A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.

To determine the role of homotypic interactions between neighboring dopaminergic amacrine (DA) cells upon dendritic morphogenesis, the morphology of single cells was examined relative to the positioning of all neighboring homotypic cells. For each labeled cell, the dendritic field was reconstructed, its Voronoi domain was calculated, and the two were related. The dendritic fields of DA cells were observed to be large, sparse, and highly irregular. Dendrites readily overlapped those of neighboring cells, showing no evidence for dendritic tiling or inter-digitation consistent with homotypic repulsion or avoidance. Furthermore, a direct comparison of dendritic field area with the Voronoi domain area of the same cell showed no evidence for dendritic growth being constrained or biased by the local distribution of homotypic neighbors in wild-type retinas. A comparison of the processes of adjacent filled cells confirmed their immediate proximity to one another within the inner plexiform layer, indicating that they do not engage in mutual avoidance by coursing at different depths. Together, these results suggest that the morphogenesis of DA cells is independent of homotypic interactions. However, in the absence of the pro-apoptotic Bax gene, which yields a fourfold increase in DA cell number, a small but significant reduction in dendritic field size was obtained, although not so great as would be predicted by the increase in density. The present results are considered in light of recent studies on the role of cell adhesion molecules expressed by developing DA cells.

Inputs from starburst amacrine cells (SACs) to ON-OFF direction selective ganglion cells (DSGCs) in the rabbit retina are themselves directional. However, the synaptic asymmetry between SACs and DSGCs required for generating direction selectivity has been controversial. We investigated dendritic contacts and distribution of inhibitory synapses between SACs and their overlapped DSGCs. Double injection of SAC/DSGC pairs and quantitative analysis revealed no obvious asymmetry of dendritic contacts between SACs and DSGCs. Furthermore, examination of the inhibitory input pattern on the dendrites of DSGCs using antibodies against GABA(A) receptors also suggested an isotropic arrangement with the overlapping SACs in both the preferred and the null directions. Therefore, the presynaptic mechanism of direction selectivity upon DSGCs may not result from a simple asymmetric arrangement with overlapping SACs. Multiple layer interactions and sophisticated synaptic connections between SACs and DSGCs are necessary.

We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol. 527:159-173, 2019. © 2016 Wiley Periodicals, Inc.

Direction selectivity in the retina has been studied as a model of dendritic computation of neural circuits. Starburst amacrine cells (SACs) have been examined as a model system of dendritic computation as they play a pivotal role in the formation of direction selectivity. Because the difference of anatomical location inside the retina made ON-SACs an easier target to record, the biophysical properties of ON-SACs have been used to predict those of OFF-SACs. In this study, we systematically compared the responses of ON- and OFF-SACs to the two principal neurotransmitters, glycine and glutamate. We found that responses to glycine were significantly larger in ON-SACs than in OFF-SACs. In contrast, ON- and OFF-SACs responded similarly to glutamate. The amplitude of glycine responses in ON-SACs increased after eye opening and the largest amplitude was observed at postnatal day 28. On the other hand, no increase in the amplitude of glycine responses in OFF-SACs was observed until postnatal day 28. Glycine-evoked currents were inhibited by the application of strychnine. Glutamate-evoked currents were mimicked by the application of AMPA or kainite, and responses to N-methyl-d-aspartate were observed in the absence of Mg(2+) block. Glutamate-evoked currents produced an increase in the frequency of GABAergic inhibitory postsynaptic currents. Our results suggest that signal processing in ON-SACs cannot be directly used to understand the properties of OFF-SACs. Therefore fully defining the physiological properties of OFF-SACs will be critical to understanding and modelling direction selectivity in the retina.

