Ganglion Cell Dendrites Research Articles

Phosphorylated α-synuclein in the retina is a biomarker of Parkinson's disease pathology severity.

PD patients often have visual alterations, for example, loss of visual acuity, contrast sensitivity or motion perception, and diminished electroretinogram responses. PD pathology is mainly characterized by the accumulation of pathological α-synuclein deposits in the brain, but little is known about how synucleinopathy affects the retina. To study the correlation between α-synuclein deposits in the retina and brain of autopsied subjects with PD and incidental Lewy body disease. We evaluated the presence of phosphorylated α-synuclein in the retina of autopsied subjects with PD (9 subjects), incidental Lewy body disease (4 subjects), and controls (6 subjects) by immunohistochemistry and compared the retinal synucleinopathy with brain disease severity indicators. Whereas controls did not show any phosphorylated α-synuclein immunoreactivity in their retina, all PD subjects and 3 of 4 incidental Lewy body disease subjects had phosphorylated α-synuclein deposits in ganglion cell perikarya, dendrites, and axons, some of them resembling brain Lewy bodies and Lewy neurites. The Lewy-type synucleinopathy density in the retina significantly correlated with Lewy-type synucleinopathy density in the brain, with the Unified Parkinson's disease pathology stage and with the motor UPDRS. These data suggest that phosphorylated α-synuclein accumulates in the retina in parallel with that in the brain, including in early stages preceding development of clinical signs of parkinsonism or dementia. Therefore, the retina may provide an in vivo indicator of brain pathology severity, and its detection could help in the diagnosis and monitoring of disease progression. © 2018 International Parkinson and Movement Disorder Society.

AB015. Metabolic stress in glaucoma engages early activation of the energy biosensor adenosine monophosphate-activated protein kinase leading to neuronal dysfunction

Background: Metabolic stress has been proposed to contribute to neuronal damage in glaucoma, but the mechanism driving this response is not understood. The adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis that becomes active at the onset of energy stress. AMPK is a potent inhibitor of the mammalian target of rapamycin complex 1 (mTORC1), which we showed is essential for the maintenance of retinal ganglion cell (RGC) dendrites, synapses, and survival. Here, we tested the hypothesis that AMPK is an early mediator of metabolic stress in glaucoma.

Detecting Structural Progression in Glaucoma with Optical Coherence Tomography

Optical coherence tomography (OCT) is increasingly used to obtain objective measurements of the retinal nerve fiber layer (RNFL), optic nerve head, and macula for assessing glaucoma progression. Although OCT has been adopted widely in clinical practice, uncertainty remains concerning its optimal role. Questions include: What is the best structure to measure? What quantity of change is significant? Are structural changes relevant to the patient? How are longitudinal measurements affected by aging? How can changes resulting from aging be differentiated from true progression? How best should OCT be used alongside visual fields, and how often should OCT be performed? Recent studies have addressed some of these questions. Important developments include appreciation of the need to use a consistent point of reference for structural measurements, leading to the introduction of Bruch's membrane opening (BMO)-based measurements, including BMO-minimum rim width and BMO-minimum rim area. Commercially available OCT devices also permit analysis of macular changes over time, for example, changes in the ganglion cell and inner plexiform layers, the sites of the retinal ganglion cell bodies and dendrites, respectively. Several longitudinal studies have compared rates of change in RNFL and macular measurements, with some suggesting that the relative value of each parameter may differ at different stages of disease. In early disease, looking for change over time also may be useful for glaucoma diagnosis, with advantages over classifying eyes using cross-sectional normative databases. Optimal glaucoma management requires information from imaging and visual fields, and efforts have been made to combine information, reducing the noise inherent in both tests to benefit from their different performances according to the stage of disease. Combining information from different structural measurements may also be useful. There is now substantial evidence that progressive structural changes are of direct clinical relevance, with progressive changes on OCT often preceding functional loss and patients with faster change on OCT at increased risk of worsening visual losses. Identification of such patients offers the possibility of commencing or escalating treatment at an earlier stage. This review appraises recent developments in the use of OCT for assessing glaucoma progression.

