Amacrine Cells Research Articles

Loss of paired immunoglobin-like type 2 receptor B gene associated with age-related macular degeneration impairs photoreceptor function in mouse retina.

Genome-wide association studies have uncovered mostly non-coding variants at over 60 genetic loci linked to susceptibility for age-related macular degeneration (AMD). To ascertain the causal gene at the PILRB/PILRA locus, we used a CRISPR strategy to produce germline deletions in the mouse paired immunoglobin-like type 2 receptor (Pilr) genes that encode highly related activating (PILRB) and inhibitory (PILRA) receptors. We show that a combined loss of Pilrb1 and Pilrb2, but not Pilra, leads to an early but relatively stationary defect as the electroretinography (ERG) amplitudes of Pilrb1/2-/- mice exhibit a marked reduction as early as postnatal day 15 and do not show additional significant decrease at 3 and 12-months. No alterations are evident in Müller glia, microglia, bipolar, amacrine and horizontal cells based on immunohistochemistry using cell-type specific markers. PILRB immunostaining is specifically detected at the proximal part of photoreceptor outer segment. Reduced expression of select calcium-regulated phototransduction and synapse-associated proteins, including GCAP1 and 2, PDE6b, AIPL1, PSD95, and CTBP1 indicates dysregulation of calcium homeostasis as a possible mechanism of retinal phenotype in Pilrb1/2-/- mice. Our studies suggest a novel function of PILRB in retinal photoreceptors and an association of PILRB, but not PILRA, with AMD pathogenesis.

Retinal ganglion cell-derived semaphorin 6A segregates starburst amacrine cell dendritic scaffolds to organize the inner retina.

To form functional circuits, neurons must settle in their appropriate cellular locations and then project and elaborate neurites to contact their target synaptic neuropils. Laminar organization within the vertebrate retinal inner plexiform layer (IPL) facilitates pre- and postsynaptic neurite targeting, yet the precise mechanisms underlying establishment of functional IPL subdomains are not well understood. Here we explore mechanisms defining the compartmentalization of OFF and ON neurites generally, and OFF and ON direction-selective neurites specifically, within the developing IPL. We show that semaphorin 6A (Sema6A), a repulsive axon guidance cue, is required for delineation of OFF versus ON circuits within the IPL: in the Sema6a null IPL, the boundary between OFF and ON domains is blurred. Furthermore, Sema6A expressed by retinal ganglion cells (RGCs) directs laminar segregation of OFF and ON starburst amacrine cell (SAC) dendritic scaffolds, which themselves serve as a substrate upon which other retinal neurites elaborate. These results demonstrate that RGCs, the first neuron-type born within the retina, play an active role in functional specialization of the IPL.

Effects of elevated intraocular pressure on alpha ganglion cells in experimental glaucoma mice

Glaucoma is a leading cause of blindness worldwide and glaucoma patients exhibit an early diffuse loss of retinal sensitivity followed by focal loss of RGCs. Combining some previous published results and some new data, this paper provides our current view on how high IOP (H-IOP) affects the light response sensitivity of a subset of RGCs, the alpha-ganglion cells (αGCs), as well as their presynaptic bipolar cells (DBCs and HBCs) and A2 amacrine cells (AIIACs) in dark-adapted mouse retinas. Our data demonstrate that H-IOP in experimental glaucoma mice significantly decreases light-evoked spike response sensitivity of sONαGCs and sOFFαGCs (i.e., raises thresholds by 1.5–2.5 log units), but not that of the tONαGCs and tOFFαGCs. The sensitivity loss in sONαGCs and sOFFαGCs is mediated by a H-IOP induced suppression of AIIAC response which is caused by a decrease of transmission efficacy of the DBCR→AIIAC synapse. We also provide evidence supporting the hypothesis that BK channels in the A17AC→DBCR feedback synapse are the H-IOP sensor that regulates the DBCR→AIIAC synaptic efficacy, as BK channel blocker IBTX mimics the action of H-IOP. Our results provide useful information for designing strategies for early detection and possible treatments of glaucoma as physiological changes occur before irreversible structural damage.