Neurons receive synaptic inputs on extensive neurite arbors. How information is organized across arbors and how local processing in neurites contributes to circuit function is mostly unknown. Here, we used two-photon Ca2+ imaging to study visual processing in VGluT3-expressing amacrine cells (VG3-ACs) in the mouse retina. Contrast preferences (ON vs. OFF) varied across VG3-AC arbors depending on the laminar position of neurites, with ON responses preferring larger stimuli than OFF responses. Although arbors of neighboring cells overlap extensively, imaging population activity revealed continuous topographic maps of visual space in the VG3-AC plexus. All VG3-AC neurites responded strongly to object motion, but remained silent during global image motion. Thus, VG3-AC arbors limit vertical and lateral integration of contrast and location information, respectively. We propose that this local processing enables the dense VG3-AC plexus to contribute precise object motion signals to diverse targets without distorting target-specific contrast preferences and spatial receptive fields.

Choline acetyltransferase (ChAT) expressing retinal amacrine cells are present across vertebrates. These interneurons play important roles in the development of retinal projections to the brain and in motion detection, specifically in generating direction-selective responses to moving stimuli. ChAT amacrine cells typically comprise two spatially segregated populations that form circuits in the 'ON' or 'OFF' synaptic layers of the inner retina. This stereotypic arrangement is also found across the adult human retina, with the notable exception that ChAT expression is evident in the ON but not OFF layer of the fovea, a region specialized for high-acuity vision. We thus investigated whether the human fovea exhibits a developmental path for ON and OFF ChAT cells that is retinal location-specific. Our analysis shows that at each retinal location, human ON and OFF ChAT cells differentiate, form their separate synaptic layers, and establish non-random mosaics at about the same time. However, unlike in the adult fovea, ChAT immunostaining is initially robust in both ON and OFF populations, up until at least mid-gestation. ChAT expression in the OFF layer in the fovea is therefore significantly reduced after mid-gestation. OFF ChAT cells in the human fovea and in the retinal periphery thus follow distinct maturational paths.

Aquaporins (AQPs) are membrane proteins that facilitate water transport across biological membranes and are essential for the proper function of neural tissue. Although AQPs have been extensively studied in mammalian retina, their presence in lower vertebrate retina is less frequently characterized. AQP4 expressed in mammalian and chick Müller cells plays a major part in maintaining retinal homeostasis. In this study, we examined the immunoreactivity of AQP4 in the adult retina of gilthead sea bream (Sparus aurata-teleost fish), during light and dark adaptation. The AQP4 expression was detected in Müller cell somas at the inner nuclear layer and in the end-feet processes near the vitreoretinal border. Moreover, AQP4 was also evident in cone photoreceptor cells and in a GABAergic subpopulation of amacrine cells (AQP4-ACs). Four different types of AQP4-ACs were characterized based on their morphology and dendrite stratification. Interestingly, a stronger AQP4 immunoreactivity was observed in the inner nuclear layer during dark adaptation, accompanied by a significant increment in AQP4-ACs cell size. Hence, AQP4 may play an important role in water distribution in the teleost fish retina.

Oxytocin, secreted by oxytocin neurons in the hypothalamus, is an endogenous neuropeptide involved in modulating multiple sensory information processing pathways, and its roles in the brain have been associated with prosocial, maternal, and feeding-related behaviors. Visual information is necessary for initiating these behaviors, with the retina consisting of the first stage in the visual system mediating external stimulus perception. Oxytocin has been detected in the mammalian retina; however, the expression and possible function of oxytocin receptors (OxtR) in the retina remain unknown. Here, we explore the role of oxytocin in regulating visual information processing in the retina.

