Gap junctions fine-tune ganglion cell signals to equalize response kinetics within a given electrically coupled array.

Retinal ganglion cells (RGCs) summate inputs and forward a spike train code to the brain in the form of either maintained spiking (sustained) or a quickly decaying brief spike burst (transient). We report diverse response transience values across the RGC population and, contrary to the conventional transient/sustained scheme, responses with intermediary characteristics are the most abundant. Pharmacological tests showed that besides GABAergic inhibition, gap junction (GJ)-mediated excitation also plays a pivotal role in shaping response transience and thus visual coding. More precisely GJs connecting RGCs to nearby amacrine and RGCs play a defining role in the process. These GJs equalize kinetic features, including the response transience of transient OFF alpha (tOFFα) RGCs across a coupled array. We propose that GJs in other coupled neuron ensembles in the brain are also critical in the harmonization of response kinetics to enhance the population code and suit a corresponding task.

Long-term Effects of the pituitary-adenylate cyclase-activating Polypeptide (PACAP38) in the Adult Mouse Retina: Microglial Activation and Induction of Neural Proliferation.

The degenerative retinal disorders characterized by progressive cell death and exacerbating inflammation lead ultimately to blindness. The ubiquitous neuropeptide, PACAP38 is a promising therapeutic agent as its proliferative potential and suppressive effect on microglia might enable cell replacement and attenuate inflammation, respectively. Our previous finding that PACAP38 caused a marked increase of the amacrine cells in the adult (1-year-old) mouse retina, served as a rationale of the current study. We aimed to determine the proliferating elements and the inflammatory status of the PACAP38-treated retina. Three months old mice were intravitreally injected with 100 pmol PACAP38 at 3 months intervals (3X). Retinas of 1-year-old animals were dissected and effects on cell proliferation, and expression of inflammatory regulators were analyzed. Interestingly, both mitogenic and anti-mitogenic actions were detected after PACAP38-treatment. Further analysis of the mitogenic effect revealed that proliferating cells include microglia, endothelial cells, and neurons of the ganglion cell layer but not amacrine cells. Furthermore, PACAP38 stimulated retinal microglia to polarize dominantly into M2-phenotype but also might cause subsequent angiogenesis. According to our results, PACAP38 might dampen pro-inflammatory responses and help tissue repair by reprogramming microglia into an M2 phenotype, nonetheless, with angiogenesis as a warning side effect.

Extrinsic and Intrinsic Factors Determine Expression Levels of Gap Junction-Forming Connexins in the Mammalian Retina.

Gap junctions (GJs) are not static bridges; instead, GJs as well as the molecular building block connexin (Cx) proteins undergo major expression changes in the degenerating retinal tissue. Various progressive diseases, including retinitis pigmentosa, glaucoma, age-related retinal degeneration, etc., affect neurons of the retina and thus their neuronal connections endure irreversible changes as well. Although Cx expression changes might be the hallmarks of tissue deterioration, GJs are not static bridges and as such they undergo adaptive changes even in healthy tissue to respond to the ever-changing environment. It is, therefore, imperative to determine these latter adaptive changes in GJ functionality as well as in their morphology and Cx makeup to identify and distinguish them from alterations following tissue deterioration. In this review, we summarize GJ alterations that take place in healthy retinal tissue and occur on three different time scales: throughout the entire lifespan, during daily changes and as a result of quick changes of light adaptation.

Traumatic Brain Injury Induces Microglial and Caspase3 Activation in the Retina.

Traumatic brain injury (TBI) is among the main causes of sudden death after head trauma. These injuries can result in severe degeneration and neuronal cell death in the CNS, including the retina, which is a crucial part of the brain responsible for perceiving and transmitting visual information. The long-term effects of mild-repetitive TBI (rmTBI) are far less studied thus far, even though damage induced by repetitive injuries occurring in the brain is more common, especially amongst athletes. rmTBI can also have a detrimental effect on the retina and the pathophysiology of these injuries is likely to differ from severe TBI (sTBI) retinal injury. Here, we show how rmTBI and sTBI can differentially affect the retina. Our results indicate an increase in the number of activated microglial cells and Caspase3-positive cells in the retina in both traumatic models, suggesting a rise in the level of inflammation and cell death after TBI. The pattern of microglial activation appears distributed and widespread but differs amongst the various retinal layers. sTBI induced microglial activation in both the superficial and deep retinal layers. In contrast to sTBI, no significant change occurred following the repetitive mild injury in the superficial layer, only the deep layer (spanning from the inner nuclear layer to the outer plexiform layer) shows microglial activation. This difference suggests that alternate response mechanisms play a role in the case of the different TBI incidents. The Caspase3 activation pattern showed a uniform increase in both the superficial and deep layers of the retina. This suggests a different action in the course of the disease in sTBI and rmTBI models and points to the need for new diagnostic procedures. Our present results suggest that the retina might serve as such a model of head injuries since the retinal tissue reacts to both forms of TBI and is the most accessible part of the human brain.

Serotonin is a gap junction-permeable neuronal tracer in the mouse retina.

