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Amacrine cells are a highly diverse group of inhibitory retinal interneurons that sculpt the responses of bipolar cells, ganglion cells, and other amacrine cells. They integrate excitatory inputs from bipolar cells and inhibitory inputs from other amacrine cells, but for most amacrine cells, little is known about the specificity and functional properties of their inhibitory inputs. Here, we have investigated GABA A receptors of the AII amacrine, a critical neuron in the rod pathway microcircuit, using patch-clamp recording in rat retinal slices. Puffer application of GABA evoked robust responses, but, surprisingly, spontaneous GABA A receptor-mediated postsynaptic currents were not observed, neither under control conditions nor following application of high-K + solution to facilitate release. To investigate the biophysical and pharmacological properties of GABA A receptors in AIIs, we therefore used nucleated patches and a fast application system. Both brief and long pulses of GABA (3 mM) evoked GABA A receptor-mediated currents with slow, multi-exponential decay kinetics. The average weighted time constant (τ w ) of deactivation was ~163 ms. Desensitization was even slower, with τ w ~330 ms. Non-stationary noise analysis of patch responses and directly observed channel gating yielded a single-channel conductance of ~23 pS. Pharmacological investigation suggested the presence of α2 and/or α3 subunits, as well as the γ2 subunit. Such subunit combinations are typical of GABA A receptors with slow kinetics. If synaptic GABA A receptors of AII amacrines have similar functional properties, the slow deactivation and desensitization kinetics will facilitate temporal summation of GABAergic inputs, allowing effective summation and synaptic integration to occur even for relatively low frequencies of inhibitory inputs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Beltrán-Matas, Hartveit and Veruki.)

Bipolar cells convey signals from photoreceptors in the outer retina to amacrine and ganglion cells in the inner retina. In mammals, there are typically 10-15 types of cone bipolar cells and one type of rod bipolar cell. Different types of cone bipolar cells are thought to code and transmit different features of a complex visual stimulus, thereby generating parallel channels that uniquely filter and transform the photoreceptor outputs. Differential synaptic connectivity and expression of ligand- and voltage-gated ion channels are thought to be important mechanisms for processing and filtering visual signals. Whereas the biophysical basis for such mechanisms has been investigated more extensively in rat retina, there is a lack of quantitative morphological data necessary for advancing the structure-function correlation in this species, as recent connectomics investigations have focused on mouse retina. Here, we performed whole-cell recordings from cone and rod bipolar cells in rat retinal slices, filled the cells with fluorescent dyes, and acquired image stacks by multiphoton excitation microscopy. Following deconvolution, we performed digital reconstruction and morphometric analysis of 25 cone and 14 rod bipolar cells. Compared to previous descriptions, the extent and complexity of branching of the axon terminal was surprisingly high. By precisely quantifying the level of stratification of the axon terminals in the inner plexiform layer, we have generated a reference system for reliable classification of individual cells in future studies focused on correlating physiological and morphological properties. The implemented workflow can be extended to the development of morphologically realistic compartmental models for these neurons., (© 2022 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

The vertebrate retina contains a large number of different types of neurons that can be distinguished by their morphological properties. Assuming that no location should be without a contribution from the circuitry and function linked to a specific type of neuron, it is expected that the dendritic trees of neurons belonging to a type will cover the retina in a regular manner. Thus, for most types of neurons, the contribution to visual processing is thought to be independent of the exact location of individual neurons across the retina. Here, we have investigated the distribution of AII amacrine cells in rat retina. The AII is a multifunctional amacrine cell found in mammals and involved in synaptic microcircuits that contribute to visual processing under both scotopic and photopic conditions. Previous investigations have suggested that AIIs are regularly distributed, with a nearest-neighbor distance regularity index of ~4. It has been argued, however, that this presumed regularity results from treating somas as points, without taking into account their actual spatial extent which constrains the location of other cells of the same type. When we simulated random distributions of cell bodies with size and density similar to real AIIs, we confirmed that the simulated distributions could not be distinguished from the distributions observed experimentally for AIIs in different regions and eccentricities of the retina. The developmental mechanisms that generate the observed distributions of AIIs remain to be investigated.

Amacrine cells, inhibitory interneurons of the retina, feature synaptic inputs and outputs in close proximity throughout their dendritic trees, making them notable exceptions to prototypical somato-dendritic integration with output transmitted via axonal action potentials. The extent of dendritic compartmentalization in amacrine cells with widely differing dendritic tree morphology, however, is largely unexplored. Combining compartmental modeling, dendritic Ca 2+ imaging, targeted microiontophoresis and multielectrode patch-clamp recording (voltage and current clamp, capacitance measurement of exocytosis), we investigated integration in the AII amacrine cell, a narrow-field electrically coupled interneuron that participates in multiple, distinct microcircuits. Physiological experiments were performed with in vitro slices prepared from retinas of both male and female rats. We found that the morphology of the AII enables simultaneous local and global integration of inputs targeted to different dendritic regions. Local integration occurs within spatially restricted dendritic subunits and narrow time windows and is largely unaffected by the strength of electrical coupling. In contrast, global integration across the dendritic tree occurs over longer time periods and is markedly influenced by the strength of electrical coupling. These integrative properties enable AII amacrines to combine local control of synaptic plasticity with location-independent global integration. Dynamic inhibitory control of dendritic subunits is likely to be of general importance for amacrine cells, including cells with small dendritic trees, as well as for inhibitory interneurons in other regions of the CNS. SIGNIFICANCE STATEMENT Our understanding of synaptic integration is based on the prototypical morphology of a neuron with multiple dendrites and a single axon at opposing ends of a cell body. Many neurons, notably retinal amacrine cells, are exceptions to this arrangement, and display input and output synapses interspersed along their dendritic branches. In the large dendritic trees of some amacrine cells, such arrangements can give rise to multiple computational subunits. Other amacrine cells, with small dendritic trees, have been assumed to operate as single computational units. Here, we report the surprising result that despite a small dendritic tree, the AII amacrine cell simultaneously performs local integration of synaptic inputs (over smaller dendritic subregions) and global integration across the entire cell., (Copyright © 2022 the authors.)