Wavy multistratified amacrine cells in the monkey retina contain immunoreactive secretoneurin

The goals of this study were to describe the morphology, neurotransmitter content and synaptic connections of neurons in primate retinas that contain the neuropeptide secretoneurin. Amacrine cells were labeled with antibodies to secretoneurin in macaque and baboon retinas. Their processes formed three distinct plexuses in the inner plexiform layer: one in the outermost stratum, one in the center and one in the innermost stratum. In light microscopic double immunolabeling experiments, GABA was colocalized with secretoneurin in these cells, but glycine transporter 1 and Substance P were not. ON bipolar cell axon terminals labeled with antibody to the cholecystokinin precursor, G6-gly, have ON responses to stimulation of short wavelength sensitive (S) cones. Axons of these bipolar cells made contacts with amacrine cell dendrites containing secretoneurin. Secretoneurin-IR amacrine cells also made contacts with retinal ganglion cell dendrites labeled with antibody to the photopigment melanopsin, which have OFF responses to stimulation of S cones. Using electron microscopic immunolabeling, 436 synapses from macaque retina were analyzed. Axons from bipolar cells were identified by their characteristic synaptic ribbons; their synaptic densities were asymmetric like those of excitatory synapses in the brain. Amacrine cells made and received conventional synapses with symmetric synaptic densities, like those of inhibitory synapses in the brain. Ganglion cell dendrites were identified by their absence of presynaptic specializations; they received inputs from both amacrine cells and bipolar cells. The majority of inputs to the secretoneurin-IR amacrine cells were from other amacrine cells, but they also received 21% of their input from bipolar cells. They directed most of their output, 54%, to amacrine cells, but there were many synapses onto bipolar cell axons and ganglion cell dendrites, as well. The synaptic connections were very similar in the three plexuses with one notable exception; output synapses to bipolar cells were significantly less common in the innermost one, where the S-ON bipolar cells terminate. Taken together, these findings suggest that the secretoneurin-IR amacrine cells in primates receive excitatory input from S-ON bipolar cells and, in turn, inhibit intrinsically photosensitive retinal ganglion cells.

Inhibition of the classical pathway of the complement cascade prevents early dendritic and synaptic degeneration in glaucoma.

BackgroundGlaucoma is a complex, multifactorial disease characterised by the loss of retinal ganglion cells and their axons leading to a decrease in visual function. The earliest events that damage retinal ganglion cells in glaucoma are currently unknown. Retinal ganglion cell death appears to be compartmentalised, with soma, dendrite and axon changes potentially occurring through different mechanisms. There is mounting evidence from other neurodegenerative diseases suggesting that neuronal dendrites undergo a prolonged period of atrophy, including the pruning of synapses, prior to cell loss. In addition, recent evidence has shown the role of the complement cascade in synaptic pruning in glaucoma and other diseases.ResultsUsing a genetic (DBA/2J mouse) and an inducible (rat microbead) model of glaucoma we first demonstrate that there is loss of retinal ganglion cell synapses and dendrites at time points that precede axon or soma loss. We next determine the role of complement component 1 (C1) in early synaptic loss and dendritic atrophy during glaucoma. Using a genetic knockout of C1qa (D2.C1qa-/- mouse) or pharmacological inhibition of C1 (in the rat bead model) we show that inhibition of C1 is sufficient to preserve dendritic and synaptic architecture.ConclusionsThis study further supports assessing the potential for complement-modulating therapeutics for the prevention of retinal ganglion cell degeneration in glaucoma.

Dendritic mitochondria reach stable positions during circuit development.

Mitochondria move throughout neuronal dendrites and localize to sites of energy demand. The prevailing view of dendritic mitochondria as highly motile organelles whose distribution is continually adjusted by neuronal activity via Ca(2+)-dependent arrests is based on observations in cultured neurons exposed to artificial stimuli. Here, we analyze the movements of mitochondria in ganglion cell dendrites in the intact retina. We find that whereas during development 30% of mitochondria are motile at any time, as dendrites mature, mitochondria all but stop moving and localize stably to synapses and branch points. Neither spontaneous nor sensory-evoked activity and Ca(2+) transients alter motility of dendritic mitochondria; and pathological hyperactivity in a mouse model of retinal degeneration elevates rather than reduces motility. Thus, our findings indicate that dendritic mitochondria reach stable positions during a critical developmental period of high motility, and challenge current views about the role of activity in regulating mitochondrial transport in dendrites.

NF-Protocadherin Regulates Retinal Ganglion Cell Axon Behaviour in the Developing Visual System.

Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into the final target area. Collectively, our results expand our understanding of the role of NFPC in RGC guidance and illustrate that this adhesion molecule contributes to axon behaviour at multiple points in the optic pathway.

In vivo imaging methods to assess glaucomatous optic neuropathy

The goal of this review is to summarize the most common imaging methods currently applied for in vivo assessment of ocular structure in animal models of experimental glaucoma with an emphasis on translational relevance to clinical studies of the human disease. The most common techniques in current use include optical coherence tomography and scanning laser ophthalmoscopy. In reviewing the application of these and other imaging modalities to study glaucomatous optic neuropathy, this article is organized into three major sections: 1) imaging the optic nerve head, 2) imaging the retinal nerve fiber layer and 3) imaging retinal ganglion cell soma and dendrites. The article concludes with a brief section on possible future directions.

Differential protection of injured retinal ganglion cell dendrites by brimonidine.

To determine whether brimonidine protects against the retraction and loss of retinal ganglion cell (RGC) dendrites after optic nerve crush (ONC). Fluorescent RGCs of mice expressing yellow fluorescent protein (YFP) under the control of the Thy-1 promoter (Thy1-YFP mice) were imaged in vivo and assigned to one of six groups according to dendrite structure. The mice then received brimonidine every other day starting 2 days before, or 2 or 6 days after, unilateral ONC. Control animals received vehicle every other day starting 2 days before ONC. Control animals received vehicle every other day starting 2 days before ONC. Total dendrite length, dendrite branching complexity, and the time until complete loss of dendrites were assessed weekly for 4 weeks. Overall, brimonidine treatment significantly slowed the complete loss of RGC dendrites and significantly slowed the reduction of total dendrite length and branching complexity. Separate analysis of each RGC group showed brimonidine significantly delayed the time until complete loss of dendrites in four of the RGC groups. These delays generally were similar when treatment started either 2 days before or 2 days after ONC, but were smaller or absent when treatment started 6 days after ONC Protection against loss of total dendrite length and loss of branching complexity was observed in three of the RGC groups. In two of these RGC groups, protective effects persisted until the end of the study. Brimonidine protects many RGC types against dendrite retraction, loss of branching complexity, and complete loss of dendrites following ONC. However, the pattern and magnitude of this protection differs substantially among different RGC types. These results indicate that requirements for RGC-protective therapies following optic nerve injury may differ among RGC types.

Protection of injured retinal ganglion cell dendrites and unfolded protein response resolution after long-term dietary resveratrol

Long-term dietary supplementation with resveratrol protects against cardiovascular disease, osteoporesis, and metabolic decline. This study determined how long-term dietary resveratrol treatment protects against retinal ganglion cell (RGC) dendrite loss after optic nerve injury and alters the resolution of the unfolded protein response. Associated changes in markers of endoplasmic reticulum stress in RGCs also were investigated. Young-adult Thy1-yellow fluorescent protein (YFP) and C57BL/6 mice received either control diet or diet containing resveratrol for approximately 1 year. Both groups then received optic nerve crush (ONC). Fluorescent RGC dendrites in the Thy1-YFP mice were imaged weekly for 4 weeks after ONC. There was progressive loss of dendrite length in all RGC types within the mice that received control diet. Resveratrol delayed loss of dendrite complexity and complete dendrite loss for most RGC types. However, there were variations in the rate of retraction among different RGC types. Three weeks after ONC, cytoplasmic binding immunoglobulin protein (BiP) suppression observed in control diet ganglion cell layer neurons was reversed in mice that received resveratrol, nuclear C/EBP homologous protein (CHOP) was near baseline in control diet eyes but was moderately increased by resveratrol; and increased nuclear X-box–binding protein-1 (XBP-1) observed in control diet eyes was reduced in eyes that received resveratrol to the same level as in control diet uncrushed eyes. These results indicate that protection of dendrites by resveratrol after ONC differs among RGC types and suggest that alterations in long-term expression of binding immunoglobulin protein, CHOP, and XBP-1 may contribute to the resveratrol-mediated protection of RGC dendrites after ONC.

Melanopsin ganglion cells extend dendrites into the outer retina during early postnatal development.