Introduction: Gap junctions are dynamically modulated bridges allowing the transcellular passage of ions and small molecules with a molecular mass of up to 1 kDa, a mechanism utilized for molecular communication purposes by living cells. This same mechanism is also exploited by scientists to reveal the existence of gap junction contacts by the cell-to-cell movement of tracers. However, multiple labeling experiments require the availability of multiple gap junction-permeable tracers. Methods: To this end, we utilized the well-known transient OFF alpha retinal ganglion cell (RGC)-coupled array as a model system to study and compare the transjunctional movement of neurobiotin (NB), a commonly used tracer, and serotonin, a recently identified tracer. Results: Although the transjunctional movement of serotonin has been established in cell cultures, here we show, for the first time, that serotonin is also a potent tracer in in vitro tissue. In addition, serotonin is lighter than the classical gap junction-permeable NB, and thus, we expected that tracer movement would be comparable to or better than that of serotonin. We found that intracellular serotonin injections result in the labeling of the coupled transient OFF alpha RGC array very similar to those of the classical NB-labeled arrays. Both serotonin and NB-injected transient OFF alpha RGCs displayed the well-known pattern with coupled RGCs and a cohort of coupled wide-field amacrine cells (ACs). Discussion: By using morphological characteristics, we confirm that the serotonin and the NB-coupled AC arrays are identical, and thereby confirm that serotonin is a potent gap junction-permeable tracer and can be readily used as an alternative to NB in in vitro tissue. Moreover, serotonin can be utilized in parallel with other dyes or tracers, enabling the use of multiple labels in the same material.

Transience of the Retinal Output Is Determined by a Great Variety of Circuit Elements.

Retinal ganglion cells (RGCs) encrypt stimulus features of the visual scene in action potentials and convey them toward higher visual centers in the brain. Although there are many visual features to encode, our recent understanding is that the ~46 different functional subtypes of RGCs in the retina share this task. In this scheme, each RGC subtype establishes a separate, parallel signaling route for a specific visual feature (e.g., contrast, the direction of motion, luminosity), through which information is conveyed. The efficiency of encoding depends on several factors, including signal strength, adaptational levels, and the actual efficacy of the underlying retinal microcircuits. Upon collecting inputs across their respective receptive field, RGCs perform further analysis (e.g., summation, subtraction, weighting) before they generate the final output spike train, which itself is characterized by multiple different features, such as the number of spikes, the inter-spike intervals, response delay, and the rundown time (transience) of the response. These specific kinetic features are essential for target postsynaptic neurons in the brain in order to effectively decode and interpret signals, thereby forming visual perception. We review recent knowledge regarding circuit elements of the mammalian retina that participate in shaping RGC response transience for optimal visual signaling.

LED-Induced Microglial Activation and Rise in Caspase3 Suggest a Reorganization in the Retina.

Vision is our primary sense as the human eye is the gateway for more than 65% of information reaching the human brain. Today's increased exposure to different wavelengths and intensities of light from light emitting diode (LED) sources could induce retinal degeneration and accompanying neuronal cell death. Damage induced by chronic phototoxic reactions occurring in the retina accumulates over years and it has been suggested as being responsible for the etiology of many debilitating ocular conditions. In this work, we examined how LED stimulation affects vision by monitoring changes in the expression of death and survival factors as well as microglial activation in LED-induced damage (LID) of the retinal tissue. We found an LED-exposure-induced increase in the mRNA levels of major apoptosis-related markers BAX, Bcl-2, and Caspase-3 and accompanying widespread microglial and Caspase-3 activation. Everyday LED light exposure was accounted for in all the described changes in the retinal tissue of mice in this study, indicating that overuse of non-filtered direct LED light can have detrimental effects on the human retina as well.

The role of gap junctions in cell death and neuromodulation in the retina.

Vision altering diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration, myopia, retinal vascular disease, traumatic brain injuries and others cripple many lives and are projected to continue to cause anguish in the foreseeable future. Gap junctions serve as an emerging target for neuromodulation and possible regeneration as they directly connect healthy and/or diseased cells, thereby playing a crucial role in pathophysiology. Since they are permeable for macromolecules, able to cross the cellular barriers, they show duality in illness as a cause and as a therapeutic target. In this review, we take recent advancements in gap junction neuromodulation (pharmacological blockade, gene therapy, electrical and light stimulation) into account, to show the gap junction's role in neuronal cell death and the possible routes of rescuing neuronal and glial cells in the retina succeeding illness or injury.

Imatinib Sets Pericyte Mosaic in the Retina.

The nervous system demands an adequate oxygen and metabolite exchange, making pericytes (PCs), the only vasoactive cells on the capillaries, essential to neural function. Loss of PCs is a hallmark of multiple diseases, including diabetes, Alzheimer's, amyotrophic lateral sclerosis (ALS) and Parkinson's. Platelet-derived growth factor receptors (PDGFRs) have been shown to be critical to PC function and survival. However, how PDGFR-mediated PC activity affects vascular homeostasis is not fully understood. Here, we tested the hypothesis that imatinib, a chemotherapeutic agent and a potent PDGFR inhibitor, alters PC distribution and thus induces vascular atrophy. We performed a morphometric analysis of the vascular elements in sham control and imatinib-treated NG2-DsRed mice. Vascular morphology and the integrity of the blood-retina barrier (BRB) were evaluated using blood albumin labeling. We found that imatinib decreased the number of PCs and blood vessel (BV) coverage in all retinal vascular layers; this was accompanied by a shrinkage of BV diameters. Surprisingly, the total length of capillaries was not altered, suggesting a preferential effect of imatinib on PCs. Furthermore, blood-retina barrier disruption was not evident. In conclusion, our data suggest that imatinib could help in treating neurovascular diseases and serve as a model for PC loss, without BRB disruption.

