A17 sculpts the feedforward signal from the RBC to the AII, a critical component of the circuitry mediating night vision. Here, we show that the two inhibitory synapses from the A17 to the RBC express kinetically distinct populations of GABA receptors: rapidly activating GABA(A)Rs are enriched at one synapse while more slowly activating GABA(C)Rs are enriched at the other. Anatomical and electrophysiological data suggest that macromolecular complexes of voltage-gated (Cav) channels and Ca(2+)-activated K(+) channels help to regulate GABA release from A17 varicosities and limit GABA(C)R activation under certain conditions. Finally, we find that selective elimination of A17-mediated feedback inhibition reduces the signal to noise ratio of responses to dim flashes recorded in the feedforward pathway (i.e., the AII amacrine cell). We conclude that A17-mediated feedback inhibition improves the signal to noise ratio of RBC-AII transmission near visual threshold, thereby improving visual sensitivity at night.

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.