AII Amacrine Cells Research Articles

Retinal bipolar cells borrow excitability from electrically coupled inhibitory interneurons to amplify excitatory synaptic transmission.

Bipolar cells of the retina carry visual information from photoreceptors in the outer retina to retinal ganglion cells (RGCs) in the inner retina. Bipolar cells express L-type voltage-gated Ca2+ channels at the synaptic terminal, but generally lack other types of channels capable of regenerative activity. As a result, the flow of information from outer to inner retina along bipolar cell processes is generally passive in nature, with no opportunity for signal boost or amplification along the way. Here we report the surprising discovery that blocking voltage-gated Na+ channels profoundly reduces the synaptic output of one class of bipolar cell, the type 6 ON bipolar cell (CBC6), despite the fact that the CBC6 itself does not express voltage-gated Na+ channels. Instead, CBC6 borrows voltage-gated Na+ channels from its neighbor, the inhibitory AII amacrine cell, with whom it is connected via an electrical synapse. Thus, an inhibitory neuron aids in amplification of an excitatory signal as it moves through the retina, ensuring that small changes in the membrane potential of bipolar cells are reliably passed onto downstream RGCs.

Biotin-cGMP and -cAMP are able to permeate through the gap junctions of some amacrine cells in the mouse retina despite their large size.

Gap junctions transmit electrical signals in neurons and serve metabolic coupling and chemical communication. Gap junctions are made of intercellular channels with large pores, allowing ions and small molecules to permeate. In the mammalian retina, intercellular coupling fulfills many vital functions in visual signal processing but is also implicated in promoting cell death after insults, such as excitotoxicity or hypoxia. Conversely, some studies also suggested a role for retinal gap junctions in neuroprotection. Recently, gap junctions were also advocated as conduits for therapeutic drug delivery in neurodegenerative disorders. This requires the permeation of rather large molecules through retinal gap junctions. However, the permeability of retinal networks for molecules >0.6 kDa has not been tested systematically. Here, we used the cut-loading method and probed gap junctional networks in the mouse retina for their permeability to cGMP and cAMP coupled to Biotin, using the well-characterized tracer Neurobiotin as control. Biotin-cGMP and -cAMP have a molecular weight of >0.8 kDa. We show that they cannot pass the gap junctions of horizontal cells but can permeate through the gap junctions of specific amacrine cells in the inner retina. These amacrine cells do not comprise AII amacrine cells and nitric oxide-releasing amacrine cells but some unknown type. In summary, we show that some retinal gap junctions are large enough to let molecules >0.8 kDa pass, making the intercellular delivery of therapeutic agents - already successfully exploited, for example, in cancer - also feasible in neurodegenerative diseases.

Characterizing the rod pathway in cone-dominated thirteen-lined ground squirrels.

AII-amacrine cells (AIIs) are widely accepted as a critical element of scotopic pathways mediating night vision in the mammalian retina and have been well-characterized in rod-dominant mice, rabbits, and non-human primates. The rod pathway is characteristic of all mammalian eyes, however, the anatomic and physiologic role of AIIs and the rod pathways in cone dominant thirteen-lined ground squirrels (TLGS) is limited. Here, we employed both immunohistochemistry and electrophysiological approaches to investigate the morphology of AIIs and functional aspects of the rod pathway in TLGS. In all TLGS retinas examined, putative AIIs were calretinin-positive and exhibited connections to rod bipolar cells with decreased cell density and expanded arborization. Notably, AIIs retained connections with each other via gap junctions labeled with Connexin36. Comparisons between single photoreceptor recordings and full-field electroretinograms revealed scotopic ERG responses were mediated by both rods and cones. Thus, the components of the rod pathway are conserved in TLGS and rod signals traverse the retina in these cone-dominant animals. AIIs are sparsely populated, matching the diminished rod and rod bipolar cell populations compared to rod-dominant species. The infrequent distribution and lateral spacing of AII's indicate that they probably do not play a significant role in cone signaling pathways that encode information at a finer spatial scale. This contrasts with the mouse retina, where they significantly contribute to cone signaling pathways. Therefore, the AII's original function is likely that of a 'rod' amacrine cell, and its role in cone pathways in the mouse retina might be an adaptive feature stemming from its rod dominance.