Loss of Stim2 in zebrafish induces glaucoma-like phenotype.

Calcium is involved in vision processes in the retina and implicated in various pathologies, including glaucoma. Rod cells rely on store-operated calcium entry (SOCE) to safeguard against the prolonged lowering of intracellular calcium ion concentrations. Zebrafish that lacked the endoplasmic reticulum Ca2+ sensor Stim2 (stim2 knockout [KO]) exhibited impaired vision and lower light perception-related gene expression. We sought to understand mechanisms that are responsible for vision impairment in stim2 KO zebrafish. The single-cell RNA (scRNA) sequencing of neuronal cells from brains of 5days postfertilization larvae distinguished 27 cell clusters, 10 of which exhibited distinct gene expression patterns, including amacrine and γ-aminobutyric acid (GABA)ergic retinal interneurons and GABAergic optic tectum cells. Five clusters exhibited significant changes in cell proportions between stim2 KO and controls, including GABAergic diencephalon and optic tectum cells. Transmission electron microscopy of stim2 KO zebrafish revealed decreases in width of the inner plexiform layer, ganglion cells, and their dendrites numbers (a hallmark of glaucoma). GABAergic neuron densities in the inner nuclear layer, including amacrine cells, as well as photoreceptors significantly decreased in stim2 KO zebrafish. Our study suggests a novel role for Stim2 in the regulation of neuronal insulin expression and GABAergic-dependent vision causing glaucoma-like retinal pathology.

Use of Electrophysiology in the Diagnosis of Optic Nerve Disorders

Visual electrodiagnostic tests are non invasive, easily applicable that enable objective evaluation of the functions of the visual pathways. The most preferred among these test are; electroretinography (ERG), pattern electroretinography (PERG), electrooculography (EOG) and visual evoked potential (VEP). ERG is used to evaluate retinal functions especially rod and cone responses. PERG is used to evaluate functions of macular and retinal ganglion cells. EOG is used to evaluate retinal pigment epithelium functions. VEP is preferred method for evaluating the visual pathways up to the optic nerve and occipital cortex as a whole. Photopic negative response (PhNR) is originates from the electrical activity of amacrine and glial cells and also mostly retinal ganglion cells. In this study, VEP, PERG and PhNR tests which are the most preferred in the diagnosis of optic nerve disorders, will be mainly mentioned.

Diminished light sensitivities of ON alpha retinal ganglion cells observed in a mouse model of hyperglycemia

This study aimed to investigate potential functional changes in retinal ganglion cells (RGCs) in a mouse model of hyperglycemia and explore possible therapeutic approaches. Hyperglycemia resembling type 1 diabetes mellitus (DM) was induced in C57BL/6 mice through intraperitoneal injection of streptozotocin (STZ). Blood glucose levels were confirmed to be elevated after 1 week and 4 weeks of injection. Mice with blood glucose levels above 350 mg/mL after 4 weeks of one-dose STZ injection were considered hyperglycemic. The light sensitivity of ON alpha (α) retinal ganglion cells (RGCs), not OFF αRGCs, was reduced in the hyperglycemic mouse model. The number of apoptotic cells, RGCs, and amacrine cells (ACs) remained unaffected at this stage. Similarly, the eletroretinogram (ERG) and optokinetic test results showed no significant differences. The application of picrotoxin (PTX) to block GABA receptors could increase the light sensitivity of ON αRGCs by 1 log unit in hyperglycemic mice. The results show that ON αRGCs may be more susceptible to microenvironmental changes caused by hyperglycemia than OFF αRGCs. This decline in light sensitivity may occur before cell apoptosis during the early stages of the hyperglycemic mouse model but has the potential to be reversed.

Role of Elavl-like RNA-binding protein in retinal development and signal transduction.

