Dark rearing alters the normal development of spatiotemporal response properties but not of contrast detection threshold in mouse retinal ganglion cells

The mouse visual system is immature when the eyes open two weeks after birth. As in other mammals, some of the maturation that occurs in the subsequent weeks is known to depend on visual experience. Development of the retina, which as the first stage of vision provides the visual information to the brain, also depends on light‐driven activity for proper development but has been less well studied than visual cortical development. The critical properties for retinal encoding of images include detection of contrast and responsiveness to the broad range of temporal stimulus frequencies present in natural stimuli. Here we show that contrast detection threshold and temporal frequency response characteristics of ON and OFF retinal ganglion cells (RGCs), which are poor at eye opening, subsequently undergo maturation, improving RGC performance. Further, we find that depriving mice of visual experience from before birth by rearing them in the dark causes ON and OFF RGCs to have smaller receptive field centers but does not affect their contrast detection threshold development. The modest developmental increase in temporal frequency responsiveness of RGCs in mice reared on a normal light cycle was inhibited by dark rearing only in ON but not OFF RGCs. Thus, these RGC response characteristics are in many ways unaffected by the experience‐dependent changes to synaptic and spontaneous activity known to occur in the mouse retina in the two weeks after eye opening, but specific differences are apparent in the ON vs. OFF RGC populations. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 692–706, 2014     

Network Oscillations Drive Correlated Spiking of ON and OFF Ganglion Cells in the rd1 Mouse Model of Retinal Degeneration

Following photoreceptor degeneration, ON and OFF retinal ganglion cells (RGCs) in the rd-1/rd-1 mouse receive rhythmic synaptic input that elicits bursts of action potentials at ∼10 Hz. To characterize the properties of this activity, RGCs were targeted for paired recording and morphological classification as either ON alpha, OFF alpha or non-alpha RGCs using two-photon imaging. Identified cell types exhibited rhythmic spike activity. Cross-correlation of spike trains recorded simultaneously from pairs of RGCs revealed that activity was correlated more strongly between alpha RGCs than between alpha and non-alpha cell pairs. Bursts of action potentials in alpha RGC pairs of the same type, i.e. two ON or two OFF cells, were in phase, while bursts in dissimilar alpha cell types, i.e. an ON and an OFF RGC, were 180 degrees out of phase. This result is consistent with RGC activity being driven by an input that provides correlated excitation to ON cells and inhibition to OFF cells. A2 amacrine cells were investigated as a candidate cellular mechanism and found to display 10 Hz oscillations in membrane voltage and current that persisted in the presence of antagonists of fast synaptic transmission and were eliminated by tetrodotoxin. Results support the conclusion that the rhythmic RGC activity originates in a presynaptic network of electrically coupled cells including A2s via a Na⁺-channel dependent mechanism. Network activity drives out of phase oscillations in ON and OFF cone bipolar cells, entraining similar frequency fluctuations in RGC spike activity over an area of retina that migrates with changes in the spatial locus of the cellular oscillator.     

Responses of Retinal Ganglion Cells to Extracellular Electrical Stimulation,from Single Cell to Population: Model-Based Analysis

Retinal ganglion cells (RGCs), which survive in large numbers following neurodegenerative diseases, could be stimulated with extracellular electric pulses to elicit artificial percepts. How do the RGCs respond to electrical stimulation at the sub-cellular level under different stimulus configurations, and how does this influence the whole-cell response? At the population level, why have experiments yielded conflicting evidence regarding the extent of passing axon activation? We addressed these questions through simulations of morphologically and biophysically detailed computational RGC models on high performance computing clusters. We conducted the analyses on both large-field RGCs and small-field midget RGCs. The latter neurons are unique to primates. We found that at the single cell level the electric potential gradient in conjunction with neuronal element excitability, rather than the electrode center location per se, determined the response threshold and latency. In addition, stimulus positioning strongly influenced the location of RGC response initiation and subsequent activity propagation through the cellular structure. These findings were robust with respect to inhomogeneous tissue resistivity perpendicular to the electrode plane. At the population level, RGC cellular structures gave rise to low threshold hotspots, which limited axonal and multi-cell activation with threshold stimuli. Finally, due to variations in neuronal element excitability over space, following supra-threshold stimulation some locations favored localized activation of multiple cells, while others favored axonal activation of cells over extended space.     