Functional maturation of the rod bipolar to AII-amacrine cell ribbon synapse in the mouse retina

Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we examined the maturation of the ribbon synapse between rod bipolar cells (RBCs) and AII-amacrine cells (AII-ACs) after eye opening (postnatal day 14) in the mouse retina at near physiological temperatures. We find that light-evoked excitatory postsynaptic currents (EPSCs) in AII-ACs exhibit a slow sustained component that increases in magnitude with advancing age, whereas a fast transient component remains unchanged. Similarly, paired recordings reveal a dual-component EPSC with a slower sustained component that increases during development, even though the miniature EPSC (mEPSC) amplitude and kinetics do not change significantly. We thus propose that the readily releasable pool of vesicles from RBCs increases after eye opening, and we estimate that a short light flash can evoke the release of ∼4,000 vesicles onto a single mature AII-AC.

The Atoh1-Cre Knock-In Allele Ectopically Labels a Subpopulation of Amacrine Cells and Bipolar Cells in Mouse Retina.

The retina has diverse neuronal cell types derived from a common pool of retinal progenitors. Many molecular drivers, mostly transcription factors, have been identified to promote different cell fates. In Drosophila, atonal is required for specifying photoreceptors. In mice, there are two closely related atonal homologs, Atoh1 and Atoh7 While Atoh7 is known to promote the genesis of retinal ganglion cells, there is no study on the function of Atoh1 in retinal development. Here, we crossed Atoh1Cre/+ mice to mice carrying a Cre-dependent TdTomato reporter to track potential Atoh1-lineage neurons in retinas. We characterized a heterogeneous group of TdTomato+ retinal neurons that were detected at the postnatal stage, including glutamatergic amacrine cells, AII amacrine cells, and BC3b bipolar cells. Unexpectedly, we did not observe TdTomato+ retinal neurons in the mice with an Atoh1-FlpO knock-in allele and a Flp-dependent TdTomato reporter, suggesting Atoh1 is not expressed in the mouse retina. Consistent with these data, conditional removal of Atoh1 in the retina did not cause any observable phenotypes. Importantly, we did not detect Atoh1 expression in the retina at multiple ages using mice with Atoh1-GFP knock-in allele. Therefore, we conclude that Atoh1Cre/+ mice have ectopic Cre expression in the retina and that Atoh1 is not required for retinal development.

Layers of inhibitory networks shape receptive field properties of AII amacrine cells

In the retina, rod and cone pathways mediate visual signals over a billion-fold range in luminance. AII ("A-two") amacrine cells (ACs) receive signals from both pathways via different bipolar cells, enabling AIIs to operate at night and during the day. Previous work has examined luminance-dependent changes in AII gap junction connectivity, but less is known about how surrounding circuitry shapes AII receptive fields across light levels. Here, we report that moderate contrast stimuli elicit surround inhibition in AIIs under all but the dimmest visual conditions, due to actions of horizontal cells and at least two ACs that inhibit presynaptic bipolar cells. Under photopic (daylight) conditions, surround inhibition transforms AII response kinetics, which are inherited by downstream ganglion cells. Ablating neuronal nitric oxide synthase type-1 (nNOS-1) ACs removes AII surround inhibition under mesopic (dusk/dawn), but not photopic, conditions. Our findings demonstrate how multiple layers of neural circuitry interact to encode signals across a wide physiological range.

A common cause for nystagmus in different congenital stationary night blindness mouse models.