Amacrine cells constitute a large and heterogeneous group of inhibitory interneurons in the retina. The A17 amacrine plays an important role for visual signalling in the rod pathway microcircuit of the mammalian retina. It receives excitatory input from rod bipolar cells and provides feedback inhibition to the same cells. However, from ultrastructural investigations, there is evidence for input to A17s from other types of amacrine cells, presumably inhibitory, but there is a lack of information about the identity and functional properties of the synaptic receptors and how inhibition contributes to the integrative properties of A17s. Here, we studied the biophysical and pharmacological properties of GABAergic spontaneous inhibitory postsynaptic currents (spIPSCs) and GABA A receptors of A17 amacrines using whole-cell and outside-out patch recordings from rat retinal slices. The spIPSCs displayed fast onsets (10%-90% rise time ~740 μs) and double-exponential decays (τ fast ~4.5 ms [43% of amplitude]; τ slow ~22 ms). Ultra-fast application of brief pulses of GABA (3 mM) to patches evoked responses with deactivation kinetics best fitted by a triple-exponential function (τ 1 ~5.3 ms [55% of amplitude]; τ 2 ~48 ms [32% of amplitude]; τ 3 ~187 ms). Non-stationary noise analysis of spIPSCs and patch responses yielded single-channel conductances of ~21 and ~25 pS, respectively. Pharmacological analysis suggested that the spIPSCs are mediated by receptors with an α1βγ2 subunit composition and the somatic receptors have an α2βγ2 and/or α3βγ2 composition. These results demonstrate the presence of synaptic GABA A receptors on A17s, which may play an important role in signal integration in these cells., (© 2022 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Signal processing within the retina is generally mediated by graded potentials, whereas output is conveyed by action potentials transmitted along optic nerve axons. Among retinal neurons, amacrine cells seem to be an exception to this general rule, as several types generate voltage-gated Na + (Na v ) channel-dependent action potentials. The AII, a narrow-field, bistratified axon-less amacrine cell found in mammalian retinas, displays a unique process that resembles an axon initial segment (AIS), with expression of Na v channels colocalized with the cytoskeletal protein ankyrin-G, and generates action potentials. As the role of spiking in AIIs is uncertain, we hypothesized that the morphological properties of the AIS-like process could provide information relevant for its functional importance, including potential pre- and/or postsynaptic connectivity. For morphological analysis, we injected AII amacrine cells in slices with fluorescent dye and immunolabeled the slices for ankyrin-G. Subsequently, this enabled us to reliably identify AII-type processes among ankyrin-G-labeled processes in wholemount retina. We systematically analyzed the laminar localization, spatial orientation, and distribution of the AIS-like processes as a function of retinal eccentricity. In the horizontal plane, the processes displayed no preferred orientation and terminal endings were randomly distributed. In the vertical plane, the processes displayed a horizontal preference, but also ascended and descended into the inner nuclear layer and proximal inner plexiform layer, respectively. These results suggest that the AII amacrine AIS-like process is unlikely to take part in conventional synaptic connections, but may instead be adapted to respond to volume neurotransmission by means of extrasynaptic receptors., (© 2021 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

The NMDA receptors (NMDARs) expressed by AII and A17 amacrine cells, the two main inhibitory interneurons of the rod pathway microcircuit in the mammalian retina, are exclusively extrasynaptic, activated by ambient levels of glutamate, and molecularly distinct, with AII and A17 amacrines expressing GluN2B- and GluN2A-containing receptors, respectively. This important sensory microcircuit thus provides a unique model to study the activation and function of extrasynaptic NMDARs. Here, we investigated the sources of glutamate and the endogenous co-agonists (d-serine or glycine) that activate these distinct populations of NMDARs. With acute slices from rat retina, we used whole-cell voltage-clamp recording and measurement of current noise to monitor levels of NMDAR activity. Pre-incubation of retina with bafilomycin A1 (an inhibitor of neurotransmitter uptake into synaptic vesicles) abolished NMDAR-mediated noise in AII, but not A17 amacrines, suggesting a vesicular source of glutamate activates AII NMDARs, whereas a non-vesicular source activates A17 NMDARs. Pre-incubation of retina with l-methionine sulfoximine (an inhibitor of glutamine synthetase) also abolished NMDAR-mediated noise in AII, but not A17 amacrines, suggesting a neuronal source of glutamate activates AII NMDARs, whereas a glial source activates A17 NMDARs. Enzymatic breakdown of d-serine reduced NMDAR-mediated noise in AII, but not A17 amacrines, suggesting d-serine is the endogenous co-agonist at AII, but not A17 NMDARs. Our results reveal unique characteristics of these two populations of extrasynaptic NMDARs. The differential and independent activation of these receptors is likely to provide specific contributions to the signal processing and plasticity of the cellular components of the rod pathway microcircuit., (© 2021 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Gap junctions are ubiquitous within the retina, but in general, it remains to be determined whether gap junction coupling between specific cell types is sufficiently strong to mediate functionally relevant coupling via electrical synapses. From ultrastructural, tracer coupling and immunolabeling studies, there is clear evidence for gap junctions between cone bipolar cells, but it is not known if these gap junctions function as electrical synapses. Here, using whole-cell voltage-clamp recording in rat (male and female) retinal slices, we investigated whether the gap junctions of bipolar cells make a measurable contribution to the membrane properties of these cells. We measured the input resistance (R N ) of bipolar cells before and after applying meclofenamic acid (MFA) to block gap junctions. In the presence of MFA, R N of ON-cone bipolar cells displayed a clear increase, paralleled by block of the electrical coupling between these cells and AII amacrine cells in recordings of coupled cell pairs. For OFF-cone and rod bipolar cells, R N did not increase in the presence of MFA. The results for rod bipolar cells are consistent with the lack of gap junctions in these cells. However, for OFF-cone bipolar cells, our results suggest that the morphologically identified gap junctions between these cells do not support a junctional conductance that is sufficient to mediate effective electrical coupling. Instead, these junctions might play a role in chemical and/or metabolic coupling between subcellular compartments.

Exocytotic release of neurotransmitter can be quantified by electrophysiological recording from postsynaptic neurons. Alternatively, fusion of synaptic vesicles with the cell membrane can be measured as increased capacitance by recording directly from a presynaptic neuron. The "Sine + DC" technique is based on recording from an unbranched cell, represented by an electrically equivalent RC-circuit. It is challenging to extend such measurements to branching neurons where exocytosis occurs at a distance from a somatic recording electrode. The AII amacrine is an important inhibitory interneuron of the mammalian retina and there is evidence that exocytosis at presynaptic lobular dendrites increases the capacitance. Here, we combined electrophysiological recording and computer simulations with realistic compartmental models to explore capacitance measurements of rat AII amacrine cells. First, we verified the ability of the "Sine + DC" technique to detect depolarization-evoked exocytosis in physiological recordings. Next, we used compartmental modeling to demonstrate that capacitance measurements can detect increased membrane surface area at lobular dendrites. However, the accuracy declines for lobular dendrites located further from the soma due to frequency-dependent signal attenuation. For sine wave frequencies ≥1 kHz, the magnitude of the total releasable pool of synaptic vesicles will be significantly underestimated. Reducing the sine wave frequency increases overall accuracy, but when the frequency is sufficiently low that exocytosis can be detected with high accuracy from all lobular dendrites (~100 Hz), strong electrical coupling between AII amacrines compromises the measurements. These results need to be taken into account in studies with capacitance measurements from these and other electrically coupled neurons., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

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 Ca 2+ imaging, we verified that activation of NMDA receptors evoked an increase of intracellular Ca 2+ 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., (Copyright © 2019 the authors 0270-6474/19/390627-24$15.00/0.)

