The rod synapse in aging wildtype and Dscaml1 mutant mice.

The retina is an intricately organized neural tissue built on cone and rod pathways for color and night vision. Genetic mutations that disrupt the proper function of the rod circuit contribute to blinding diseases including retinitis pigmentosa and congenital stationary night blindness (CSNB). Down Syndrome cell adhesion molecule like 1 (Dscaml1) is expressed by rods, rod bipolar cells (RBCs), and sub-populations of amacrine cells, and has been linked to a middle age onset of CSNB in humans. However, how Dscaml1 contributes to this visual deficit remains unexplored. Here, we probed Dscaml1's role in the maintenance of the rod-to-RBC synapse using a loss of function mouse model. We used immunohistochemistry to investigate the anatomical formation and maintenance of the rod-to-RBC synapse in the young, adult, and aging retina. We generated 3D reconstructions, using serial electron micrographs, of rod spherules and RBCs to measure the number of invaginating neurites, RBC dendritic tip number, and RBC mitochondrial morphology. We find that while rod-to-RBC synapses form and are maintained, similar to wildtype, that there is an increase in the number of invaginating neurites in rod spherules, a reduction in RBC dendritic tips, and reduced mitochondrial volume and complexity in the Dscaml1 mutant retina compared to controls. We also observed precocious sprouting of RBC dendrites into the outer nuclear layer (ONL) of the Dscaml1 mutant retina compared to controls. These results contribute to our knowledge of Dscaml1's role in rod circuit development and maintenance and give additional insight into possible genetic therapy targets for blinding diseases and disorders like CSNB.

DSCAM gene triplication causes excessive GABAergic synapses in the neocortex in Down syndrome mouse models.

Down syndrome (DS) is caused by the trisomy of human chromosome 21 (HSA21). A major challenge in DS research is to identify the HSA21 genes that cause specific symptoms. Down syndrome cell adhesion molecule (DSCAM) is encoded by a HSA21 gene. Previous studies have shown that the protein level of the Drosophila homolog of DSCAM determines the size of presynaptic terminals. However, whether the triplication of DSCAM contributes to presynaptic development in DS remains unknown. Here, we show that DSCAM levels regulate GABAergic synapses formed on neocortical pyramidal neurons (PyNs). In the Ts65Dn mouse model for DS, where DSCAM is overexpressed due to DSCAM triplication, GABAergic innervation of PyNs by basket and chandelier interneurons is increased. Genetic normalization of DSCAM expression rescues the excessive GABAergic innervations and the increased inhibition of PyNs. Conversely, loss of DSCAM impairs GABAergic synapse development and function. These findings demonstrate excessive GABAergic innervation and synaptic transmission in the neocortex of DS mouse models and identify DSCAM overexpression as the cause. They also implicate dysregulated DSCAM levels as a potential pathogenic driver in related neurological disorders.

Student remote and distance research in neuroanatomy: Mapping Dscaml1 expression with a LacZ gene trap in mouse brain.

Undergraduate student engagement in research increases retention and degree completion, especially for students who are underrepresented in science. Several approaches have been adopted to increase research opportunities including curriculum based undergraduate research opportunities (CUREs), in which research is embedded into course content. Here we report on efforts to tackle a different challenge: providing research opportunities to students engaged in remote learning or who are learning at satellite campuses or community colleges with limited research infrastructure. In our project we engaged students learning remotely or at regional centers to map gene expression in the mouse brain. In this project we mapped expression of the Down syndrome cell adhesion molecule like 1 (Dscaml1) gene in mouse brain using a LacZ expression reporter line. Identifying where Dscaml1 is expressed in the brain is an important next step in determining if its roles in development and function in the retina are conserved in the rest of the brain. Students working remotely reconstruct brain montages and annotated Dscaml1 expression in the brain of mice carrying one or two copies of the gene trap. We built on these findings by further characterizing Dscaml1 expression in inhibitory neurons of the visual pathway. These results build on and extend previous findings and demonstrate the utility of including distance learners in an active research group for both the student learners and the research team. We conclude with best practices we have developed based on this and other distance learner focused projects.

Stakeholder perceptions of the use of a rapidly deployed modified ECHO to train and prepare healthcare providers for COVID-19.