In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), synchronous oscillating retinal ganglion cells (RGCs) lead to oscillatory eye movements, i.e. nystagmus. Given the specific expression of mGluR6 and Cav 1.4 in the photoreceptor to bipolar cell synapses, as well as their clinical association with CSNB, we hypothesize that Grm6nob3 and Cav 1.4-KO mutants show, like the Nyxnob mouse, oscillations in both their RGC activity and eye movements. Using multi-electrode array recordings of RGCs and measurements of the eye movements, we demonstrate that Grm6nob3 and Cav 1.4-KO mice also show oscillations of their RGCs as well as a nystagmus. Interestingly, the preferred frequencies of RGC activity as well as the eye movement oscillations of the Grm6nob3 , Cav 1.4-KO and Nyxnob mice differ among mutants, but the neuronal activity and eye movement behaviour within a strain remain aligned in the same frequency domain. Model simulations indicate that mutations affecting the photoreceptor-bipolar cell synapse can form a common cause of the nystagmus of CSNB by driving oscillations in RGCs via AII amacrine cells. KEY POINTS: In Nyxnob mice, a model for congenital nystagmus associated with congenital stationary night blindness (CSNB), their oscillatory eye movements (i.e. nystagmus) are caused by synchronous oscillating retinal ganglion cells. Here we show that the same mechanism applies for two other CSNB mouse models - Grm6nob3 and Cav 1.4-KO mice. We propose that the retinal ganglion cell oscillations originate in the AII amacrine cells. Model simulations show that by only changing the input to ON-bipolar cells, all phenotypical differences between the various genetic mouse models can be reproduced.

Nfia Is Critical for AII Amacrine Cell Production: Selective Bipolar Cell Dependencies and Diminished ERG.

The nuclear factor one (NFI) transcription factor genes Nfia, Nfib, and Nfix are all enriched in late-stage retinal progenitor cells, and their loss has been shown to retain these progenitors at the expense of later-generated retinal cell types. Whether they play any role in the specification of those later-generated fates is unknown, but the expression of one of these, Nfia, in a specific amacrine cell type may intimate such a role. Here, Nfia conditional knockout (Nfia-CKO) mice (both sexes) were assessed, finding a massive and largely selective absence of AII amacrine cells. There was, however, a partial reduction in type 2 cone bipolar cells (CBCs), being richly interconnected to AII cells. Counts of dying cells showed a significant increase in Nfia-CKO retinas at postnatal day (P)7, after AII cell numbers were already reduced but in advance of the loss of type 2 CBCs detected by P10. Those results suggest a role for Nfia in the specification of the AII amacrine cell fate and a dependency of the type 2 CBCs on them. Delaying the conditional loss of Nfia to the first postnatal week did not alter AII cell number nor differentiation, further suggesting that its role in AII cells is solely associated with their production. The physiological consequences of their loss were assessed using the ERG, finding the oscillatory potentials to be profoundly diminished. A slight reduction in the b-wave was also detected, attributed to an altered distribution of the terminals of rod bipolar cells, implicating a role of the AII amacrine cells in constraining their stratification.SIGNIFICANCE STATEMENT The transcription factor NFIA is shown to play a critical role in the specification of a single type of retinal amacrine cell, the AII cell. Using an Nfia-conditional knockout mouse to eliminate this population of retinal neurons, we demonstrate two selective bipolar cell dependencies on the AII cells; the terminals of rod bipolar cells become mis-stratified in the inner plexiform layer, and one type of cone bipolar cell undergoes enhanced cell death. The physiological consequence of this loss of the AII cells was also assessed, finding the cells to be a major contributor to the oscillatory potentials in the electroretinogram.

Optogenetic targeting of AII amacrine cells restores retinal computations performed by the inner retina

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of inner retinal neurons is a promising avenue to restore vision in retinas having lost their photoreceptors. Expression of optogenetic proteins in surviving ganglion cells, the retinal output, allows them to take on the lost photoreceptive function. Nonetheless, this creates an exclusively ON retina by expression of depolarizing optogenetic proteins in all classes of ganglion cells, whereas a normal retina extracts several features from the visual scene, with different ganglion cells detecting light increase (ON) and light decrease (OFF). Refinement of this therapeutic strategy should thus aim at restoring these computations. Here we used a vector that targets gene expression to a specific interneuron of the retina called the AII amacrine cell. AII amacrine cells simultaneously activate the ON pathway and inhibit the OFF pathway. We show that the optogenetic stimulation of AII amacrine cells allows restoration of both ON and OFF responses in the retina, but also mediates other types of retinal processing such as sustained and transient responses. Targeting amacrine cells with optogenetics is thus a promising avenue to restore better retinal function and visual perception in patients suffering from retinal degeneration.