Melanopsin ganglion cells express the photopigment melanopsin and are the first functional photoreceptors to develop in the mammalian retina. They have been shown to play a variety of important roles in visual development and behavior in the early postnatal period (Johnson et al., 2010; Kirkby and Feller, 2013; Rao et al., 2013; Renna et al., 2011). Here, we probed the maturation of the dendritic arbors of melanopsin ganglion cells during this developmental period in mice. We found that some melanopsin ganglion cells (mainly the M1-subtype) transiently extend their dendrites not only into the inner plexiform layer (where they receive synaptic inputs from bipolar and amacrine cells) but also into the outer plexiform layer, where in mature retina, rod and cone photoreceptors are thought to contact only bipolar and horizontal cells. Thus, some immature melanopsin ganglion cells are biplexiform. This feature is much less common although still present in the mature retina. It reaches peak incidence 8-12 days after birth, before the eyes open and bipolar cells are sufficiently mature to link rods and cones to ganglion cells. At this age, some outer dendrites of melanopsin ganglion cells lie in close apposition to the axon terminals of cone photoreceptors and express a postsynaptic marker of glutamatergic transmission, postsynaptic density-95 protein (PSD-95). These findings raise the possibility of direct, monosynaptic connections between cones and melanopsin ganglion cells in the early postnatal retina. We provide a detailed description of the developmental profile of these processes and consider their possible functional and evolutionary significance.

Short-term increases in transient receptor potential vanilloid-1 mediate stress-induced enhancement of neuronal excitation.

Progression of neurodegeneration in disease and injury is influenced by the response of individual neurons to stressful stimuli and whether this response includes mechanisms to counter declining function. Transient receptor potential (TRP) cation channels transduce a variety of disease-relevant stimuli and can mediate diverse stress-dependent changes in physiology, both presynaptic and postsynaptic. Recently, we demonstrated that knock-out or pharmacological inhibition of the TRP vanilloid-1 (TRPV1) capsaicin-sensitive subunit accelerates degeneration of retinal ganglion cell neurons and their axons with elevated ocular pressure, the critical stressor in the most common optic neuropathy, glaucoma. Here we probed the mechanism of the influence of TRPV1 on ganglion cell survival in mouse models of glaucoma. We found that induced elevations of ocular pressure increased TRPV1 in ganglion cells and its colocalization at excitatory synapses to their dendrites, whereas chronic elevation progressively increased ganglion cell Trpv1 mRNA. Enhanced TRPV1 expression in ganglion cells was transient and supported a reversal of the effect of TRPV1 on ganglion cells from hyperpolarizing to depolarizing, which was also transient. Short-term enhancement of TRPV1-mediated activity led to a delayed increase in axonal spontaneous excitation that was absent in ganglion cells from Trpv1(-/-) retina. In isolated ganglion cells, pharmacologically activated TRPV1 mobilized to discrete nodes along ganglion cell dendrites that corresponded to sites of elevated Ca(2+). These results suggest that TRPV1 may promote retinal ganglion cell survival through transient enhancement of local excitation and axonal activity in response to ocular stress.

REDD2-mediated inhibition of mTOR promotes dendrite retraction induced by axonal injury.

Dendritic defects occur in neurodegenerative diseases accompanied by axonopathy, yet the mechanisms that regulate these pathologic changes are poorly understood. Using Thy1-YFPH mice subjected to optic nerve axotomy, we demonstrate early retraction of retinal ganglion cell (RGC) dendrites and selective loss of mammalian target of rapamycin (mTOR) activity, which precede soma loss. Axonal injury triggered rapid upregulation of the stress-induced protein REDD2 (regulated in development and DNA damage response 2), a potent inhibitor of mTOR. Short interfering RNA-mediated REDD2 knockdown restored mTOR activity and rescued dendritic length, area and branch complexity in a rapamycin-dependent manner. Whole-cell recordings demonstrated that REDD2 depletion leading to mTOR activation in RGCs restored their light response properties. Lastly, we show that REDD2-dependent mTOR activity extended RGC survival following axonal damage. These results indicate that injury-induced stress leads to REDD2 upregulation, mTOR inhibition and dendrite pathology causing neuronal dysfunction and subsequent cell death.