Background: Innovative approaches to deliver timely information to rural healthcare providers are necessary with the COVID-19 pandemic. Project Extension for Community Healthcare Outcomes (ECHO) is a telementoring program designed to provide practitioners in rural communities with opportunities to engage in specialty training. We examined participant perceptions of a rapidly deployed, single continuing education session to improve healthcare provider preparedness for COVID-19 in Idaho. Methods: A modified Project ECHO session was developed to inform providers about emergency preparedness, treatment, testing, and resources for COVID-19. A post-session survey examined session impact and barriers on clinical practice. Results: Respondents believed the modified ECHO session increased COVID-19 knowledge and would improve their clinical practice and preparedness. Respondents were satisfied with the session and identified content, interdisciplinary collaboration, and format as beneficial; perceived barriers for utilizing session information included a lack of relevance of content and clinical applicability, and time constraints. Conclusions: A rapidly deployed modified Project ECHO session was perceived as an effective mechanism to foster collaboration and relay information to promote best practices at the start of the COVID-19 pandemic. An established Project ECHO network may be useful to rapidly exchange knowledge and information during a health emergency.

Migrating pyramidal neurons require DSCAM to bypass the border of the developing cortical plate.

During mammalian neocortex development, nascent pyramidal neurons migrate along radial glial cells and overtake earlier-born neurons to terminate at the front of the developing cortical plate (CP), leading to the outward expansion of the CP border. While much has been learned about the cellular and molecular mechanisms that underlie the migration of pyramidal neurons, how migrating neurons bypass the preceding neurons at the end of migration to reach their final positions remains poorly understood. Here, we report that Down syndrome cell adhesion molecule (DSCAM) is required for migrating neurons to bypass their post-migratory predecessors during the expansion of the upper cortical layers. DSCAM is a type I transmembrane cell adhesion molecule. It has been linked to Down syndrome through its location in the Down syndrome critical region of Chromosome 21 trisomy and to autism spectrum disorders through loss-of-function mutations. Ex vivo time-lapse imaging demonstrates that DSCAM is required for migrating neurons to bypass their post-migratory predecessors, crossing the CP border to expand the upper cortical layers. In DSCAM-deficient cortices, migrating neurons stop prematurely under the CP border, leading to thinner and denser upper cortical layers. We further show that DSCAM weakens cell adhesion mediated by N-cadherin in the upper cortical plate, allowing migrating neurons to traverse the CP border and expand the CP. These findings suggest that DSCAM is required for proper migratory termination and final positioning of nascent pyramidal neurons, which may provide insight into brain disorders that exhibit thinner upper layers of the cerebral cortex without neuronal loss.SIGNIFICANCE STATEMENTNewly born neurons in the developing mammalian neocortex migrate outward towards the cortical surface, bypassing earlier born neurons to expand the developing cortex. How migrating neurons bypass the preceding neurons and terminate at the front of the expanding cortex remains poorly understood. We demonstrate that Down syndrome cell adhesion molecule (DSCAM), linked to Down syndrome and autism spectrum disorder, is required by migrating neurons to bypass their post-migratory predecessors and terminate migration in the outwardly expanding cortical layer. Migrating neurons deficient in DSCAM stop prematurely, failing to expand the cortex. We further show that DSCAM likely mediates migratory termination by weakening cell-adhesion mediated by N-cadherin.

Nicotinamide provides neuroprotection in glaucoma by protecting against mitochondrial and metabolic dysfunction.

Nicotinamide adenine dinucleotide (NAD) is a REDOX cofactor and metabolite essential for neuronal survival. Glaucoma is a common neurodegenerative disease in which neuronal levels of NAD decline. We assess the effects of nicotinamide (a precursor to NAD) on retinal ganglion cells (the affected neuron in glaucoma) in normal physiological conditions and across a range of glaucoma relevant insults including mitochondrial stress and axon degenerative insults. We demonstrate retinal ganglion cell somal, axonal, and dendritic neuroprotection by nicotinamide in rodent models which represent isolated ocular hypertensive, axon degenerative, and mitochondrial degenerative insults. We performed metabolomics enriched for small molecular weight metabolites for the retina, optic nerve, and superior colliculus which demonstrates that ocular hypertension induces widespread metabolic disruption, including consistent changes to α-ketoglutaric acid, creatine/creatinine, homocysteine, and glycerophosphocholine. This metabolic disruption is prevented by nicotinamide. Nicotinamide provides further neuroprotective effects by increasing oxidative phosphorylation, buffering and preventing metabolic stress, and increasing mitochondrial size and motility whilst simultaneously dampening action potential firing frequency. These data support continued determination of the utility of long-term nicotinamide treatment as a neuroprotective therapy for human glaucoma.