Sources of Calcium at Connexin 36 Gap Junctions in the Retina.

Synaptic plasticity is a fundamental feature of the CNS that controls the magnitude of signal transmission between communicating cells. Many electrical synapses exhibit substantial plasticity that modulates the degree of coupling within groups of neurons, alters the fidelity of signal transmission, or even reconfigures functional circuits. In several known examples, such plasticity depends on calcium and is associated with neuronal activity. Calcium-driven signaling is known to promote potentiation of electrical synapses in fish Mauthner cells, mammalian retinal AII amacrine cells, and inferior olive neurons, and to promote depression in thalamic reticular neurons. To measure local calcium dynamics in situ, we developed a transgenic mouse expressing a GCaMP calcium biosensor fused to Connexin 36 (Cx36) at electrical synapses. We examined the sources of calcium for activity-dependent plasticity in retina slices using confocal or Super-Resolution Radial Fluctuations imaging. More than half of Cx36-GCaMP gap junctions responded to puffs of glutamate with transient increases in fluorescence. The responses were strongly dependent on NMDA receptors, in keeping with known activity-dependent signaling in some amacrine cells. We also found that some responses depended on the activity of voltage-gated calcium channels, representing a previously unrecognized source of calcium to control retinal electrical synaptic plasticity. The high prevalence of calcium signals at electrical synapses in response to glutamate application indicates that a large fraction of electrical synapses has the potential to be regulated by neuronal activity. This provides a means to tune circuit connectivity dynamically based on local activity.

Functional properties of GABAA receptors of AII amacrine cells of the rat retina.

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 GABAA 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 GABAA 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 GABAA receptors in AIIs, we therefore used nucleated patches and a fast application system. Both brief and long pulses of GABA (3 mM) evoked GABAA receptor-mediated currents with slow, multi-exponential decay kinetics. The average weighted time constant (τw) of deactivation was ~163 ms. Desensitization was even slower, with τw ~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 GABAA receptors with slow kinetics. If synaptic GABAA 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.

Loss of Müller cell glutamine synthetase immunoreactivity is associated with neuronal changes in late-stage retinal degeneration.

A hallmark of photoreceptor degenerations is progressive, aberrant remodeling of the surviving retinal neurons and glia following photoreceptor loss. The exact relationship between neurons and glia remodeling in this late stage of retinal degeneration, however, is unclear. This study assessed this by examining Müller cell dysfunction via glutamine synthetase immunoreactivity and its spatial association with retinal neuron subpopulations through various cell markers. Aged Rd1 mice retinae (P150 - P536, n = minimum 5 per age) and control heterozygous rd1 mice retinae (P536, n = 5) were isolated, fixed and cryosectioned. Fluorescent immunolabeling of glutamine synthetase was performed and retinal areas quantified as having low glutamine synthetase immunoreactivity if proportion of labeled pixels in an area was less than two standard deviations of the mean of the total retina. Other Müller cell markers such as Sox9 and Glial fibrillary acidic protein along with neuronal cell markers Calbindin, Calretinin, recoverin, Protein kinase C-α, Glutamic acid decarboxylase 67, and Islet-1 were then quantified within areas of low and normal synthetase immunoreactivity. Glutamine synthetase immunoreactivity was lost as a function of age in the rd1 mouse retina (P150 - P536). Immunoreactivity of other Müller cell markers, however, were unaffected suggesting Müller cells were still present in these low glutamine synthetase immunoreactive regions. Glutamine synthetase immunoreactivity loss affected specific neuronal populations: Type 2, Type 8 cone, and rod bipolar cells, as well as AII amacrine cells based on reduced recoverin, protein kinase Ca and parvalbumin immunoreactivity, respectively. The number of cell nuclei within regions of low glutamine synthetase immunoreactivity was also reduced suggesting possible neuronal loss rather than reduced cell marker immunoreactivity. These findings further support a strong interplay between glia-neuronal alterations in late-stage degeneration and highlight a need for future studies and consideration in intervention development.