In the present study, we investigated the topographical distribution of ganglion cells and displaced amacrine cells in the retina of the collared peccary (Pecari tajacu), a diurnal neotropical mammal of the suborder Suina (Order Artiodactyla) widely distributed across central and mainly South America. Retinas were prepared and processed following the Nissl staining method. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from three animals. The average density of ganglion cells was 351.822 ± 31.434 GC/mm2. The peccary shows a well-developed visual streak. The average peak density was 6,767 GC/mm2 and located within the visual range and displaced temporally as an area temporalis. Displaced amacrine cells have an average density of 300 DAC/mm2, but the density was not homogeneous along the retina, closer to the center of the retina the number of cells decreases and when approaching the periphery the density increases, in addition, amacrine cells do not form retinal specialization like ganglion cells. Outside the area temporalis, amacrine cells reach up to 80% in the ganglion cell layer. However, in the region of the area temporalis, the proportion of amacrine cells drops to 32%. Thus, three retinal specializations were found in peccary's retina by ganglion cells: visual streak, area temporalis and dorsotemporal extension. The topography of the ganglion cells layer in the retina of the peccary resembles other species of Order Artiodactyla already described and is directly related to its evolutionary history and ecology of the species.

Immunoelectron microscopy was used to investigate the ultrastructural features and synaptic relationships of serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina. Serotonin-positive somas exhibited an evenly distributed peroxidase reaction product throughout their cytoplasm. Their nuclei were unstained and possessed indented nuclear membranes. Serotonin-immunoreactive processes were generally stained throughout with the exception of their mitochondria, whose morphology was often disrupted by the staining reaction. They were further characterized by an occasional dense-cored vesicle/s in addition to a generally homogeneous population of small, round, clear synaptic vesicles. Serotonin-immunoreactive amacrine cell processes formed conventional synapses that were characterized by symmetrical synaptic membrane densities. A total of 222 synaptic arrangements were observed that involved the immunostained processes of serotonin-amacrine cells. As presynaptic elements, they primarily contacted amacrine cells processes (37.8%). They also provided substantial synaptic input to processes that lacked synaptic vesicles (16.2%) and whose origin was unidentified. Serotonin-processes provided a far fewer number of synaptic contacts onto the processes of bipolar cells (1.4%) and the somas of cells in the amacrine cell layer (0.5%). As postsynaptic elements, they received synaptic inputs from amacrine cells (27.9%) and bipolar cells (16.2%). With the exception of their synapses onto bipolar cells and the somas of cells in the amacrine cell layer, each of the synaptic relationships of serotonin-amacrine cells was observed in each of sublayers 1-5 of the inner plexiform layer.

Brain-derived neurotrophic-factor (BDNF) is expressed in the retina and controls the development of subtypes of amacrine cells. In the present study we investigated the effects of BDNF on amacrine cells expressing vasoactive intestinal polypeptide (VIP). Rats received three intraocular injections of BDNF on postnatal days (P) 16, 18, and 20. The animals were sacrificed on P22, P40, P60, P80, and P120, and VIP expression in their retinas was detected by immunohistochemistry (P22, P40) and by radioimmunoassay (RIA; P22, P40, P60, P80, P120) to assess the time course of BDNF effects on VIP. A significant increase in the density of VIP-positive amacrine cells was detected in BDNF-treated retinas, and VIP concentration was up-regulated by 150% both at P22 and at P40 with respect to untreated controls. VIP concentration then slowly declined in the treated retinas over a period of 3 months; however, a statistically significant increase of 50% was still detectable on P120. The impact of endogenous BDNF on the regulation of VIP expression in the retina was analyzed in mice homozygous for a targeted deletion of the BDNF gene locus (bdnf-/-). VIP immunohistochemistry revealed a marked reduction of VIP-positive amacrine cells and of VIP-immunopositive processes in the inner plexiform layer of the BDNF knockout mice. Mice lacking BDNF expressed only 5% of the VIP protein in their retinas compared with the retinas of wild-type mice as measured by RIA. Our data show that BDNF is a major regulator of VIP expression in retinal amacrine cells and exerts long-lasting effects on VIP content.