Intra-articular Injections of the Hip and Knee With Triamcinolone vs Ketorolac: A Randomized Controlled Trial.

BACKGROUND: Clinicians commonly utilize intra-articular injections to treat symptomatic primary arthritis. Steroid injections are common yet have immune-modulating effects and can alter gene expression which may delay definitive arthroplasty and further damage cartilage. Nonsteroidal anti-inflammatory injections may offer a safer profile due to their differing mechanism of action; however, there is a relative dearth of information regarding their efficacy. This noninferiority study compares the effectiveness of triamcinolone vs ketorolac in treating symptoms of moderate to advanced primary osteoarthritis of the hip and knee. METHODS: In total, 110 patients (52 hips and 58 knees) with moderate to severe radiographic primary osteoarthritis of the hip or knee were randomized in a double-blinded study to receive an ultrasound-guided intra-articular injection of ketorolac or triamcinolone. Patient-reported outcome measures were collected pre-injection and at 1 week, 1 month, and 3 months. RESULTS: For hips and knees, intra-articular injections with either ketorolac or triamcinolone led to statistically significant improvements in patient-reported outcome measures. The treatment effect size was largest at 1 week and decreased over time. Primary analysis of variance comparisons revealed no significant differences between ketorolac and triamcinolone. For knee injections, post hoc secondary analysis suggests slight added durability in the triamcinolone group. Adverse effects were minimal with both interventions. CONCLUSION: Intra-articular ketorolac injections provide comparable improvement to triamcinolone for primary hip and knee osteoarthritis. Ketorolac is an additional low-cost option for conservative management of primary osteoarthritis, and due to its differing mechanism of action, it may not propagate additional cartilage damage or preclude from early surgical intervention if unsuccessful. TRIAL REGISTRATION NUMBER: NCT04441112.

OFF bipolar cell density varies by subtype, eccentricity, and along the dorsal ventral axis in the mouse retina.

The neural retina is organized along central-peripheral, dorsal-ventral, and laminar planes. Cellular density and distributions vary along the central-peripheral and dorsal-ventral axis in species including primates, mice, fish, and birds. Differential distribution of cell types within the retina is associated with sensitivity to different types of damage that underpin major retinal diseases, including macular degeneration and glaucoma. Normal variation in retinal distribution remains unreported for multiple cell types in widely used research models, including mouse. Here we map the distribution of all known OFF bipolar cell (BC) populations and horizontal cells. We report significant variation in the distribution of OFF BC populations and horizontal cells along the dorsal-ventral and central-peripheral axes of the retina. Distribution patterns are much more pronounced for some populations of OFF BC cells than others and may correspond to the cell type's specialized functions.

Increased density and age-related sharing of synapses at the cone to OFF bipolar cell synapse in the mouse retina.

Neural circuits in the adult nervous system are characterized by stable, cell type-specific patterns of synaptic connectivity. In many parts of the nervous system these patterns are established during development through initial over-innervation by multiple pre- or postsynaptic targets, followed by a process of refinement that takes place during development and is in many instances activity dependent. Here we report on an identified synapse in the mouse retina, the cone photoreceptor➔type 4 bipolar cell (BC4) synapse, and show that its development is distinctly different from the common motif of over-innervation followed by refinement. Indeed, the majority of cones are contacted by single BC4 throughout development, but are contacted by multiple BC4s through ongoing dendritic elaboration between 1 and 6 months of age-well into maturity. We demonstrate that cell density drives contact patterns downstream of single cones in Bax null mice and may serve to maintain constancy in both the dendritic and axonal projective field.

Expression patterns of dscam and sdk gene paralogs in developing zebrafish retina.