Alterations of the synapse of the inner retinal layers after chronic intraocular pressure elevation in glaucoma animal model

BackgroundDendrites of retinal ganglion cells (RGCs) synapse with axon terminals of bipolar cells in the inner plexiform layer (IPL). Changes in RGC dendrites and synapses between bipolar cells in the inner retinal layer may critically alter the function of RGCs in glaucoma. Recently, synaptic plasticity has been observed in the adult central nervous system, including the outer retinal layers. However, few studies have focused on changes in the synapses between RGCs and bipolar cells in glaucoma. In the present study, we used a rat model of ocular hypertension induced by episcleral vein cauterization to investigate changes in synaptic structure and protein expression in the inner retinal layer at various time points after moderate intraocular pressure (IOP) elevation.ResultsSynaptophysin, a presynaptic vesicle protein, increased throughout the IPL, outer plexiform layer, and outer nuclear layer after IOP elevation. Increased synaptophysin after IOP elevation was expressed in bipolar cells in the innermost IPL. The RGC marker, SMI-32, co-localized with synaptophysin in RGC dendrites and were significantly increased at 1 week and 4 weeks after IOP elevation. Both synaptophysin and postsynaptic vesicle protein, PSD-95, were increased after IOP elevation by western blot analysis. Ribbon synapses in the IPL were quantified and structurally evaluated in retinal sections by transmission electron microscopy. After IOP elevation the total number of ribbon synapses decreased. There were increases in synapse diameter and synaptic vesicle number and decreases in active zone length and the number of docked vesicles after IOP elevation.ConclusionsAlthough the total number of synapses decreased as RGCs were lost after IOP elevation, there are attempts to increase synaptic vesicle proteins and immature synapse formation between RGCs and bipolar cells in the inner retinal layers after glaucoma induction.

Kainate Receptors Mediate Signaling in Both Transient and Sustained OFF Bipolar Cell Pathways in Mouse Retina

A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.

Rapid Contrast Adaptation in Glaucoma and in Aging

The visual system rapidly adapts to contrast changes, often with each fixation. One key anatomical site underpinning contrast adaptation is the retinal ganglion cell dendrites, where degenerative changes occur in glaucoma. This study investigated the effects of early glaucoma and aging on rapid contrast adaptation. Contrast detection and discrimination thresholds were measured in central vision for briefly presented (94 ms) Gabor patches with and without adaptation to 50% contrast Gabor patches (1000 ms). Fourteen people with glaucoma (aged 58-77 years), 17 age-similar controls (aged 50-72 years), and 19 younger adults (aged 20-31 years) participated. Detection thresholds were measured at various time points (47, 106, 200, 400, 600, and 1000 ms) post adaptation. Discrimination thresholds were measured post adaptation relative to a reference contrast below (30%), equivalent to (50%), or above (70%) the adaptor. The glaucoma group demonstrated elevated unadapted detection (P < 0.0001) and discrimination (P = 0.01) thresholds relative to age-similar controls. In normal observers, aging elevated unadapted thresholds (detection: P < 0.0001; discrimination: P < 0.0001). At 47 ms post adaptation, the glaucoma group demonstrated reduced effects of adaptation relative to controls (P = 0.009). Adaptation was also reduced when the reference contrast (50%) was equivalent to the adaptor (P = 0.02). Aging did not alter adaptation of normal observers. Glaucoma alters rapid contrast adaptation while aging does not. Contrast adaptation is key to visual processing in natural visual environments. Our results imply that glaucoma produces abnormalities in natural visual experiences in central vision.

Identification of a Pathway from the Retina to Koniocellular Layer K1 in the Lateral Geniculate Nucleus of Marmoset

Three well characterized pathways in primate vision (midget-parvocellular, parasol-magnocellular, bistratified-koniocellular) have been traced from the first synapse in the retina, through the visual thalamus (lateral geniculate nucleus, LGN), to the visual cortex. Here we identify a pathway from the first synapse in the retina to koniocellular layer K1 in marmoset monkeys (Callithrix jacchus). Particle-mediated gene transfer of an expression plasmid for the postsynaptic density 95-green fluorescent protein (PSD95-GFP) was used to label excitatory synapses on retinal ganglion cells and combined with immunofluorescence to identify the presynaptic bipolar cells. We found that axon terminals of one type of diffuse bipolar cell (DB6) provide dominant synaptic input to the dendrites of narrow thorny ganglion cells. Retrograde tracer injections into the LGN and photofilling of retinal ganglion cells showed that narrow thorny cells were preferentially labeled when koniocellular layer K1 was targeted. Layer K1 contains cells with high sensitivity for rapid movement, and layer K1 sends projections to association visual areas as well as to primary visual cortex. We hypothesize that the DB6-narrow thorny-koniocellular pathway contributes to residual visual functions ("blindsight") that survive injury to primary visual cortex in adult or early life.