RNA-binding proteins (RBPs) play central roles in post-transcriptional gene regulation. However, the function of RBP in retinal progenitor cell differentiation and synaptic signal transmission are largely unexplored. Previously we have shown that Elavl2 regulates amacrine cell (AC) differentiation during retinogenesis, by directly binding to Nr4a2 and Barhl2. Elavl2 is expressed in early neuronal progenitors to mature neurons, and Elavl4 expression begins slightly later, during cortical neuron development as a paralog. Here, Retinal-specific Elavl2 and Elavl4 double knockout mice were made to further explore the role of Elavl2 and Elavl4 in retinal development and signal transduction. We disclose that Elavl4 binds to Satb1 to regulate Neurod1, then promoting retinal progenitor and amacrine cells differentiation. We were also surprised to find that Elavl2 interacted with GABAB receptors at the RNA and protein levels. In conclusion, Elavl2 and Elavl4 regulate amacrine cells differentiation through different pathways, leading to decreased scotopic vision. Our findings reveal the roles of Elavl2 and Elavl4 in retinal amacrine cells differentiation in modulating visual functions.

Presynaptic Proteins and Their Roles in Visual Processing by the Retina.

The sense of vision begins in the retina, where light is detected and processed through a complex series of synaptic connections into meaningful information relayed to the brain via retinal ganglion cells. Light responses begin as tonic and graded signals in photoreceptors, later emerging from the retina as a series of spikes from ganglion cells. Processing by the retina extracts critical features of the visual world, including spatial frequency, temporal frequency, motion direction, color, contrast, and luminance. To achieve this, the retina has evolved specialized and unique synapse types. These include the ribbon synapses of photoreceptors and bipolar cells, the dendritic synapses of amacrine and horizontal cells, and unconventional synaptic feedback from horizontal cells to photoreceptors. We review these unique synapses in the retina with a focus on the presynaptic molecules and physiological properties that shape their capabilities.

Characterizing Presumed Displaced Retinal Ganglion Cells in the Living Human Retina of Healthy and Glaucomatous Eyes.

The purpose of this study was to investigate the large somas presumed to be displaced retinal ganglion cells (dRGCs) located in the inner nuclear layer (INL) of the living human retina. Whereas dRGCs have previously been studied in mammals and human donor tissue, they have never been investigated in the living human retina. Five young, healthy subjects and three subjects with varying types of glaucoma were imaged at multiple locations in the macula using adaptive optics optical coherence tomography. In the acquired volumes, bright large somas at the INL border with the inner plexiform layer were identified, and the morphometric biomarkers of soma density, en face diameter, and spatial distribution were measured at up to 13 degrees retinal eccentricity. Susceptibility to glaucoma was assessed. In the young, healthy individuals, mean density of the bright, large somas was greatest foveally (550 and 543 cells/mm2 at 2 degrees temporal and nasal, respectively) and decreased with increasing retinal eccentricity (38 cells/mm2 at 13 degrees temporal, the farthest we measured). Soma size distribution showed the opposite trend with diameters and size variation increasing with retinal eccentricity, from 12.7 ± 1.8 µm at 2 degrees to 15.7 ± 3.5 µm at 13 degrees temporal, and showed evidence of a bimodal distribution in more peripheral locations. Within and adjacent to the arcuate defects of the subjects with glaucoma, density of the bright large somas was significantly lower than found in the young, healthy individuals. Our results suggest that the bright, large somas at the INL border are likely comprised of dRGCs but amacrine cells may contribute too. These somas appear highly susceptible to glaucomatous damage.

Comprehensive Characterization of a Subfamily of Ca2+-Binding Proteins in Mouse and Human Retinal Neurons at Single-Cell Resolution.