Purpose: The differential adhesion hypothesis states that a cell adhesion code provides cues that direct the specificity of nervous system development. The Down syndrome cell adhesion molecule (DSCAM) and sidekick (SDK) proteins belong to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and provide both attractive and repulsive cues that help to organize the nervous system during development, according to the differential adhesion hypothesis. The zebrafish genome is enriched in dscam and sdk genes, making the zebrafish an excellent model system to further test this hypothesis. The goal of this study is to describe the phylogenetic relationships of the paralogous CAM genes and their spatial expression and co-expression patterns in the embryonic zebrafish retina. Methods: Exon-intron structures, karyotypic locations, genomic context, and amino acid sequences of the zebrafish CAM genes (dscama, dscamb, dscaml1, sdk1a, sdk1b, sdk2a, and sdk2b) were obtained from the Ensembl genome database. The Prosite and SMART programs were used to determine the number and identity of protein domains for each CAM gene. The randomized axelerated maximum likelihood (RaxML) program was used to perform a phylogenetic analysis of the zebrafish CAM genes and orthologs in other vertebrates. A synteny analysis of regions surrounding zebrafish CAM paralogs was performed. Digoxigenin (dig)-labeled cRNA probes for each CAM gene were generated to perform in situ hybridization of retinal cryosections from zebrafish embryos and larvae. Dual in situ hybridization of retinal cryosections from zebrafish larvae was performed with dig- and fluorescein-labeled cRNA probes. Results: We found the studied zebrafish CAM genes encode similar protein domain structures as their corresponding orthologs in mammals and possess similar intron-exon organizations. CAM paralogs were located on different chromosomes. Phylogenetic and synteny analyses provided support for zebrafish dscam and sdk2 paralogs having originated during the teleost genome duplication. We found that dscama and dscamb are co-expressed in the ganglion cell layer (GCL) and the basal portion of the inner nuclear layer (INL), with weak expression in the photoreceptor-containing outer nuclear layer (ONL). Of the dscam genes, only dscamb was strongly expressed in ONL. Sdk1a and sdk1b were co-expressed in the GCL and the basal portion of the INL. Sdk2a and sdk2b also showed co-expression in the GCL and basal portion of the INL. All Sdk genes were expressed in the ciliary marginal zone (CMZ). Dual in situ hybridizations revealed alternating patterns of co-expression and exclusive expression for the dscam and sdk1 paralogs in cells of the GCL and the INL. The same alternating pattern was observed between dscam and sdk2 paralogs and between sdk1 and sdk2 paralogs. The expression of dscaml1 was observed in the INL and the GCL, with some cells in the basal portion of the INL showing co-expression of dscaml1 and dscama. Conclusions: These findings suggest that zebrafish dscam and sdk2 paralogs were likely the result of the teleost whole genome duplication and that all CAM duplicates show some differential expression patterns. We also demonstrate that the comparative expression patterns of CAM genes in the zebrafish are distinct from the exclusive expression patterns observed in chick retina, in which retinal ganglion cells express one of the four chick Dscam or Sdk genes only. The patterns in zebrafish are more similar to those of mice, in which co-expression of Dscam and Sdk genes is observed. These findings provide the groundwork for future functional analysis of the roles of the CAM paralogs in zebrafish.

Analysis of Retinal Vascular Plexuses and Interplexus Connections.

The retina is a highly organized neural tissue consisting of three neural layers and two synaptic layers. Blood vessels that nourish the mouse and human neural retina mirror this organization consisting of three plexus layers, or plexuses, that run parallel within the retina, connected by interplexus vessels to create a closed vascular network. Here, we describe a methodology to describe this organization that can be used to interrogate factors mediating retinal vessel patterning including: coverage of the vascular plexuses, branching and orientation of the interplexus connections, and digital reconstruction of the retinal vasculature to measure vessel length and density. The methodology focuses on the mouse retina, but can easily be adapted to study retinal vessels of other species.

DSCAM-mediated control of dendritic and axonal arbor outgrowth enforces tiling and inhibits synaptic plasticity.