Amacrine cells are critical for processing of visual signals, but little is known about their electrotonic structure and passive membrane properties. AII amacrine cells are multifunctional interneurons in the mammalian retina and essential for both rod- and cone-mediated vision. Their dendrites are the site of both input and output chemical synapses and gap junctions that form electrically coupled networks. This electrical coupling is a challenge for developing realistic computer models of single neurons. Here, we combined multiphoton microscopy and electrophysiological recording from dye-filled AII amacrine cells in rat retinal slices to develop morphologically accurate compartmental models. Passive cable properties were estimated by directly fitting the current responses of the models evoked by voltage pulses to the physiologically recorded responses, obtained after blocking electrical coupling. The average best-fit parameters (obtained at - 60 mV and ~ 25 °C) were 0.91 µF cm -2 for specific membrane capacitance, 198 Ω cm for cytoplasmic resistivity, and 30 kΩ cm 2 for specific membrane resistance. We examined the passive signal transmission between the cell body and the dendrites by the electrotonic transform and quantified the frequency-dependent voltage attenuation in response to sinusoidal current stimuli. There was significant frequency-dependent attenuation, most pronounced for signals generated at the arboreal dendrites and propagating towards the soma and lobular dendrites. In addition, we explored the consequences of the electrotonic structure for interpreting currents in somatic, whole-cell voltage-clamp recordings. The results indicate that AII amacrines cannot be characterized as electrotonically compact and suggest that their morphology and passive properties can contribute significantly to signal integration and processing.

Loss of neurons due to injury or neurodegeneration can lead to dramatically altered neural circuits, resulting in reduced or lost function. One mechanism to preserve function could be to re-establish the stereotypic connectivity among the remnant neurons. In the mammalian retina, such a selective re-wiring has now been described., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In chemical synapses, neurotransmitter molecules released from presynaptic vesicles activate populations of postsynaptic receptors that vary in functional properties depending on their subunit composition. Differential expression and localization of specific receptor subunits are thought to play fundamental roles in signal processing, but our understanding of how that expression is adapted to the signal processing in individual synapses and microcircuits is limited. At ribbon synapses, glutamate release is independent of action potentials and characterized by a high and rapidly changing rate of release. Adequately translating such presynaptic signals into postsynaptic electrical signals poses a considerable challenge for the receptor channels in these synapses. Here, we investigated the functional properties of AMPA receptors of AII amacrine cells in rat retina that receive input at spatially segregated ribbon synapses from OFF-cone and rod bipolar cells. Using patch-clamp recording from outside-out patches, we measured the concentration dependence of response amplitude and steady-state desensitization, the single-channel conductance and the maximum open probability. The GluA4 subunit seems critical for the functional properties of AMPA receptors in AII amacrines and immunocytochemical labeling suggested that GluA4 is located at synapses made by both OFF-cone bipolar cells and rod bipolar cells. Finally, we used a series of experimental observables to develop kinetic models for AII amacrine AMPA receptors and subsequently used the models to explore the behavior of the receptors and responses generated by glutamate concentration profiles mimicking those occurring in synapses. These models will facilitate future in silico modeling of synaptic signaling and processing in AII amacrine cells.

Background: Accurate reconstruction of the morphology of single neurons is important for morphometric studies and for developing compartmental models. However, manual morphological reconstruction can be extremely time-consuming and error-prone and algorithms for automatic reconstruction can be challenged when applied to neurons with a high density of extensively branching processes., New Method: We present a procedure for semi-automatic reconstruction specifically adapted for densely branching neurons such as the AII amacrine cell found in mammalian retinas. We used whole-cell recording to fill AII amacrine cells in rat retinal slices with fluorescent dyes and acquired digital image stacks with multi-photon excitation microscopy. Our reconstruction algorithm combines elements of existing procedures, with segmentation based on adaptive thresholding and reconstruction based on a minimal spanning tree. We improved this workflow with an algorithm that reconnects neuron segments that are disconnected after adaptive thresholding, using paths extracted from the image stacks with the Fast Marching method., Results: By reducing the likelihood that disconnected segments were incorrectly connected to neighboring segments, our procedure generated excellent morphological reconstructions of AII amacrine cells., Comparison With Existing Methods: Reconstructing an AII amacrine cell required about 2h computing time, compared to 2-4days for manual reconstruction. To evaluate the performance of our method relative to manual reconstruction, we performed detailed analysis using a measure of tree structure similarity (DIADEM score), the degree of projection area overlap (Dice coefficient), and branch statistics., Conclusions: We expect our procedure to be generally useful for morphological reconstruction of neurons filled with fluorescent dyes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

AII amacrine cells have been found in all mammalian retinas examined and play an important role for visual processing under both scotopic and photopic conditions. Whereas ultrastructural investigations have provided a detailed understanding of synaptic connectivity, there is little information available with respect to quantitative properties and variation of cellular morphology. Here, we performed whole-cell recordings from AII amacrine cells in rat retinal slices and filled the cells with fluorescent dyes. Multi-photon excitation microscopy was used to acquire image stacks and after deconvolution, we performed quantitative morphological reconstruction by computer-aided manual tracing. We reconstructed and performed morphometric analysis on 43 AII amacrine cells, with a focus on branching pattern, dendritic lengths and diameters, surface area, and number and distribution of dendritic varicosities. Compared to previous descriptions, the most surprising result was the considerable extent of branching, with the maximum branch order ranging from approximately 10-40. We found that AII amacrine cells conform to a recently described general structural design principle for neural arbors, where arbor density decreases proportionally to increasing territory size. We confirmed and quantified the bi-stratified morphology of AII amacrine cells by analyzing the arborizations as a function of retinal localization or with Sholl spheres. Principal component and cluster analysis revealed no evidence for morphological subtypes of AII amacrines. These results establish a database of morphometric properties important for studies of development, regeneration, degeneration, and disease processes, as well as a workflow compatible with compartmental modeling., Competing Interests: The authors declare that they have no conflict of interest.