Ca2+-binding proteins (CaBPs; CaBP1-5) are a subfamily of neuronal Ca2+ sensors with high homology to calmodulin. Notably, CaBP4, which is exclusively expressed in rod and cone photoreceptors, is crucial for maintaining normal retinal functions. However, the functional roles of CaBP1, CaBP2, and CaBP5 in the retina remain elusive, primarily due to limited understanding of their expression patterns within inner retinal neurons. In this study, we conducted a comprehensive transcript analysis using single-cell RNA sequencing datasets to investigate the gene expression profiles of CaBPs in mouse and human retinal neurons. Our findings revealed notable similarities in the overall expression patterns of CaBPs across both species. Specifically, nearly all amacrine cell, ganglion cell, and horizontal cell types exclusively expressed CaBP1. In contrast, the majority of bipolar cell types, including rod bipolar (RB) cells, expressed distinct combinations of CaBP1, CaBP2, and CaBP5, rather than a single CaBP as previously hypothesized. Remarkably, mouse rods and human cones exclusively expressed CaBP4, whereas mouse cones and human rods coexpressed both CaBP4 and CaBP5. Our single-cell reverse transcription polymerase chain reaction analysis confirmed the coexpression CaBP1 and CaBP5 in individual RBs from mice of either sex. Additionally, all three splice variants of CaBP1, primarily L-CaBP1, were detected in mouse RBs. Taken together, our study offers a comprehensive overview of the distribution of CaBPs in mouse and human retinal neurons, providing valuable insights into their roles in visual functions.

Gamma-protocadherins regulate dendrite self-recognition and dynamics to drive self-avoidance

Neurons form cell-type-specific morphologies that are shaped by cell-surface molecules and their cellular events governing dendrite growth. One growth rule is dendrite self-avoidance, whereby dendrites distribute uniformly within a neuron's territory by avoiding sibling branches. In mammalian neurons, dendrite self-avoidance is regulated by a large family of cell-recognition molecules called the clustered protocadherins (cPcdhs). Genetic and molecular studies suggest that the cPcdhs mediate homophilic recognition and repulsion between self-dendrites. However, this model has not been tested through direct investigation of self-avoidance during development. Here, we performed live imaging and four-dimensional (4D) quantifications of dendrite morphogenesis to define the dynamics and cPcdh-dependent mechanisms of self-avoidance. We focused on the mouse retinal starburst amacrine cell (SAC), which requires the gamma-Pcdhs (Pcdhgs) and self/non-self-recognition to establish a stereotypic radial morphology while permitting dendritic interactions with neighboring SACs. Through morphogenesis, SACs extend dendritic protrusions that iteratively fill the growing arbor and contact and retract from nearby self-dendrites. Compared to non-self-contacting protrusions, self-contacting events have longer lifetimes, and a subset persists as loops. In the absence of the Pcdhgs, non-self-contacting dynamics are unaffected but self-contacting retractions are significantly diminished. Self-contacting bridges accumulate, leading to the bundling of dendritic processes and disruption to the arbor shape. By tracking dendrite self-avoidance in real time, our findings establish that the γ-Pcdhs mediate self-recognition and retraction between contacting sibling dendrites. Our results also illustrate how self-avoidance shapes stochastic and space-filling dendritic outgrowth for robust pattern formation in mammalian neurons.

Age-related effects of optineurin deficiency in the mouse eye

Optineurin (OPTN) is a gene associated with familial normal tension glaucoma (NTG). While NTG involves intraocular pressure (IOP)-independent neurodegeneration of the visual pathway that progresses with age, how OPTN dysfunction leads to NTG remains unclear. Here, we generated an OPTN knockout mouse (Optn−/−) model to test the hypothesis that a loss-of-function mechanism induces structural and functional eye deterioration with aging. Eye anatomy, visual function, IOP, retinal histology, and retinal ganglion cell survival were compared to littermate wild-type (WT) control mice. Consistent with OPTN’s role in NTG, loss of OPTN did not increase IOP or alter gross eye anatomy in young (2–3 months) or aged (12 months) mice. When retinal layers were quantitated, young Optn−/− mice had thinner retina in the peripheral regions than young WT mice, primarily due to thinner ganglion cell-inner plexiform layers. Despite this, visual function in Optn−/− mice was not severely impaired, even with aging. We also assessed relative abundance of retinal cell subtypes, including amacrine cells, bipolar cells, cone photoreceptors, microglia, and astrocytes. While many of these cellular subtypes were unaffected by Optn deletion, more dopaminergic amacrine cells were observed in aged Optn−/− mice. Taken together, our findings showed that complete loss of Optn resulted in mild retinal changes and less visual function impairment, supporting the possibility that OPTN-associated glaucoma does not result from a loss-of-function disease mechanism. Further research using these Optn mice will elucidate detailed molecular pathways involved in NTG and identify clinical or environmental risk factors that can be targeted for glaucoma treatment.