Spatial Expression Pattern of the Major Ca2+-Buffer Proteins in Mouse Retinal Ganglion Cells.

The most prevalent Ca2+-buffer proteins (CaBPs: parvalbumin-PV; calbindin-CaB; calretinin-CaR) are widely expressed by various neurons throughout the brain, including the retinal ganglion cells (RGCs). Even though their retinal expression has been extensively studied, a coherent assessment of topographical variations is missing. To examine this, we performed immunohistochemistry (IHC) in mouse retinas. We found variability in the expression levels and cell numbers for CaR, with stronger and more numerous labels in the dorso-central area. CaBP+ cells contributed to RGCs with all soma sizes, indicating heterogeneity. We separated four to nine RGC clusters in each area based on expression levels and soma sizes. Besides the overall high variety in cluster number and size, the peripheral half of the temporal retina showed the greatest cluster number, indicating a better separation of RGC subtypes there. Multiple labels showed that 39% of the RGCs showed positivity for a single CaBP, 30% expressed two CaBPs, 25% showed no CaBP expression, and 6% expressed all three proteins. Finally, we observed an inverse relation between CaB and CaR expression levels in CaB/CaR dual- and CaB/CaR/PV triple-labeled RGCs, suggesting a mutual complementary function.

Response Latency Tuning by Retinal Circuits Modulates Signal Efficiency.

In the visual system, retinal ganglion cells (RGCs) of various subtypes encode preprocessed photoreceptor signals into a spike output which is then transmitted towards the brain through parallel feature pathways. Spike timing determines how each feature signal contributes to the output of downstream neurons in visual brain centers, thereby influencing efficiency in visual perception. In this study, we demonstrate a marked population-wide variability in RGC response latency that is independent of trial-to-trial variability and recording approach. RGC response latencies to simple visual stimuli vary considerably in a heterogenous cell population but remain reliable when RGCs of a single subtype are compared. This subtype specificity, however, vanishes when the retinal circuitry is bypassed via direct RGC electrical stimulation. This suggests that latency is primarily determined by the signaling speed through retinal pathways that provide subtype specific inputs to RGCs. In addition, response latency is significantly altered when GABA inhibition or gap junction signaling is disturbed, which further supports the key role of retinal microcircuits in latency tuning. Finally, modulation of stimulus parameters affects individual RGC response delays considerably. Based on these findings, we hypothesize that retinal microcircuits fine-tune RGC response latency, which in turn determines the context-dependent weighing of each signal and its contribution to visual perception.

Expression of Ca2+-Binding Buffer Proteins in the Human and Mouse Retinal Neurons.

Ca2+-binding buffer proteins (CaBPs) are widely expressed by various neurons throughout the central nervous system (CNS), including the retina. While the expression of CaBPs by photoreceptors, retinal interneurons and the output ganglion cells in the mammalian retina has been extensively studied, a general description is still missing due to the differences between species, developmental expression patterns and study-to-study discrepancies. Furthermore, CaBPs are occasionally located in a compartment-specific manner and two or more CaBPs can be expressed by the same neuron, thereby sharing the labor of Ca2+ buffering in the intracellular milieu. This article reviews this topic by providing a framework on CaBP functional expression by neurons of the mammalian retina with an emphasis on human and mouse retinas and the three most abundant and extensively studied buffer proteins: parvalbumin, calretinin and calbindin.

Strategic Positioning of Connexin36 Gap Junctions Across Human Retinal Ganglion Cell Dendritic Arbors.

Connexin36 (Cx36) subunits form gap junctions (GJ) between neurons throughout the central nervous system. Such GJs of the mammalian retina serve the transmission, averaging and correlation of signals prior to conveying visual information to the brain. Retinal GJs have been exhaustively studied in various animal species, however, there is still a perplexing paucity of information regarding the presence and function of human retinal GJs. Particularly little is known about GJ formation of human retinal ganglion cells (hRGCs) due to the limited number of suitable experimental approaches. Compared to the neuronal coupling studies in animal models, where GJ permeable tracer injection is the gold standard method, the post-mortem nature of scarcely available human retinal samples leaves immunohistochemistry as a sole approach to obtain information on hRGC GJs. In this study Lucifer Yellow (LY) dye injections and Cx36 immunohistochemistry were performed in fixed short-post-mortem samples to stain hRGCs with complete dendritic arbors and locate dendritic Cx36 GJs. Subsequent neuronal reconstructions and morphometric analyses revealed that Cx36 plaques had a clear tendency to form clusters and particularly favored terminal dendritic segments.