At many glutamatergic synapses, non-N-methyl-d-aspartate (NMDA) and NMDA receptors are coexpressed postsynaptically. In the mammalian retina, glutamatergic rod bipolar cells are presynaptic to two rod amacrine cells (AII and A17) that constitute dyad postsynaptic partners opposite each presynaptic active zone. Whereas there is strong evidence for expression of non-NMDA receptors by both AII and A17 amacrines, the expression of NMDA receptors by the pre- and postsynaptic neurons in this microcircuit has not been resolved. In this study, using patch-clamp recording from visually identified cells in rat retinal slices, we investigated the expression and functional properties of NMDA receptors in these cells with a combination of pharmacological and biophysical methods. Pressure application of NMDA did not evoke a response in rod bipolar cells, but for both AII and A17 amacrines, NMDA evoked responses that were blocked by a competitive antagonist (CPP) applied extracellularly and an open channel blocker (MK-801) applied intracellularly. NMDA-evoked responses also displayed strong Mg(2+)-dependent voltage block and were independent of gap junction coupling. With low-frequency application (60-s intervals), NMDA-evoked responses remained stable for up to 50 min, but with higher-frequency stimulation (10- to 20-s intervals), NMDA responses were strongly and reversibly suppressed. We observed strong potentiation when NMDA was applied in nominally Ca(2+)-free extracellular solution, potentially reflecting Ca(2+)-dependent NMDA receptor inactivation. These results indicate that expression of functional (i.e., conductance-increasing) NMDA receptors is common to both AII and A17 amacrine cells and suggest that these receptors could play an important role for synaptic signaling, integration, or plasticity in the rod pathway., (Copyright © 2016 the American Physiological Society.)

There is increasing evidence that diabetic retinopathy is a primary neuropathological disorder that precedes the microvascular pathology associated with later stages of the disease. Recently, we found evidence for altered functional properties of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in A17, but not AII, amacrine cells in the mammalian retina, and the observed changes were consistent with an upregulation of the GluA2 subunit, a key determinant of functional properties of AMPA receptors, including Ca(2+) permeability and current-voltage (I-V) rectification properties. Here, we have investigated functional changes of extrasynaptic AMPA receptors in AII amacrine cells evoked by diabetes. With patch-clamp recording of nucleated patches from retinal slices, we measured Ca(2+) permeability and I-V rectification in rats with ∼3 wk of streptozotocin-induced diabetes and age-matched, noninjected controls. Under bi-ionic conditions (extracellular Ca(2+) concentration = 30 mM, intracellular Cs(+) concentration = 171 mM), the reversal potential (Erev) of AMPA-evoked currents indicated a significant reduction of Ca(2+) permeability in diabetic animals [Erev = -17.7 mV, relative permeability of Ca(2+) compared with Cs(+) (PCa/PCs) = 1.39] compared with normal animals (Erev = -7.7 mV, PCa/PCs = 2.35). Insulin treatment prevented the reduction of Ca(2+) permeability. I-V rectification was examined by calculating a rectification index (RI) as the ratio of the AMPA-evoked conductance at +40 and -60 mV. The degree of inward rectification in patches from diabetic animals (RI = 0.48) was significantly reduced compared with that in normal animals (RI = 0.30). These results suggest that diabetes evokes a change in the functional properties of extrasynaptic AMPA receptors of AII amacrine cells. These changes could be representative for extrasynaptic AMPA receptors elsewhere in AII amacrine cells and suggest that synaptic and extrasynaptic AMPA receptors are differentially regulated., (Copyright © 2015 the American Physiological Society.)

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., (Copyright © 2015 the authors 0270-6474/15/355422-12$15.00/0.)

Adaptation enables the visual system to operate across a large range of background light intensities. There is evidence that one component of this adaptation is mediated by modulation of gap junctions functioning as electrical synapses, thereby tuning and functionally optimizing specific retinal microcircuits and pathways. The AII amacrine cell is an interneuron found in most mammalian retinas and plays a crucial role for processing visual signals in starlight, twilight and daylight. AII amacrine cells are connected to each other by gap junctions, potentially serving as a substrate for signal averaging and noise reduction, and there is evidence that the strength of electrical coupling is modulated by the level of background light. Whereas there is extensive knowledge concerning the retinal microcircuits that involve the AII amacrine cell, it is less clear which signaling pathways and intracellular transduction mechanisms are involved in modulating the junctional conductance between electrically coupled AII amacrine cells. Here we review the current state of knowledge, with a focus on the recent evidence that suggests that the modulatory control involves activity-dependent changes in the phosphorylation of the gap junction channels between AII amacrine cells, potentially linked to their intracellular Ca(2+) dynamics. This article is part of a Special Issue entitled Electrical Synapses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tunneling nanotubes (TNTs) are recently discovered conduits for a previously unrecognized form of cell-to-cell communication. These nanoscale, F-actin-containing membrane tubes connect cells over long distances and facilitate the intercellular exchange of small molecules and organelles. Using optical membrane-potential measurements combined with mechanical stimulation and whole-cell patch-clamp recording, we demonstrate that TNTs mediate the bidirectional spread of electrical signals between TNT-connected normal rat kidney cells over distances of 10 to 70 μm. Similar results were obtained for other cell types, suggesting that electrical coupling via TNTs may be a widespread characteristic of animal cells. Strength of electrical coupling depended on the length and number of TNT connections. Several lines of evidence implicate a role for gap junctions in this long-distance electrical coupling: punctate connexin 43 immunoreactivity was frequently detected at one end of TNTs, and electrical coupling was voltage-sensitive and inhibited by meclofenamic acid, a gap-junction blocker. Cell types lacking gap junctions did not show TNT-dependent electrical coupling, which suggests that TNT-mediated electrical signals are transmitted through gap junctions at a membrane interface between the TNT and one cell of the connected pair. Measurements of the fluorescent calcium indicator X-rhod-1 revealed that TNT-mediated depolarization elicited threshold-dependent, transient calcium signals in HEK293 cells. These signals were inhibited by the voltage-gated Ca(2+) channel blocker mibefradil, suggesting they were generated via influx of calcium through low voltage-gated Ca(2+) channels. Taken together, our data suggest a unique role for TNTs, whereby electrical synchronization between distant cells leads to activation of downstream target signaling.