Mature mammalian neurons have a limited ability to extend neurites and make new synaptic connections, but the mechanisms that inhibit such plasticity remain poorly understood. Here, we report that OFF-type retinal bipolar cells in mice are an exception to this rule, as they form new anatomical connections within their tiled dendritic fields well after retinal maturity. The Down syndrome cell-adhesion molecule (Dscam) confines these anatomical rearrangements within the normal tiled fields, as conditional deletion of the gene permits extension of dendrite and axon arbors beyond these borders. Dscam deletion in the mature retina results in expanded dendritic fields and increased cone photoreceptor contacts, demonstrating that DSCAM actively inhibits circuit-level plasticity. Electrophysiological recordings from Dscam-/- OFF bipolar cells showed enlarged visual receptive fields, demonstrating that expanded dendritic territories comprise functional synapses. Our results identify cell-adhesion molecule-mediated inhibition as a regulator of circuit-level neuronal plasticity in the adult retina.

DSCAM promotes axon fasciculation and growth in the developing optic pathway.

Although many aspects of optic pathway development are beginning to be understood, the mechanisms promoting the growth of retinal ganglion cell (RGC) axons toward visual targets remain largely unknown. Down syndrome cell adhesion molecule (Dscam) is expressed by mouse RGCs shortly after they differentiate at embryonic day 12 and is essential for multiple aspects of postnatal visual system development. Here we show that Dscam is also required during embryonic development for the fasciculation and growth of RGC axons. Dscam is expressed along the developing optic pathway in a pattern consistent with a role in regulating RGC axon outgrowth. In mice carrying spontaneous mutations in Dscam (Dscamdel17 ; Dscam2J), RGC axons pathfind normally, but growth from the chiasm toward their targets is impaired, resulting in a delay in RGC axons reaching the dorsal thalamus compared with that seen in wild-type littermates. Conversely, Dscam gain of function results in exuberant growth into the dorsal thalamus. The growth of ipsilaterally projecting axons is particularly affected. Axon organization in the optic chiasm and tract and RGC growth cone morphologies are also altered in Dscam mutants. In vitro DSCAM promotes RGC axon growth and fasciculation, and can act independently of cell contact. In vitro and in situ DSCAM is required both in the RGC axons and in their environment for the promotion of axon outgrowth, consistent with a homotypic mode of action. These findings identify DSCAM as a permissive signal that promotes the growth and fasciculation of RGC axons, controlling the timing of when RGC axons reach their targets.

Characterization and Evolution of the Spotted Gar Retina.

In this study, we characterize the retina of the spotted gar, Lepisosteus oculatus, a ray-finned fish. Gar did not undergo the whole genome duplication event that occurred at the base of the teleost fish lineage, which includes the model species zebrafish and medaka. The divergence of gars from the teleost lineage and the availability of a high-quality genome sequence make it a uniquely useful species to understand how genome duplication sculpted features of the teleost visual system, including photoreceptor diversity. We developed reagents to characterize the cellular organization of the spotted gar retina, including representative markers for all major classes of retinal neurons and Müller glia. We report that the gar has a preponderance of predicted short-wavelength shifted (SWS) opsin genes, including a duplicated set of SWS1 (ultraviolet) sensitive opsin encoding genes, a SWS2 (blue) opsin encoding gene, and two rod opsin encoding genes, all of which were expressed in retinal photoreceptors. We also report that gar SWS1 cones lack the geometric organization of photoreceptors observed in teleost fish species, consistent with the crystalline photoreceptor mosaic being a teleost innovation. Of note the spotted gar expresses both exo-rhodopsin (RH1-1) and rhodopsin (RH1-2) in rods. Exo-rhodopsin is an opsin that is not expressed in the retina of zebrafish and other teleosts, but rather is expressed in regions of the brain. This study suggests that exo-rhodopsin is an ancestral actinopterygian (ray finned fish) retinal opsin, and in teleosts its expression has possibly been subfunctionalized to the pineal gland.

Replacing the PDZ-interacting C-termini of DSCAM and DSCAML1 with epitope tags causes different phenotypic severity in different cell populations.

Different types of neurons in the retina are organized vertically into layers and horizontally in a mosaic pattern that helps ensure proper neural network formation and information processing throughout the visual field. The vertebrate Dscams (DSCAM and DSCAML1) are cell adhesion molecules that support the development of this organization by promoting self-avoidance at the level of cell types, promoting normal developmental cell death, and directing vertical neurite stratification. To understand the molecular interactions required for these activities, we tested the functional significance of the interaction between the C-terminus of the Dscams and multi-PDZ domain-containing scaffolding proteins in mouse. We hypothesized that this PDZ-interacting domain would mediate a subset of the Dscams' functions. Instead, we found that in the absence of these interactions, some cell types developed almost normally, while others resembled complete loss of function. Thus, we show differential dependence on this domain for Dscams' functions in different cell types.