Pathway-specific genetic attenuation of glutamate release alters select features of competition-based visual circuit refinement

A hallmark of mammalian neural circuit development is the refinement of initially imprecise connections by competitive activity-dependent processes. In the developing visual system retinal ganglion cell (RGC) axons from the two eyes undergo activity-dependent competition for territory in the dorsal lateral geniculate nucleus (dLGN). The direct contributions of synaptic transmission to this process, however, remain unclear. We used a genetic approach to reduce glutamate release selectively from ipsilateral-projecting RGCs and found that their release-deficient axons failed to exclude competing axons from the ipsilateral eye territory in the dLGN. Nevertheless, the release-deficient axons consolidated and maintained their normal amount of dLGN territory, even in the face of fully active competing axons. These results show that during visual circuit refinement glutamatergic transmission plays a direct role in excluding competing axons from inappropriate target regions, but they argue that consolidation and maintenance of axonal territory are largely insensitive to alterations in synaptic activity levels.     

Timing and specificity of feed-forward inhibition within the LGN

Local interneurons provide feed-forward inhibition from retinal ganglion cells (RGCs) to thalamocortical (TC) neurons, but questions remain regarding the timing, magnitude, and functions of this inhibition. Here, we identify two types of inhibition that are suited to play distinctive roles. We recorded excitatory and inhibitory postsynaptic currents (EPSCs/IPSCs) in TC neurons in mouse brain slices and activated individual RGC inputs. In 34% of TC neurons, we identified EPSCs and IPSCs with identical thresholds that were tightly correlated, indicating activation by the same RGC. Such "locked" IPSCs occurred 1 ms after EPSC onset. The remaining neurons had only "nonlocked" inhibition, in which EPSCs and IPSCs had different thresholds, indicating activation by different RGCs. Nonlocked inhibition may refine receptive fields within the LGN by providing surround inhibition. In contrast, dynamic-clamp recordings suggest that locked inhibition improves the precision of synaptically evoked responses in individual TC neurons by eliminating secondary spikes.     

‘Communication’ in weakly electric fish, Gnathonemus niger (Mormyridae) I. Variation of electric organ discharge (EOD) frequency elicited by controlled electric stimuli

The weakly electric fish, Gathonemus niger, discharged with a frequency of 4 to 8 Hz during the day and 10 to 16 Hz during the night. The frequency of superimposed burst discharges (32 to 56 Hz) was independent of diurnal factors. The variation of the electric organ discharge frequency during the day was investigated in response to controlled electric stimulus patterns: (a) A free running stimulus frequency of 4 Hz, simulating the resting frequency of another fish, and different stimulus intensities, simulating different distances between two fish. (b) Free running frequencies of 4, 8, 16, …, 128 Hz and two particular stimulus intensities. (c) Discharge coupled stimuli (each discharge triggered an electric stimulus with a fixed delay) and different stimulus intensities.All three kinds of stimuli elicited defined and predictable response discharge patterns supporting the assumption that an electric fish would respond to a particular discharge pattern of another fish also in a similar and predictable manner. Low stimulus intensities (0·04 to 0·2 mV per cm) caused cessation of the discharge activity, a ‘hiding’ or ‘listening’ response. The discharge rate increased linearly with the logarithm of the stimulus intensity. The fish was particularly sensitive to stimulus frequencies which simulated its burst activity (32 to 56 Hz). Discharge coupled stimuli showed that the fish responded to about eight times lower stimulus intensities if the stimulus occurred between two discharges (15 to 30 m-s after the fish's discharge) than if the stimulus occurred within or immediately after the discharge. All suprathreshold stimuli elicited a typical discharge pattern: The irregular resting discharge activity became significantly regular. The degree of regularity was even improved during maintained stimulation. The regularisation of the discharge activity is thought to be involved in the fish's electrolocating system whereas frequency variations are considered as being involved in both the locating system and as communication signals among weakly electric fish.     

Modeling activity and target-dependent developmental cell death of mouse retinal ganglion cells ex vivo

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death.     

The role of laminin and the laminin/fibronectin receptor complex in the outgrowth of retinal ganglion cell axons

Chick embryo retinal ganglion cell (RGC) axons grow to the optic tectum along a stereotyped route, as if responding to cues distributed along the pathway. We showed previously that, in culture, RGCs from embryonic Day 6 retina are responsive to the neurite-promoting effects of the extracellular matrix glycoprotein laminin and that this response is lost by RGCs at a later stage of development. Here we report that, before axon outgrowth is initiated in vivo, laminin, is expressed along the optic pathway at nonbasal lamina sites that are accessible to the growth cones of RGC axons. The distribution of laminin within the pathway is consistent with its localization at the end-feet of neuroepithelial cells that line the route, and it continues to be expressed at these marginal sites during the first week of embryonic development. At later stages, concomitant with the loss of response by RGCs in culture, laminin becomes restricted to basal laminae at the retinal inner limiting membrane and pial surface of the optic pathway. Neurofilament-positive RGC axons bind a monoclonal antibody, JG22, which recognizes the laminin/fibronectin receptor complex, and continue to do so throughout embryonic development. We show that, in vitro, the JG22 antigen expressed by RGCs appears to function as a laminin receptor, by demonstrating that JG22 antibody blocks neurite outgrowth on a substrate of laminin. These findings are consistent with the possibility that laminin defines a transient performed pathway specifically recognized by early RGC growth cones as they navigate toward their central target.     