Accurate measurement of the junctional conductance (G(j)) between electrically coupled cells can provide important information about the functional properties of coupling. With the development of tight-seal, whole-cell recording, it became possible to use dual, single-electrode voltage-clamp recording from pairs of small cells to measure G(j). Experiments that require reduced perturbation of the intracellular environment can be performed with high-resistance pipettes or the perforated-patch technique, but an accompanying increase in series resistance (R(s)) compromises voltage-clamp control and reduces the accuracy of G(j) measurements. Here, we present a detailed analysis of methodologies available for accurate determination of steady-state G(j) and related parameters under conditions of high R(s), using continuous or discontinuous single-electrode voltage-clamp (CSEVC or DSEVC) amplifiers to quantify the parameters of different equivalent electrical circuit model cells. Both types of amplifiers can provide accurate measurements of G(j), with errors less than 5% for a wide range of R(s) and G(j) values. However, CSEVC amplifiers need to be combined with R(s)-compensation or mathematical correction for the effects of nonzero R(s) and finite membrane resistance (R(m)). R(s)-compensation is difficult for higher values of R(s) and leads to instability that can damage the recorded cells. Mathematical correction for R(s) and R(m) yields highly accurate results, but depends on accurate estimates of R(s) throughout an experiment. DSEVC amplifiers display very accurate measurements over a larger range of R(s) values than CSEVC amplifiers and have the advantage that knowledge of R(s) is unnecessary, suggesting that they are preferable for long-duration experiments and/or recordings with high R(s)., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

AII amacrine cells in the mammalian retina are connected via electrical synapses to on-cone bipolar cells and to other AII amacrine cells. To understand synaptic integration in these interneurons, we need information about the junctional conductance (g(j)), the membrane resistance (r(m)), the membrane capacitance (C(m)), and the cytoplasmic resistivity (R(i)). Due to the extensive electrical coupling, it is difficult to obtain estimates of r(m), as well as the relative contribution of the junctional and nonjunctional conductances to the total input resistance of an AII amacrine cell. Here we used dual voltage-clamp recording of pairs of electrically coupled AII amacrine cells in an in vitro slice preparation from rat retina and applied meclofenamic acid (MFA) to block the electrical coupling and isolate single AII amacrines electrically. In the control condition, the input resistance (R(in)) was approximately 620 Mohms and the apparent r(m) was approximately 760 Mohms. After block of electrical coupling, determined by estimating g(j) in the dual recordings, R(in) and r(m) were approximately 4,400 Mohms, suggesting that the nongap junctional conductance of an AII amacrine cell is approximately 16% of the total input conductance. Control experiments with nucleated patches from AII amacrine cells suggested that MFA had no effect on the nongap junctional membrane of these cells. From morphological reconstructions of AII amacrine cells filled with biocytin, we obtained a surface area of approximately 900 microm(2) which, with a standard value for C(m) of 0.01 pF/microm(2), corresponds to an average capacitance of approximately 9 pF and a specific membrane resistance of approximately 41 kohms cm(2). Together with information concerning synaptic connectivity, these data will be important for developing realistic compartmental models of the network of AII amacrine cells.

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.

Rod bipolar cells transmit visual signals from their dendrites, where they receive input from rod photoreceptors, to their axon terminals, where they synapse onto amacrine cells. Little is known, however, about the transmission and possible transformation of these signals. We have combined axon terminal recording in retinal slices, quantitative, light-microscopic morphological reconstruction and computer modelling to obtain detailed compartmental models of rat rod bipolar cells. Passive cable properties were estimated by directly fitting the current responses of the models evoked by voltage pulses to the physiologically recorded responses. At a holding potential of -60 mV, the average best-fit parameters were 1.1 microF cm(-2) for specific membrane capacitance (C(m)), 130 Omega cm for cytoplasmic resistivity (R(i)), and 24 kOmega cm(2) for specific membrane resistance (R(m)). The passive integration of excitatory and inhibitory synaptic inputs was examined by computer modelling with physiologically realistic synaptic conductance waveforms. For both transient and steady-state synaptic inhibition, the inhibitory effect was relatively insensitive to the location of the inhibition. For transient synaptic inhibition, the time window of effective inhibition depended critically on the relative timing of inhibition and excitation. The passive signal transmission between soma and axon terminal was examined by the electrotonic transform and quantified as the frequency-dependent voltage attenuation of sinusoidal voltage waveforms. For the range of parameters explored (axon diameter and length, R(i)), the lowest cutoff frequency observed was approximately 300 Hz, suggesting that realistic scotopic visual signals will be faithfully transmitted from soma to axon terminal, with minimal passive attenuation along the axon.

AII amacrine cells form a network of electrically coupled interneurons in the mammalian retina and tracer coupling studies suggest that the junctional conductance (G(j)) can be modulated. However, the dynamic range of G(j) and the functional consequences of varying G(j) over the dynamic range are unknown. Here we use whole cell recordings from pairs of coupled AII amacrine cells in rat retinal slices to provide direct evidence for physiological modulation of G(j), appearing as a time-dependent increase from about 500 pS to a maximum of about 3,000 pS after 30-90 min of recording. The increase occurred in recordings with low- but not high-resistance pipettes, suggesting that it was related to intracellular washout and perturbation of a modulatory system. Computer simulations of a network of electrically coupled cells verified that our recordings were able to detect and quantify changes in G(j) over a large range. Dynamic-clamp electrophysiology, with insertion of electrical synapses between AII amacrine cells, allowed us to finely and reversibly control G(j) within the same range observed for physiologically coupled cells and to examine the quantitative relationship between G(j) and steady-state coupling coefficient, synchronization of subthreshold membrane potential fluctuations, synchronization and transmission of action potentials, and low-pass filter characteristics. The range of G(j) values over which signal transmission was modulated depended strongly on the specific functional parameter examined, with the largest range observed for action potential transmission and synchronization, suggesting that the full range of G(j) values observed during spontaneous run-up of coupling could represent a physiologically relevant dynamic range.