Pou4f2 knock-in Cre mouse: A multifaceted genetic tool for vision researchers.

PURPOSE: A transgenic mouse that expresses Cre recombinase under control of the Pou4f2-promoter (also referred to as Brn-3b and Brn-3.2) was characterized. Pou4f2 expression has been reported in a subset of retinal ganglion cells (RGCs) in the retina, in the midbrain, and in the germline. In this study, we characterize the expression pattern of this Cre-recombinase line and report its utility in targeted deletion, temporal deletion, RGC depletion, and germline targeting, which can be regulated by the sex of the Cre-carrying mouse. METHODS: Pou4f2(Cre) was mapped by using a combination of PCR and sequencing of PCR products to better understand the construct and to locate where it was inserted within the Pou4f2 locus. Cre expression patterns were examined by crossing Pou4f2(Cre/+) mice to Cre reporter mice. Immunohistochemistry was used to further define the pattern of Cre expression and Cre-mediated recombination within the retina, brain, and other tissues. RESULTS: An internal ribosome entry site (IRES)-Cre cassette was inserted into the Pou4f2 gene disrupting normal gene function, as verified by the depletion of RGCs in mice homozygous for the insert. Pou4f2(Cre) expression was observed in the retina, brain, peripheral neurons, and male germ cells. Germline recombination was observed when the sire carried the Cre and the target for recombination. In all other breeding schemes, recombination was observed within subsets of cells within the retina, brain, intestines, heart, and gonads. In the retina, Cre efficiently targets recombination in neurons within the RGC layer (RGL), the inner nuclear layer (INL), and a small percentage of photoreceptors, activity that has not been previously reported. Unlike most other Cre lines active in the inner retina, recombination in Müller and other glia was not observed in mice carrying Pou4f2(Cre) . Within the visual centers of the brain, Cre targets recombination in about 15% of cells within the superchiasmatic nucleus, lateral geniculate nucleus, and superior colliculus. CONCLUSIONS: Pou4f2(Cre) provides multiple uses for the vision researcher's genetic toolkit. First, Pou4f2(Cre) is a knock-in allele that can be used to eliminate Pou4f2, resulting in depletion of RGCs. Second, expression of Cre in male germ cells makes this strain an efficient germline activator of recombination, for example, to target LoxP-flanked sequences in the whole mouse. Third, Pou4f2(Cre) efficiently targets RGCs, amacrine cells, bipolar cells, horizontal cells, and a small number of photoreceptors within the retina, as well as the visual centers in the brain. Unlike other Cre recombinase lines that target retinal neurons, no recombination was observed in Müller or other retinal glia. These properties make this Cre recombinase line a useful tool for vision researchers.

Defective Angiogenesis and Intraretinal Bleeding in Mouse Models With Disrupted Inner Retinal Lamination.

PURPOSE: Abnormal retinal angiogenesis leads to visual impairment and blindness. Understanding how retinal vessels develop normally has dramatically improved treatments for people with retinal vasculopathies, but additional information about development is required. Abnormal neuron patterning in the outer retina has been shown to result in abnormal vessel development and blindness, for example, in people and mouse models with Crumbs homologue 1 (CRB1) mutations. In this study, we report and characterize a mouse model of inner retinal lamination disruption and bleeding, the Down syndrome cell adhesion molecule (Dscam) mutant, and test how neuron-neurite placement within the inner retina guides development of intraretinal vessels. METHODS: Bax mutant mice (increased neuron cell number), Dscam mutant mice (increased neuron cell number, disorganized lamination), Fat3 mutant mice (disorganized neuron lamination), and Dscam gain-of-function mice (Dscam(GOF)) (decreased neuron cell number) were used to manipulate neuron placement and number. Immunohistochemistry was used to assay organization of blood vessels, glia, and neurons. In situ hybridization was used to map the expression of angiogenic factors. RESULTS: Significant changes in the organization of vessels within mutant retinas were found. Displaced neurons and microglia were associated with the attraction of vessels. Using Fat3 mutant and Dscam(GOF) retinas, we provide experimental evidence that vessel branching is induced at the neuron-neurite interface, but that other factors are required for full plexus layer formation. We further demonstrate that the displacement of neurons results in the mislocalization of angiogenic factors. CONCLUSIONS: Inner retina neuron lamination is required for development of intraretinal vessels.