Origin of Retinal Oscillatory Potentials in the Mouse, a Tool to Specifically Locate Retinal Damage

To determine the origin of oscillatory potentials (OPs), binocular electroretinogram (ERG) recordings were performed under light and dark adaptation on adult healthy C57BL/6J mice. In the experimental group, 1 μL of PBS was injected into the left eye, while the right eye was injected with 1 μL of PBS containing different agents: APB, GABA, Bicuculline, TPMPA, Glutamate, DNQX, Glycine, Strychnine, or HEPES. The OP response depends on the type of photoreceptors involved, showing their maximum response amplitude in the ERG induced by mixed rod/cone stimulation. The oscillatory components of the OPs were affected by the injected agents, with some drugs inducing the complete abolition of oscillations (APB, GABA, Glutamate, or DNQX), whereas other drugs merely reduced the oscillatory amplitudes (Bicuculline, Glycine, Strychnine, or HEPES) or did not even affect the oscillations (TPMPA). Assuming that rod bipolar cells (RBC) express metabotropic Glutamate receptors, GABAA, GABAC, and Glycine receptors and that they release glutamate mainly on Glycinergic AII amacrine cells and GABAergic A17 amacrine cells, which are differently affected by the mentioned drugs, we propose that RBC-AII/A17 reciprocal synapses are responsible for the OP generation in the ERG recordings in the mice. We conclude that the reciprocal synapses between RBC and AII/A17 are the basis of the ERG OP oscillations of the light response, and this fact must be taken into consideration in any ERG test that shows a decrease in the OPs' amplitude.

Quantitative trait loci on chromosomes 9 and 19 modulate AII amacrine cell number in the mouse retina.

Sequence variants modulating gene function or expression affect various heritable traits, including the number of neurons within a population. The present study employed a forward-genetic approach to identify candidate causal genes and their sequence variants controlling the number of one type of retinal neuron, the AII amacrine cell. Data from twenty-six recombinant inbred (RI) strains of mice derived from the parental C57BL/6J (B6/J) and A/J laboratory strains were used to identify genomic loci regulating cell number. Large variation in cell number is present across the RI strains, from a low of ∼57,000 cells to a high of ∼87,000 cells. Quantitative trait locus (QTL) analysis revealed three prospective controlling genomic loci, on Chromosomes (Chrs) 9, 11, and 19, each contributing additive effects that together approach the range of variation observed. Composite interval mapping validated two of these loci, and chromosome substitution strains, in which the A/J genome for Chr 9 or 19 was introgressed on a B6/J genetic background, showed increased numbers of AII amacrine cells as predicted by those two QTL effects. Analysis of the respective genomic loci identified candidate controlling genes defined by their retinal expression, their established biological functions, and by the presence of sequence variants expected to modulate gene function or expression. Two candidate genes, Dtx4 on Chr 19, being a regulator of Notch signaling, and Dixdc1 on Chr 9, a modulator of the WNT-β-catenin signaling pathway, were explored in further detail. Postnatal overexpression of Dtx4 was found to reduce the frequency of amacrine cells, while Dixdc1 knockout retinas contained an excess of AII amacrine cells. Sequence variants in each gene were identified, being the likely sources of variation in gene expression, ultimately contributing to the final number of AII amacrine cells.

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Calcium-permeable AMPA receptors on AII amacrine cells mediate sustained signaling in the On-pathway of the primate retina.