The functional properties of glycine receptors were analysed in different types of wide-field amacrine cells, narrowly stratifying cells considered to play a role in larger-scale integration across the retina. The patch-clamp technique was used to record spontaneous IPSCs (spIPSCs) and glycine-evoked patch responses from mature rat retinal slices (4-7 weeks postnatal). Glycinergic spIPSCs were blocked reversibly by strychnine (300 nM). Compared to previously described spIPSCs in AII amacrine cells, the spIPSCs in wide-field amacrine cells displayed a very slow decay time course (tau(fast) approximately 15 ms; tau(slow) approximately 57 ms). The kinetic properties of spIPSCs in whole-cell recordings were paralleled by even slower deactivation kinetics of responses evoked by brief pulses of glycine (3 mm) to outside-out patches from wide-field amacrine cells (tau(fast) approximately 45 ms; tau(slow) approximately 350 ms). Non-stationary noise analysis of patch responses and spIPSCs yielded similar average single-channel conductances (approximately 31 and approximately 34 pS, respectively). Similar, as well as both lower- and higher-conductance levels could be identified from directly observed single-channel gating during the decay phase of spIPSCs and patch responses. These results suggest that the slow glycinergic spIPSCs in wide-field amacrine cells involve alpha2beta heteromeric receptors. Taken together with previous work, the kinetic properties of glycine receptors in different types of amacrine cells display a considerable range that is probably a direct consequence of differential expression of receptor subunits. Unique kinetic properties are likely to differentially shape the glycinergic input to different types of amacrine cells and thereby contribute to distinct integrative properties among these cells.

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.

Chemical synaptic transmission depends on neurotransmitter-gated ion channels concentrated in the postsynaptic membrane of specialized synaptic contacts. The functional characteristics of these neurotransmitter receptor channels are important for determining the properties of synaptic transmission. Whole-cell recording of postsynaptic currents (PSCs) and outside-out patch recording of transmitter-evoked currents are important tools for estimating the single-channel conductance and the number of receptors contributing to the PSC activated by a single transmitter quantum. When single-channel activity cannot be directly resolved, non-stationary noise analysis is a valuable tool for determining these parameters. Peak-scaled non-stationary noise analysis can be used to compensate for quantal variability in synaptic currents. Here, we present detailed protocols for conventional and peak-scaled non-stationary noise analysis of spontaneous PSCs and responses in outside-out patches. In addition, we include examples of computer code for individual functions used in the different stages of non-stationary noise analysis. These analysis procedures require 3-8 h.

Whereas glutamate transporters in glial cells and postsynaptic neurons contribute significantly to re-uptake of synaptically released transmitter, the functional role of presynaptic glutamate transporters is poorly understood. Here, we used electrophysiological recording to examine the functional properties of a presynaptic glutamate transporter in rat retinal rod bipolar cells and its role in regulating glutamatergic synaptic transmission between rod bipolar cells and amacrine cells. Release of glutamate activated the presynaptic transporter with a time course that suggested a perisynaptic localization. The transporter was also activated by spillover of glutamate from neighboring rod bipolar cells. By recording from pairs of rod bipolar cells and AII amacrine cells, we demonstrate that activation of the transporter-associated anion current hyperpolarizes the presynaptic terminal and thereby inhibits synaptic transmission by suppressing transmitter release. Given the evidence for presynaptic glutamate transporters, similar mechanisms could be of general importance for transmission in the nervous system.

AII amacrine cells play a crucial role in retinal signal transmission under scotopic conditions. We have used rat retinal slices to investigate the functional properties of inhibitory glycine receptors on AII cells by recording spontaneous IPSCs (spIPSCs) in whole cells and glycine-evoked responses in outside-out patches. Glycinergic spIPSCs displayed fast kinetics with an average 10-90% rise time of approximately 500 mus, and a decay phase best fitted by a double-exponential function with tau(fast) approximately 4.8 ms (97.5% amplitude contribution) and tau(slow) approximately 33 ms. Decay kinetics were voltage dependent. Ultrafast application of brief ( approximately 2-5 ms) pulses of glycine (3 mm) to patches, evoked responses with fast deactivation kinetics best fitted with a double-exponential function with tau(fast) approximately 4.6 ms (85% amplitude contribution) and tau(slow) approximately 17 ms. Double-pulse experiments indicated recovery from desensitization after a 100-ms pulse of glycine with a double-exponential time course (tau(fast) approximately 71 ms and tau(slow) approximately 1713 ms). Non-stationary noise analysis of spIPSCs and patch responses, and directly observed channel gating yielded similar single-channel conductances ( approximately 41 to approximately 47 pS). In addition, single-channel gating occurred at approximately 83 pS. These results suggest that the fast glycinergic spIPSCs in AII cells are probably mediated by alpha1beta heteromeric receptors with a contribution from alpha1 homomeric receptors. We hypothesize that glycinergic synaptic input may target the arboreal dendrites of AII cells, and could serve to shunt excitatory input from rod bipolar cells and transiently uncouple the transcellular current through electrical synapses between AII cells and between AII cells and ON-cone bipolar cells.

The properties of neurotransmitter receptor channels are important for determining synaptic transmission in the nervous system. The presence of quantal variability complicates the use of conventional non-stationary noise analysis for determining the unitary conductance and number of channels involved in synaptic currents. Peak-scaled non-stationary noise analysis has been used to compensate for quantal variability, but there is evidence that the resulting variance versus mean relationships can be transformed from parabolic to skewed. We have used computer modelling based on experimentally derived kinetic schemes to investigate such relationships and demonstrate that their shape is a consequence of the temporal structure of the fluctuations during synaptic responses. Covariance analysis showed that peak-scaling generates a skewed relationship when the covariance function decays rapidly (compared to the average response waveform), corresponding to a low correlation between fluctuations at the peak and in neighbouring regions of the decay phase. A parabolic relationship is obtained when the covariance function decays more slowly, corresponding to a higher correlation. Irrespective of a skewed or parabolic relationship, we demonstrate that the unitary current can be reliably estimated, with a coefficient of variation (CV) as low as 0.05 and bias as low as +/-2% under ideal conditions. While the shape of the variance versus mean curve after peak-scaled non-stationary noise analysis is ultimately a consequence of the kinetic properties of the channels, inadequate alignment of individual waveforms can transform the relationship from parabolic to skewed, and low-pass filtering can transform the relationship from skewed to parabolic. These findings have important implications for analysis of experimental data.