Vision drives correlated activity without patterned spontaneous activity in developing Xenopus retina

Developing amphibians need vision to avoid predators and locate food before visual system circuits fully mature. Xenopus tadpoles can respond to visual stimuli as soon as retinal ganglion cells (RGCs) innervate the brain, however, in mammals, chicks and turtles, RGCs reach their central targets many days, or even weeks, before their retinas are capable of vision. In the absence of vision, activity-dependent refinement in these amniote species is mediated by waves of spontaneous activity that periodically spread across the retina, correlating the firing of action potentials in neighboring RGCs. Theory suggests that retinorecipient neurons in the brain use patterned RGC activity to sharpen the retinotopy first established by genetic cues. We find that in both wild type and albino Xenopus tadpoles, RGCs are spontaneously active at all stages of tadpole development studied, but their population activity never coalesces into waves. Even at the earliest stages recorded, visual stimulation dominates over spontaneous activity and can generate patterns of RGC activity similar to the locally correlated spontaneous activity observed in amniotes. In addition, we show that blocking AMPA and NMDA type glutamate receptors significantly decreases spontaneous activity in young Xenopus retina, but that blocking GABA(A) receptor blockers does not. Our findings indicate that vision drives correlated activity required for topographic map formation. They further suggest that developing retinal circuits in the two major subdivisions of tetrapods, amphibians and amniotes, evolved different strategies to supply appropriately patterned RGC activity to drive visual circuit refinement.     

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.     

Effect of p75NTR on the regulation of naturally occurring cell death and retinal ganglion cell number in the mouse eye

Neurotrophins induce neural cell survival and differentiation during retinal development and regeneration through the high-affinity tyrosine kinase (Trk) receptors. On the other hand, nerve growth factor (NGF) binding to the low-affinity neurotrophin receptor p75 (p75(NTR)) might induce programmed cell death (PCD) in the early phase of retinal development. In the present study, we examined the retinal cell types that experience p75(NTR)-induced PCD and identify them to be postmitotic retinal ganglion cells (RGCs). However, retinal morphology, RGC number, and BrdU-positive cell number in p75(NTR) knockout (KO) mouse were normal after embryonic day 15 (E15). In chick retina, migratory RGCs express p75(NTR), whereas layered RGCs express the high-affinity NGF receptor TrkA, which may switch the pro-apoptotic signaling of p75(NTR) into a neurotrophic one. In contrast to the chick model, migratory RGCs express TrkA, while stratified RGCs express p75(NTR) in mouse retina. However, RGC number in TrkA KO mouse was also normal at birth. We next examined the expression of transforming growth factor beta (TGFbeta) receptor, which modulates chick RGC number in combination with p75(NTR), but was absent in mouse RGCs. p75(NTR) and TrkA seem to be involved in the regulation of mouse RGC number in the early phase of retinal development, but the number may be later adjusted by other molecules. These results suggest the different mechanism of RGC number control between mouse and chick retina.     

Developmental mechanisms that regulate retinal ganglion cell dendritic morphology

One of the fundamental features of retinal ganglion cells (RGCs) is that dendrites of individual RGCs are confined to one or a few narrow strata within the inner plexiform layer (IPL), and each RGC synapses only with a small group of presynaptic bipolar and amacrine cells with axons/dendrites ramified in the same strata to process distinct visual features. The underlying mechanisms which control the development of this laminar-restricted distribution pattern of RGC dendrites have been extensively studied, and it is still an open question whether the dendritic pattern of RGCs is determined by molecular cues or by activity-dependent refinement. Accumulating evidence suggests that both molecular cues and activity-dependent refinement might regulate RGC dendrites in a cell subtype-specific manner. However, identification of morphological subtypes of RGCs before they have achieved their mature dendritic pattern is a major challenge in the study of RGC dendritic development. This problem is now being circumvented through the use of molecular markers in genetically engineered mouse lines to identify RGC subsets early during development. Another unanswered fundamental question in the study of activity-dependent refinement of RGC dendrites is how changes in synaptic activity lead to the changes in dendritic morphology. Recent studies have started to shed light on the molecular basis of activity-dependent dendritic refinement of RGCs by showing that some molecular cascades control the cytoskeleton reorganization of RGCs.     