Artificial Amacrine Retinal Circuits.

Event-based imaging represents a new paradigm in visual information processing that addresses the speed and energy efficiency shortcomings inherently present in the current complementary metal oxide semiconductor-based machine vision. Realizing such imaging systems has previously been sought using very large-scale integration technologies that have complex circuitries consisting of many photodiodes, differential amplifiers, capacitors, and resistors. Here, we demonstrate that event-driven sensing can be achieved using a simple one-resistor, one-capacitor (1R1C) circuit, where the capacitor is modified with colloidal quantum dots (CQDs) to have a photoresponse. This sensory circuit emulates the motion-tracking function of the biological retina, in which the amacrine cells in the bipolar-to-ganglion synaptic pathway produce a transient spiking signal only in response to changes in light intensity but remain inactive under constant illumination. When extended to a 2D imaging array, the individual sensors work independently and output signals only when a change in the light intensity is detected; hence, the concept of the frame in image processing is thereby removed. In this work, we present the fabrication and characterization of a CQD photocapacitor-based 1R1C circuit that has a spectral response at 1550 nm in the short-wave infrared (SWIR). We report on the key performance parameters including peak responsivity, noise, and optical noise equivalent power and discuss the operating mechanism that is responsible for spiking responses in these artificial retinal circuits. The present work sets the foundation for expanding the bioinspired vision sensor capability toward midwave infrared (MWIR) and long-wave infrared (LWIR) spectral regions that are invisible to human eyes and mainstream semiconductor technologies.

Retinal neuroanatomy of two emerging model organisms, the spiny mouse (Acomys dimidiatus) and the Mongolian gerbil (Meriones unguiculatus)

Current research using animal models to investigate retinal cell biology and model retinal degenerative diseases largely utilize small mammals that are nocturnal and lack the ability to restore lost vision. In contrast, the Mongolian gerbil (Meriones) is a diurnal rodent with good photopic vision, and the spiny mouse (Acomys) is a small desert-dwelling rodent with remarkable regenerative capabilities. The goal of this study was to identify antibodies that detect retinal cell classes in Meriones and Acomys, and to describe the retinal anatomy of these two species in comparison to outbred laboratory mice (Mus musculus). Immunohistochemistry was performed on retinal sections with antibodies for various retinal cell types. Sections were imaged by light, fluorescence, and confocal microscopy. Cell density, morphology, and placement were compared between species qualitatively and quantitatively. Our analyses revealed a classic assembly of retinal cells in Meriones and Acomys, with a few deviations compared to Mus. Meriones displayed the highest density of cones and Acomys the lowest. A higher density of bipolar cell bodies in the proximal portion of the inner nuclear layer was observed in both Acomys and Meriones compared to Mus, and both species exhibited an increase in amacrine cell density compared to Mus. Our results provide a foundation for future research into the visual system adaptations of these interesting species.

Activation of retinoid X receptors protects retinal neurons and pigment epithelial cells from BMAA-induced death

Exposure to the non-protein amino acid cyanotoxin β–N-methylamino-L-alanine (BMAA), released by cyanobacteria found in many water reservoirs has been associated with neurodegenerative diseases. We previously demonstrated that BMAA induced cell death in both retina photoreceptors (PHRs) and amacrine neurons by triggering different molecular pathways, as activation of NMDA receptors and formation of carbamate-adducts was only observed in amacrine cell death. We established that activation of Retinoid X Receptors (RXR) protects retinal cells, including retina pigment epithelial (RPE) cells from oxidative stress-induced apoptosis. We now investigated the mechanisms underlying BMAA toxicity in these cells and those involved in RXR protection.BMAA addition to rat retinal neurons during early development in vitro increased reactive oxygen species (ROS) generation and polyADP ribose polymers (PAR) formation, while pre-treatment with serine (Ser) before BMAA addition decreased PHR death. Notably, RXR activation with the HX630 agonist prevented BMAA-induced death in both neuronal types, reducing ROS generation, preserving mitochondrial potential, and decreasing TUNEL-positive cells and PAR formation. This suggests that BMAA promoted PHR death by substituting Ser in polypeptide chains and by inducing polyADP ribose polymerase activation. BMAA induced cell death in ARPE-19 cells, a human epithelial cell line; RXR activation prevented this death, decreasing ROS generation and caspase 3/7 activity.These findings suggest that RXR activation prevents BMAA harmful effects on retinal neurons and RPE cells, supporting this activation as a broad-spectrum strategy for treating retina degenerations.