The effect of photoreceptor degeneration on ganglion cell morphology

Retinitis pigmentosa refers to a family of inherited photoreceptor degenerations resulting in blindness. During and after photoreceptor loss, neurons of the inner retina are known to undergo plastic changes. Here, we have investigated in detail whether ganglion cells are altered at late stages of degeneration, well after the total loss of photoreceptors. We used mice, rd1-Thy1, that carry a mutation in the β-subunit of phosphodiesterase 6 and a fluorescent protein that labels a subset of ganglion cells and B6-Thy1 control mice. Retinal wholemounts from mice aged 3-11 months were processed for immunohistochemistry and analyzed. Ganglion cells were classified based on soma area, dendritic field size, and branching of dendrites. The dendritic fields of some ganglion cells were further analyzed for their length, area and quantity of branching points. There was a decrease in size and level of branching of A2, B1, and D type ganglion cells in the degenerated retina at 11 months of age. In contrast, C1 ganglion cells remained unchanged. In addition, there was a shift in the proportion of ganglion cells ramifying in the different layers of the inner plexiform layer. Careful analysis of the dendrites of ganglion cells revealed some projecting to new, more distal regions of the inner plexiform layer. We propose that these changes in ganglion cell morphology could impact the function of individual cells as well as the retinal circuitry in the degenerated retina.

Spatial Distribution of Excitatory Synapses on the Dendrites of Ganglion Cells in the Mouse Retina

Excitatory glutamatergic inputs from bipolar cells affect the physiological properties of ganglion cells in the mammalian retina. The spatial distribution of these excitatory synapses on the dendrites of retinal ganglion cells thus may shape their distinct functions. To visualize the spatial pattern of excitatory glutamatergic input into the ganglion cells in the mouse retina, particle-mediated gene transfer of plasmids expressing postsynaptic density 95-green fluorescent fusion protein (PSD95-GFP) was used to label the excitatory synapses. Despite wide variation in the size and morphology of the retinal ganglion cells, the expression of PSD95 puncta was found to follow two general rules. Firstly, the PSD95 puncta are regularly spaced, at 1–2 µm intervals, along the dendrites, whereby the presence of an excitatory synapse creates an exclusion zone that rules out the presence of other glutamatergic synaptic inputs. Secondly, the spatial distribution of PSD95 puncta on the dendrites of diverse retinal ganglion cells are similar in that the number of excitatory synapses appears to be less on primary dendrites and to increase to a plateau on higher branch order dendrites. These observations suggest that synaptogenesis is spatially regulated along the dendritic segments and that the number of synaptic contacts is relatively constant beyond the primary dendrites. Interestingly, we also found that the linear puncta density is slightly higher in large cells than in small cells. This may suggest that retinal ganglion cells with a large dendritic field tend to show an increased connectivity of excitatory synapses that makes up for their reduced dendrite density. Mapping the spatial distribution pattern of the excitatory synapses on retinal ganglion cells thus provides explicit structural information that is essential for our understanding of how excitatory glutamatergic inputs shape neuronal responses.

The First Stage of Cardinal Direction Selectivity Is Localized to the Dendrites of Retinal Ganglion Cells

Inferring the direction of image motion is a fundamental component of visual computation and essential for visually guided behavior. In the retina, the direction of image motion is computed in four cardinal directions, but it is not known at which circuit location along the flow of visual information the cardinal direction selectivity first appears. We recorded the concerted activity of the neuronal circuit elements of single direction-selective (DS) retinal ganglion cells at subcellular resolution by combining GCaMP3-functionalized transsynaptic viral tracing and two-photon imaging. While the visually evoked activity of the dendritic segments of the DS cells were direction selective, direction-selective activity was absent in the axon terminals of bipolar cells. Furthermore, the glutamate input to DS cells, recorded using a genetically encoded glutamate sensor, also lacked direction selectivity. Therefore, the first stage in which extraction of a cardinal motion direction occurs is the dendrites of DS cells.