The functional properties of spontaneous, glutamatergic EPSCs and non-NMDA receptors in AII amacrine cells were studied in whole cells and patches from slices of the rat retina using single and dual electrode voltage clamp recording. Pharmacological analysis verified that the EPSCs (Erev approximately 0 mV) were mediated exclusively by AMPA-type receptors. EPSCs displayed a wide range of waveforms, ranging from simple monophasic events to more complex multiphasic events. Amplitude distributions of EPSCs were moderately skewed towards larger amplitudes (modal peak 23 pA). Interevent interval histograms were best fitted with a double exponential function. Monophasic, monotonically rising EPSCs displayed very fast kinetics with an average 10-90 % rise time of approximately 340 micro s and a decay phase well fitted by a single exponential (taudecay approximately 760 micro s). The specific AMPA receptor modulator cyclothiazide markedly slowed the decay phase of spontaneous EPSCs (taudecay approximately 3 ms). An increase in temperature decreased both 10-90 % rise time and taudecay with Q10 values of 1.3 and 1.5, respectively. The decay kinetics were slower at positive membrane potentials compared to negative membrane potentials (205 mV/e-fold change in taudecay). Step depolarization of individual presynaptic rod bipolar cells or OFF-cone bipolar cells evoked transient, CNQX-sensitive responses in AII amacrine cells with average peak amplitudes of approximately 330 pA. Ultrafast application of brief (approximately 1 ms) or long (approximately 500 ms) pulses of glutamate to outside-out patches evoked strongly desensitizing responses with very fast deactivation and desensitization kinetics, well fitted by single (taudecay approximately 1.1 ms) and double exponential (tau1 approximately 3.5 ms; tau2 approximately 21 ms) functions, respectively. Double-pulse experiments indicated fast recovery from desensitization (tau approximately 12.4 ms). Our results indicate that spontaneous, AMPA receptor-mediated EPSCs in AII amacrine cells have very fast, voltage-dependent kinetics that can be well accounted for by the kinetic properties of the AMPA receptors themselves

In the retina, AII (rod) amacrine cells are essential for integrating rod signals into the cone pathway. In addition to being interconnected via homologous gap junctions, these cells make extensive heterologous gap junctions with ON-cone bipolar cells (BCs). These gap junctions are the pathway for transfer of rod signals to the ON-system. To investigate the functional properties of these gap junctions, we performed simultaneous whole-cell recordings from pairs of AII amacrine cells and ON-cone bipolar cells in the in vitro slice preparation of the rat retina. We demonstrate strong electrical coupling with symmetrical junction conductance (approximately 1.2 nS) and very low steady-state voltage sensitivity. However, signal transmission is more effective in the direction from AII amacrine cells to ON-cone bipolar cells than in the other direction. This functional rectification can be explained by a corresponding difference in membrane input resistance between the two cell types. Signal transmission has low-pass filter characteristics with increasing attenuation and phase shift for increasing stimulus frequency. Action potentials in AII amacrine cells evoke distinct electrical postsynaptic potentials in ON-cone bipolar cells. Strong and temporally precise synchronization of subthreshold membrane potential fluctuations are commonly observed.

The properties of non-NMDA glutamate receptor channels in AII amacrine cells were studied by patch-clamp recording from rat retinal slices. Application of AMPA or kainate to intact cells evoked currents with no apparent desensitization (EC50 of 118 microM for AMPA and 169 microM for kainate). Application of AMPA to patches evoked desensitizing responses with an EC50 of 217 and 88 microM for the peak and steady-state responses, respectively. Kainate-evoked responses of patches displayed no desensitization (EC50 = 162 microM). Cyclothiazide strongly potentiated AMPA-evoked responses and the AMPA-receptor antagonist GYKI 53655 inhibited both AMPA- and kainate-evoked responses (IC50 = 0.5-1.7 microM). Pre-equilibration with GYKI 53655 completely blocked the response to kainate and pretreatment with concanavalin A did not unmask a response mediated by kainate receptors. AMPA- and kainate-evoked currents reversed close to 0 mV. AMPA-evoked peak and steady-state response components in patches displayed linear and outwardly rectifying I-V relations with an RI (ratio of the slope conductances at +40 mV and -60 mV) of 0.96 +/- 0.11 and 5.6 +/- 1.3, respectively. AMPA-evoked currents displayed a voltage-dependent relaxation after steps to positive or negative membrane potentials, indicating that the outward rectification of the steady-state response is caused by a voltage-dependent kinetic parameter of channel gating. Under bi-ionic conditions ([Ca2+](out) = 30 mM, [Cs+)(in) = 171 mM), the reversal potentials of AMPA- and kainate-evoked currents indicated channels with significant Ca2+ permeability (P(Ca)/P(Cs) = 1.9-2.1). Stationary noise analysis indicated that kainate activated channels with an apparent chord conductance of approximately 9 pS. Non-stationary noise analysis indicated that AMPA and glutamate activated channels with apparent chord conductances of approximately 9, approximately 15, approximately 23 and approximately 38 pS. Discrete single-channel gating corresponding to chord conductances of approximately 23 pS could be directly observed in some responses. Thus, our results indicate expression of high-affinity, voltage-sensitive AMPA receptors with significant Ca2+ permeability and relatively large single-channel chord conductances in AII amacrine cells.

AII (rod) amacrine cells in the mammalian retina are reciprocally connected via gap junctions, but there is no physiological evidence that demonstrates a proposed function as electrical synapses. In whole-cell recordings from pairs of AII amacrine cells in a slice preparation of the rat retina, bidirectional, nonrectifying electrical coupling was observed in all pairs with overlapping dendritic trees (average conductance approximately 700 pS). Coupling displayed characteristics of a low-pass filter, with no evidence for amplification of spike-evoked electrical postsynaptic potentials by active conductances. Coincidence detection, as well as precise temporal synchronization of subthreshold membrane potential oscillations and TTX-sensitive spiking, was commonly observed. These results indicate a unique mode of operation and integrative capability of the network of AII amacrine cells.

The cellular and subcellular expression of the monocarboxylate transporters MCT1, MCT2 and MCT4 [corresponding to MCT3 of Price N. T. et al. (1998) Biochem. J. 329, 321-328] were investigated in the pigment epithelium and outer retina of rats. Immunofluorescence and postembedding immunogold analyses revealed strong MCT1 labelling in the apical membrane of the pigment epithelial and no detectable signal in the basolateral membrane. In contrast, antibodies to the glucose transporter GLUT1 produced intense labelling in both membranes. Neither MCT1 nor GLUT1 was enriched in intracellular compartments. The monocarboxylate transporter MCT4 was very weakly expressed in the retinal pigment epithelium of adult animals, but occurred at higher concentrations at this site in 14-day-old rats. However, even at the latter stage, the immunolabelling of MCT4 was weak compared to that of MCT1. In the neural retina, the data were consistent with a predominant glial localization of MCT1. Specifically, immunogold particles signalling MCT1 occurred in Müller cell microvilli and in the velate processes between the photoreceptors. No labelling was obtained with antibodies to MCT2. Taken together with previous biochemical analyses, the present findings indicate that MCT1 is involved in the outward transport of lactate through the retinal pigment epithelial cells, and in the transfer of lactate between Müller cells and photoreceptors.