Modulating amacrine cell-derived dopamine signaling promotes optic nerve regeneration and preserves visual function.

As part of the central nervous system, the optic nerve, composed of axons from retinal ganglion cells (RGCs), generally fails to regenerate on its own when injured in adult mammals. An innovative approach to promoting optic nerve regeneration involves manipulating the interactions between amacrine cells (ACs) and RGCs. Here, we identified a unique AC subtype, dopaminergic ACs (DACs), that responded early after optic nerve crush by down-regulating neuronal activity and reducing retinal dopamine (DA) release. Activating DACs or augmenting DA release with levodopa demonstrated neuroprotective effects and modestly enhanced axon regeneration. Within this context, we pinpointed the DA receptor D1 (DRD1) as a critical mediator of DAC-derived DA and showed that RGC-specific Drd1 overexpression effectively overcame subtype-specific barriers to regeneration. This strategy markedly boosted RGC survival and axon regeneration after crush and preserved vision in a glaucoma model. This study unveils the crucial role of DAC-derived DA signaling in optic nerve regeneration, holding promise for therapeutic insights into neural repair.

Impaired dopamine signaling in early diabetic retina: Insights from D1R and D4R agonist effects on whole retina responses

The retina has low dopamine levels early in diabetes. To determine how low dopamine levels affected dopamine signaling, the effects of dopamine receptor agonists and mRNA localization were measured after 6 weeks of diabetes. Whole retina ex vivo electroretinogram (ERG) recordings were used to analyze how dopamine type 1 receptor (D1R) and type 4 (D4R) agonists change the light-evoked retinal responses of non-diabetic and 6-week diabetic (STZ injected) mouse retinas. Fluorescence in situ hybridization was utilized to analyze D4R and D1R mRNA locations and expression levels. D4R activation reduced A- and B-wave ERG amplitudes and increased B-wave implicit time and rise-time in the non-diabetic group without a corresponding change in the diabetic group. D1R activation increased B-wave rise-time and oscillatory potential peak time in the non-diabetic group also with no change in the diabetic group. The lack of responsivity to D1R or D4R agonists shows an impairment of dopamine signaling in the diabetic retina. D4R mRNA was found primarily in the outer nuclear layer where photoreceptor cell bodies reside. D1R mRNA was found in the inner nuclear layer and ganglion cell layer that contain bipolar, amacrine, horizontal and ganglion cells. There was no change in D4R or D1R mRNA expression between the non-diabetic and diabetic retinas. This suggests that the significant dopamine signaling changes observed were not from lower receptor expression levels but could be due to changes in dopamine receptor activity or protein levels. These studies show that changes in retinal dopamine signaling could be an important mechanism of diabetic retinal dysfunction.

Retinal neurons establish mosaic patterning by excluding homotypic somata from their dendritic territories

In vertebrate retina, individual neurons of the same type are distributed regularly across the tissue in a pattern known as a mosaic. Establishment of mosaics during development requires cell-cell repulsion among homotypic neurons, but the mechanisms underlying this repulsion remain unknown. Here, we show that two mouse retinal cell types, OFF and ON starburst amacrine cells, establish mosaic spacing by using their dendritic arbors to repel neighboring homotypic somata. Using transgenic tools and single-cell labeling, we identify a developmental period when starburst somata are contacted by neighboring starburst dendrites; these serve to exclude somata from settling within the neighbor's dendritic territory. Dendrite-soma exclusion is mediated by MEGF10, a cell-surface molecule required for starburst mosaic patterning. Our results implicate dendrite-soma exclusion as a key mechanism underlying starburst mosaic spacing and raise the possibility that this could be a general mechanism for mosaic patterning across many cell types and species.