Retinal amacrine cells of the same class in cyprinid fish are homotypically connected by gap junctions. The permeability of their gap junctions examined by the diffusion of Neurobiotin into neighboring amacrine cells under application of dopamine or cyclic nucleotides to elucidate whether electrical synapses between the cells are regulated by internal messengers. Neurobiotin injected intracellularly into amacrine cells in isolated retinas of goldfish, and passage currents through the electrical synapses investigated by dual whole-patch clamp recordings under similar application of their ligands. Control conditions led us to observe large passage currents between connected cells and adequate transjunctional conductance between the cells (2.02±0.82nS). Experimental results show that high level of intracellular cyclic AMP within examined cells block transfer of Neurobiotin and suppress electrical synapses between the neighboring cells. Transjunctional conductance between examined cells reduced to 0.23nS. However, dopamine, 8-bromo-cyclic AMP or high elevation of intracellular cyclic GMP leaves gap junction channels of the cells permeable to Neurobiotin as in the control level. Under application of dopamine (1.25±0.06nS), 8-bromo-cyclic AMP (1.79±0.51nS) or intracellular cyclic GMP (0.98±0.23nS), the transjunctional conductance also remains as in the control level. These results demonstrate that channel opening of gap junctions between cyprinid retinal amacrine cells is regulated by high level of intracellular cyclic AMP.

Somatostatin (SRIF) modulates neurotransmitter release by activating the specific receptors (sst1-sst5). Our previous study showed that sst5 receptors are expressed in rat retinal GABAergic amacrine cells. Here, we investigated modulation of GABA release by SRIF in cultured amacrine cells, using patch-clamp techniques. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the amacrine cells was significantly reduced by SRIF, which was partially reversed by BIM 23056, an sst5 receptor antagonist, and was further rescued by addition of CYN-154806, an sst2 receptor antagonist. Both nimodipine, an L-type Ca2+ channel blocker, and ω-conotoxin GVIA, an N-type Ca2+ channel blocker, suppressed the sIPSC frequency, and in the presence of nimodipine and ω-conotoxin GVIA, SRIF failed to further suppress the sIPSC frequency. Extracellular application of forskolin, an activator of adenylate cyclase, increased the sIPSC frequency, while the membrane permeable protein kinase A (PKA) inhibitor Rp-cAMP reduced it, and in the presence of Rp-cAMP, SRIF did not change sIPSCs. However, SRIF persisted to suppress the sIPSCs in the presence of KT5823, a protein kinase G (PKG) inhibitor. Moreover, pre-incubation with Bis IV, a protein kinase C (PKC) inhibitor, or pre-application of xestospongin C, an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor, SRIF still suppressed the sIPSC frequency. All these results suggest that SRIF suppresses GABA release from the amacrine cells by inhibiting presynaptic Ca2+ channels, in part through activating sst5/sst2 receptors, a process that is mediated by the intracellular cAMP-PKA signaling pathway.

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.

AP-2 transcription factors play important roles in the regulation of gene expression during development. Four of the five members of the AP-2 family (AP-2α, AP-2β, AP-2γ and AP-2δ) have previously been shown to be expressed in developing retina. Mouse knockouts have revealed roles for AP-2α, AP-2β and AP-2δ in retinal cell specification and function. Here, we show that the fifth member of the AP-2 family, AP-2ε, is also expressed in amacrine cells in developing mammalian and chicken retina. Our data indicate that there are considerably fewer AP-2ε-positive cells in the developing mouse retina compared to AP-2α, AP-2β and AP-2γ-positive cells, suggesting a specialized role for AP-2ε in a subset of amacrine cells. AP-2ε, which is restricted to the GABAergic amacrine lineage, is most commonly co-expressed with AP-2α and AP-2β, especially at early stages of retinal development. Co-expression of AP-2ε and AP-2γ increases with differentiation. Analysis of previously published Drop-seq data from single retinal cells supports co-expression of multiple AP-2s in the same cell. Since AP-2s bind to their target sequences as either homodimers or heterodimers, our work suggests spatially- and temporally-coordinated roles for combinations of AP-2 transcription factors in amacrine cells during retinal development.