The water permeability of cell membranes differs by orders of magnitude, and most of this variability reflects the differential expression of aquaporin water channels. We have recently found that the CNS contains a member of the aquaporin family, aquaporin-4 (AQP4). As a prerequisite for understanding the cellular handling of water during neuronal activity, we have investigated the cellular and subcellular expression of AQP4 in the retina and optic nerve where activity-dependent ion fluxes have been studied in detail. In situ hybridization with digoxigenin-labeled riboprobes and immunogold labeling by a sensitive postembedding procedure demonstrated that AQP4 and AQP4 mRNA were restricted to glial cells, including MHller cells in the retina and fibrous astrocytes in the optic nerve. A quantitative immunogold analysis of the MHller cells showed that these cells exhibited three distinct membrane compartments with regard to AQP4 expression. End feet membranes (facing the vitreous body or blood vessels) were 10-15 times more intensely labeled than non-end feet membranes, whereas microvilli were devoid of AQP4. These data suggest that MHller cells play a prominent role in the water handling in the retina and that they direct osmotically driven water flux to the vitreous body and vessels rather than to the subretinal space. Fibrous astrocytes in the optic nerve similarly displayed a differential compartmentation of AQP4. The highest expression of AQP4 occurred in end feet membranes, whereas the membrane domain facing the nodal axolemma was associated with a lower level of immunoreactivity than the rest of the membrane. This arrangement may allow transcellular water redistribution to occur without inducing inappropriate volume changes in the perinodal extracellular space.

The localization of dopamine D2 and D3 receptors in the rat retina was studied using a polyclonal antibody raised against a peptide sequence common to the dopamine D2 and D3 receptors (D2/3). The D2/3 receptor antibody labelled a small number of somata in the innermost part of the inner nuclear layer and in the ganglion cell layer and a small number of photoreceptor outer segments. Processes in both plexiform layers were also labelled. Double-labelling experiments with the antibody against the D2/3 receptor and an antibody against tyrosine hydroxylase to label dopaminergic neurons resulted in the co-localization of the two antibodies. This demonstrates directly that dopaminergic neurons in the retina express D2/3 receptors. As previous biochemical and physiological studies have demonstrated that activation of D2-like receptors inhibits the release of dopamine in the retina, the present results suggest that the D2/3 receptors on dopaminergic neurons function as autoreceptors.

NMDA and non-NMDA receptors mediate the majority of fast excitatory synaptic transmission in the CNS. AII amacrine cells in the mammalian retina receive glutamatergic input from rod bipolar cells and are known to express non-NMDA receptors. We have investigated the expression of NMDA receptors in these cells by recording responses to exogenously applied NMDA in whole-cell recordings in slices of rat retina. Most cells displayed clear responses to NMDA. The responses could be blocked by a specific NMDA receptor antagonist and were characterized by voltage-dependent block with outward rectification. These results suggest that NMDA receptors could play a role in mediating excitatory synaptic input to AII amacrine cells.

We have localized the dopamine D1 receptor in rat retina using a subtype-specific monoclonal antibody. Immunolabelling can be detected in the inner and outer plexiform layers and in a number of cells in the inner nuclear layer. In the inner plexiform layer, labelled processes form four distinct horizontal bands and a series of patches. In order further to characterize the labelling pattern of the D1 receptor antibody, double-labelling experiments were performed with antibodies against population-specific neuronal markers in the retina. Antibodies against tyrosine hydroxylase, choline acetyltransferase, calretinin, calbindin, the glutamate transporter GLT-1, protein kinase C, recoverin and parvalbumin were co-applied with the D1 receptor antibody. With these cell markers we demonstrate that horizontal cells, at least three types of cone bipolar cells and a small number of amacrine cells are immunolabelled for the D1 receptor. In the inner plexiform layer, processes labelled by the D1 receptor antibody are co-stratified with processes labelled by the GLT-1 antibody. D1 receptor-labelled processes are not co-localized with the processes of amacrine cells and ganglion cells labelled by antibodies against tyrosine hydroxylase, choline acetyltransferase or calretinin. Our results indicate that dopamine D1 receptors are localized predominantly to horizontal cells and cone bipolar cells. Furthermore, the spatial disparity between dopaminergic processes and the site of the majority of D1 receptors supports the idea that in the retina dopamine acts as a neuromodulator that diffuses through extracellular space. The localization of D1 receptors to a number of identified cell types enables future physiological work to be directed towards specific synaptic circuits within the retina.

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.

Vasoactive intestinal polypeptide (VIP) has been shown to potentiate current responses elicited by activation of the GABAA receptor (IGABA) in freshly dissociated ganglion cells of the rat retina. Here we tested the hypothesis that this heteroreceptor cross talk is mediated by an intracellular cascade of events that includes the sequential activation of a stimulatory guanine nucleotide binding (Gs) protein and adenylate cyclase, the subsequent increase in levels of cyclic AMP and, finally, the action of the cyclic AMP-dependent protein kinase (PKA). Intracellular dialysis of freshly dissociated ganglion cells with GTP gamma s irreversibly potentiated IGABA, while GDP beta s either decreased or had no effect on IGABA. Additionally, GDP beta s blocked the potentiation of IGABA by VIP. Cholera toxin rendered VIP ineffective in potentiating IGABA, while pertussis toxin had no effect on the VIP-induced potentiation of IGABA. Extracellular application of either forskolin or 8-bromo-cyclic AMP potentiated IGABA, as did the introduction of cyclic AMP directly into the intracellular compartment through the recording pipet. Intracellular application of cyclic AMP-dependent protein kinase (PKA) potentiated IGABA, while a PKA inhibitor blocked the potentiating effect of VIP. These results lead us to conclude that activation of a cyclic AMP-dependent second-messenger system mediates the modulation of GABAA receptor function by VIP in retinal ganglion cells.

1. The effect of vasoactive intestinal polypeptide (VIP) on bipolar cells and ganglion cells freshly dissociated from the rat retina was studied under voltage clamp with the use of patch-clamp recording in the whole-cell configuration. 2. Application of VIP (1-100 microM) by itself resulted in no detectable current response in either bipolar cells or ganglion cells. However, gamma-aminobutyric acid (GABA)-activated macroscopic current responses elicited in both neuronal populations were potentiated on superimposed exposure to the neuropeptide. 3. GABA-activated chloride currents and muscimol-induced current responses were similarly potentiated on exposure to VIP, suggesting a synergistic interaction between VIP and GABAA receptor mechanisms. 4. We postulate that VIP plays a neuromodulatory role by regulating the excitability of inner retinal neurons and in this way modulates the efficacy of synaptic transmission in the retina.