Formation of functional synapses by regenerating adult rat retinal ganglion cell axons in midbrain target regions in vitro

The ability of adult rat retinal ganglion cell (RGC) axons to reinnervate normal target regions was examined in vitro. In co-culture experiments, adult rat retinal explants were placed adjacent to fetal rat midbrain sections that contained the superior colliculus (SC) which is the main target for RGC axons. Adult rat RGCs regrew axons over more than 500 μm on a polylysine-laminin substrate to reach the co-cultured explants. By using neurofilament immunohistochemistry and the fluorescent dye Dil for anterograde and retrograde tracing, it was shown that (1) adult rat RGCs with a stereotyped morphology survived in explant cultures for more than 4 weeks in the presence of fetal midbrain explants, (2) regenerating RGC axons preferentially terminated within midbrain target regions, and (3) RGCs formed functional synapses. In addition, the maturation of the SC region in midbrain explants was examined histologically and ultrastructurally to demonstrate appropiate target development. © 1993 John Wiley & Sons, Inc.     

In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.     

Light-induced plasticity of synaptic AMPA receptor composition in retinal ganglion cells

Light-evoked responses of all three major classes of?retinal ganglion cells (RGCs) are mediated by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Although synaptic activity at RGC synapses is highly dynamic, synaptic plasticity has not been observed in adult RGCs. Here, using patch-clamp recordings in dark-adapted mouse retina, we report a retina-specific form of AMPAR plasticity. Both chemical and light activation of NMDARs caused the selective endocytosis of GluA2-containing, Ca(2+)-impermeable AMPARs on RGCs and replacement with GluA2-lacking, Ca(2+)-permeable AMPARs. The plasticity was expressed in ON but not OFF RGCs and was restricted solely to the ON responses in ON-OFF RGCs. Finally, the plasticity resulted in a shift in the light responsiveness of ON RGCs. Thus, physiologically relevant light stimuli can induce a change in synaptic receptor composition of ON RGCs, providing a mechanism by which the sensitivity of RGC responses may be modified under scotopic conditions.     

VIP-mediated increase in cAMP prevents tetrodotoxin-induced retinal ganglion cell death in vitro

Afferent influences on natural cell death were modeled in retinal cultures derived from neonatal rats. Tetrodotoxin (TTX) blockade of electrical activity produced a significant reduction in surviving retinal ganglion cell (RGC) neurons during a critical period of development, similar in magnitude to the reduction observed during natural cell death in the intact retina at a similar developmental stage. The addition of vasoactive intestinal peptide (VIP) protected the RGCs from the lethal action of TTX. This effect was specific, since the related peptides PHI-27 and secretin produced no significant increase in RGC survival. Radioimmunoassay of cyclic nucleotides showed that TTX decreased culture levels of cAMP and that this trend was reversed by VIP. Decreases in RGC survival associated with TTX electrical blockade were prevented by 8-bromo:cAMP or forskolin. Furthermore, VIP10-28, the C-terminal fragment that inhibits VIP stimulation of adenylate cyclase, reduced the number of surviving RGCs. Thus, our results suggest that VIP, acting by increasing cAMP, has a neurotrophic effect on electrically blocked RGCs and may be an endogenous factor modulating normal cell death in the retina.     

Rhythmic Ganglion Cell Activity in Bleached and Blind Adult Mouse Retinas

In retinitis pigmentosa – a degenerative disease which often leads to incurable blindness- the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor’s dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the understanding of the degeneration process and may guide future rescue strategies.     

Low response variability in simultaneously recorded retinal, thalamic, and cortical neurons

The response of a cortical cell to a repeated stimulus can be highly variable from one trial to the next. Much lower variability has been reported of retinal cells. We recorded visual responses simultaneously from three successive stages of the cat visual system: retinal ganglion cells (RGCs), thalamic (LGN) relay cells, and simple cells in layer 4 of primary visual cortex. Spike count variability was lower than that of a Poisson process at all three stages but increased at each stage. Absolute and relative refractory periods largely accounted for the reliability at all three stages. Our results show that cortical responses can be more reliable than previously thought. The differences in reliability in retina, LGN, and cortex can be explained by (1) decreasing firing rates and (2) decreasing absolute and relative refractory periods.