Midget and parasol ganglion cells (GCs) represent the major output channels from the primate eye to the brain. On-type midget and parasol GCs exhibit a higher background spike rate and thus can respond more linearly to contrast changes than their Off-type counterparts. Here, we show that a calcium-permeable AMPA receptor (CP-AMPAR) antagonist blocks background spiking and sustained light-evoked firing in On-type GCs while preserving transient light responses. These effects are selective for On-GCs and are occluded by a gap-junction blocker suggesting involvement of AII amacrine cells (AII-ACs). Direct recordings from AII-ACs, cobalt uptake experiments, and analyses of transcriptomic data confirm that CP-AMPARs are expressed by primate AII-ACs. Overall, our data demonstrate that under some background light levels, CP-AMPARs at the rod bipolar to AII-AC synapse drive sustained signaling in On-type GCs and thus contribute to the more linear contrast signaling of the primate On- versus Off-pathway.

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

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

Localization of neuropeptide receptor NPY4R in rat retina

Neuropeptide Y (NPY) is a significant neuromodulator implicated in a multitude of physiological functions via activating NPY receptors which belong to seven transmembrane G-protein-coupled receptors (GPCRs). However, the detailed cellular expression of NPY receptors in retina has been scarcely investigated. In this study, the expression of the special NPY4R receptor in rat retina was assessed using Western blot analysis and immunofluorescent staining. The detailed cellular localization of NPY4R receptor was studied using double immunofluorescent staining and laser-scanning confocal microscopy. Our data demonstrated that NPY4R receptor was weakly expressed in the inner segment of outer photoreceptors and extensively expressed in the outer segment of S-opsin-positive blue cones, L/M-opsin-positive red/green cones and in the somata of CB-positive horizontal cells, GAD65-positive GABAnergic amacrine cells, ChAT-positive cholinergic amacrine cells, TH-positive dopaminergic CA1 amacrine cells and CA2 amacrine cells, PV-positive AII amacrine cells, Brn3a-positive conventional ganglion cells and melanopsin-containing ipRGCs. In addition, NPY4R receptor was diffusely distributed throughout the full thickness of the inner plexiform layer and outer plexiform layer. However, the outer segment of Rho4D2-positive rods, the somata of ChX10-positive bipolar cells and CRALBP-positive Müller glial cells seemed to lack immunoreactivity of NPY4R receptor. The new finding that multiple types of retinal cell express NPY4R receptor provides new neurobiological basis for the participation of NPY in the regulation of retinal functions through activating NPY4R receptor.

Cell numbers, cell ratios, and developmental plasticity in the rod pathway of the mouse retina.

The precise specification of cellular fate is thought to ensure the production of the correct number of neurons within a population. Programmed cell death may be an additional mechanism controlling cell number, believed to refine the proper ratio of pre- to post-synaptic neurons for a given species. Here, we consider the size of three different neuronal populations in the rod pathway of the mouse retina: rod photoreceptors, rod bipolar cells, and AII amacrine cells. Across a collection of 28 different strains of mice, large variation in the numbers of all three cell types is present. The variation in their numbers is not correlated, so that the ratio of rods to rod bipolar cells, as well as rod bipolar cells to AII amacrine cells, varies as well. Establishing connectivity between such variable pre- and post-synaptic populations relies upon plasticity that modulates process outgrowth and morphological differentiation, which we explore experimentally for both rod bipolar and AII amacrine cells in a mouse retina with elevated numbers of each cell type. While both rod bipolar dendritic and axonal arbors, along with AII lobular arbors, modulate their areal size in relation to local homotypic cell densities, the dendritic appendages of the AII amacrine cells do not. Rather, these processes exhibit a different form of plasticity, regulating the branching density of their overlapping arbors. Each form of plasticity should ensure uniformity in retinal coverage in the presence of the independent specification of afferent and target cell number.

Dendritic Morphology of an Inhibitory Retinal Interneuron Enables Simultaneous Local and Global Synaptic Integration.

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 Ca2+ 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.