Metabolic Deficits in the Retina of a Familial Dysautonomia Mouse Model.

Neurodegenerative retinal diseases such as glaucoma, diabetic retinopathy, Leber's hereditary optic neuropathy (LHON), and dominant optic atrophy (DOA) are marked by progressive death of retinal ganglion cells (RGC). This decline is promoted by structural and functional mitochondrial deficits, including electron transport chain (ETC) impairments, increased oxidative stress, and reduced energy (ATP) production. These cellular mechanisms associated with progressive optic nerve atrophy have been similarly observed in familial dysautonomia (FD) patients, who experience gradual loss of visual acuity due to the degeneration of RGCs, which is thought to be caused by a breakdown of mitochondrial structures, and a disruption in ETC function. Retinal metabolism plays a crucial role in meeting the elevated energetic demands of this tissue, and recent characterizations of FD patients' serum and stool metabolomes have indicated alterations in central metabolic processes and potential systemic deficits of taurine, a small molecule essential for retina and overall eye health. The present study sought to elucidate metabolic alterations that contribute to the progressive degeneration of RGCs observed in FD. Additionally, a critical subpopulation of retinal interneurons, the dopaminergic amacrine cells, mediate the integration and modulation of visual information in a time-dependent manner to RGCs. As these cells have been associated with RGC loss in the neurodegenerative disease Parkinson's, which shares hallmarks with FD, a targeted analysis of the dopaminergic amacrine cells and their product, dopamine, was also undertaken. One dimensional (1D) proton (1H) nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and retinal histology methods were employed to characterize retinae from the retina-specific Elp1 conditional knockout (CKO) FD mouse model (Pax6-Cre; Elp1LoxP/LoxP). Metabolite alterations correlated temporally with progressive RGC degeneration and were associated with reduced mitochondrial function, alterations in ATP production through the Cahill and mini-Krebs cycles, and phospholipid metabolism. Dopaminergic amacrine cell populations were reduced at timepoints P30-P90, and dopamine levels were 25-35% lower in CKO retinae compared to control retinae at P60. Overall, this study has expanded upon our current understanding of retina pathology in FD. This knowledge may apply to other retinal diseases that share hallmark features with FD and may help guide new avenues for novel non-invasive therapeutics to mitigate the progressive optic neuropathy in FD.

Dopaminergic amacrine cells express HCN channels in the developing and adult mouse retina.

To determine the molecular and functional expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in developing and mature dopaminergic amacrine cells (DACs), the sole source of ocular dopamine that plays a vital role in visual function and eye development. HCN channels are encoded by isoforms 1-4. HCN1, HCN2, and HCN4 were immunostained in retinal slices obtained from mice at postnatal day 4 (P4), P8, and P12 as well as in adults. Each HCN channel isoform was also immunostained with tyrosine hydroxylase, a marker for DACs, at P12 and adult retinas. Genetically-marked DACs were recorded in flat-mount retina preparation using a whole-cell current-clamp technique. HCN1 was expressed in rods/cones, amacrine cells, and retinal ganglion cells (RGCs) at P4, along with bipolar cells by P12. Different from HCN1, HCN2 and HCN4 were each expressed in amacrine cells and RGCs at P4, along with bipolar cells by P8, and in rods/cones by P12. Double immunostaining shows that each of the three isoforms was expressed in approximately half of DACs at P12 but in almost all DACs in adults. Electrophysiology results demonstrate that HCN channel isoforms form functional HCN channels, and the pharmacological blockade of HCN channels reduced the spontaneous firing frequency in most DACs. Each class of retinal neurons may use different isoforms of HCN channels to function during development. HCN1, HCN2, and HCN4 form functional HCN channels in DACs, which appears to modulate their spontaneous firing activity.