The present study examined changes in retinal tyrosine hydroxylase (TH) expression in rats having undergone optic nerve transection and housed under a normal day/night cycle or in the dark. The aim was to investigate the effects of amacrine cells on axonal regeneration in retinal ganglion cells and on the synapses that transmit visual signals. The results revealed that retinal TH expression gradually decreased following optic nerve transection in rats housed under a normal day/night cycle, reaching a minimum at 5 days. In contrast, retinal TH expression decreased to a minimum at 1 day following optic nerve transection in dark reared rats, gradually increasing afterward and reaching a normal level at 5-7 days. The number of TH-positive synaptic particles correlated with the TH levels, indicating that dark rearing can help maintain TH expression during the synaptic degeneration stage (5-7 days after optic nerve injury) in retinal amacrine cells.

Retinal degeneration (RD) encompasses a family of diseases that lead to photoreceptor death and visual impairment. Visual decline due to photoreceptor cell loss is further compromised by emerging spontaneous hyperactivity in inner retinal cells. This aberrant activity acts as a barrier to signals from the remaining photoreceptors, hindering therapeutic strategies to restore light sensitivity in RD. Gap junctions, particularly those expressed in AII amacrine cells, have been shown to be integral to the generation of aberrant activity. It is unclear whether gap junction expression and coupling are altered in RD. To test this, we evaluated the expression and phosphorylation state of connexin36 (Cx36), the gap junction subunit predominantly expressed in AII amacrine cells, in two mouse models of RD, rd10 (slow degeneration) and rd1 (fast degeneration). Using Ser293-P antibody, which recognizes a phosphorylated form of connexin36, we found that phosphorylation of connexin36 in both slow and fast RD models was significantly greater than in wildtype controls. This elevated phosphorylation may underlie the increased gap junction coupling of AII amacrine cells exhibited by RD retina.

Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine >> RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

Postsynaptic cytosolic Cl- concentration determines whether GABAergic and glycinergic synapses are inhibitory or excitatory. We have shown that nitric oxide (NO) initiates the release of Cl- from acidic internal stores into the cytosol of retinal amacrine cells (ACs) thereby elevating cytosolic Cl-. In addition, we found that cystic fibrosis transmembrane conductance regulator (CFTR) expression and Ca2+ elevations are necessary for the transient effects of NO on cytosolic Cl- levels, but the mechanism remains to be elucidated. Here, we investigated the involvement of TMEM16A as a possible link between Ca2+ elevations and cytosolic Cl- release. TMEM16A is a Ca2+-activated Cl- channel that is functionally coupled with CFTR in epithelia. Both proteins are also expressed in neurons. Based on this and its Ca2+ dependence, we test the hypothesis that TMEM16A participates in the NO-dependent elevation in cytosolic Cl- in ACs. Chick retina ACs express TMEM16A as shown by Western blot analysis, single-cell PCR, and immunocytochemistry. Electrophysiology experiments demonstrate that TMEM16A functions in amacrine cells. Pharmacological inhibition of TMEM16A with T16inh-AO1 reduces the NO-dependent Cl- release as indicated by the diminished shift in the reversal potential of GABAA receptor-mediated currents. We confirmed the involvement of TMEM16A in the NO-dependent Cl- release using CRISPR/Cas9 knockdown of TMEM16A. Two different modalities targeting the gene for TMEM16A (ANO1) were tested in retinal amacrine cells: an all-in-one plasmid vector and crRNA/tracrRNA/Cas9 ribonucleoprotein. The all-in-one CRISPR/Cas9 modality did not change the expression of TMEM16A protein and produced no change in the response to NO. However, TMEM16A-specific crRNA/tracrRNA/Cas9 ribonucleoprotein effectively reduces both TMEM16A protein levels and the NO-dependent shift in the reversal potential of GABA-gated currents. These results show that TMEM16A plays a role in the NO-dependent Cl- release from retinal ACs.