Morphological Diversity of the Rod Spherule: A Study of Serially Reconstructed Electron Micrographs.

PURPOSE: Rod spherules are the site of the first synaptic contact in the retina's rod pathway, linking rods to horizontal and bipolar cells. Rod spherules have been described and characterized through electron micrograph (EM) and other studies, but their morphological diversity related to retinal circuitry and their intracellular structures have not been quantified. Most rod spherules are connected to their soma by an axon, but spherules of rods on the surface of the Mus musculus outer plexiform layer often lack an axon and have a spherule structure that is morphologically distinct from rod spherules connected to their soma by an axon. Retraction of the rod axon and spherule is often observed in disease processes and aging, and the retracted rod spherule superficially resembles rod spherules lacking an axon. We hypothesized that retracted spherules take on an axonless spherule morphology, which may be easier to maintain in a diseased state. To test our hypothesis, we quantified the spatial organization and subcellular structures of rod spherules with and without axons. We then compared them to the retracted spherules in a disease model, mice that overexpress Dscam (Down syndrome cell adhesion molecule), to gain a better understanding of the rod synapse in health and disease. METHODS: We reconstructed serial EM images of wild type and DscamGoF (gain of function) rod spherules at a resolution of 7 nm in the X-Y axis and 60 nm in the Z axis. Rod spherules with and without axons, and retracted spherules in the DscamGoF retina, were reconstructed. The rod spherule intracellular organelles, the invaginating dendrites of rod bipolar cells and horizontal cell axon tips were also reconstructed for statistical analysis. RESULTS: Stereotypical rod (R1) spherules occupy the outer two-thirds of the outer plexiform layer (OPL), where they present as spherical terminals with large mitochondria. This spherule group is highly uniform and composed more than 90% of the rod spherule population. Rod spherules lacking an axon (R2) were also described and characterized. This rod spherule group consists of a specific spatial organization that is strictly located at the apical OPL-facing layer of the Outer Nuclear Layer (ONL). The R2 spherule displays a large bowl-shaped synaptic terminal that hugs the rod soma. Retracted spherules in the DscamGoF retina were also reconstructed to test if they are structurally similar to R2 spherules. The misplaced rod spherules in DscamGoF have a gross morphology that is similar to R2 spherules but have significant disruption in internal synapse organization. CONCLUSION: We described a morphological diversity within Mus musculus rod spherules. This diversity is correlated with rod location in the ONL and contributes to the intracellular differences within spherules. Analysis of the DscamGoF retina indicated that their R2 spherules are not significantly different than wild type R2 spherules, but that their retracted rod spherules have abnormal synaptic organization.

IPLaminator: an ImageJ plugin for automated binning and quantification of retinal lamination.

BACKGROUND: Information in the brain is often segregated into spatially organized layers that reflect the function of the embedded circuits. This is perhaps best exemplified in the layering, or lamination, of the retinal inner plexiform layer (IPL). The neurites of the retinal ganglion, amacrine and bipolar cell subtypes that form synapses in the IPL are precisely organized in highly refined strata within the IPL. Studies focused on developmental organization and cell morphology often use this layered stratification to characterize cells and identify the function of genes in development of the retina. A current limitation to such analysis is the lack of standardized tools to quantitatively analyze this complex structure. Most previous work on neuron stratification in the IPL is qualitative and descriptive. RESULTS: In this study we report the development of an intuitive platform to rapidly and reproducibly assay IPL lamination. The novel ImageJ based software plugin we developed: IPLaminator, rapidly analyzes neurite stratification patterns in the retina and other neural tissues. A range of user options allows researchers to bin IPL stratification based on fixed points, such as the neurites of cholinergic amacrine cells, or to define a number of bins into which the IPL will be divided. Options to analyze tissues such as cortex were also added. Statistical analysis of the output then allows a quantitative value to be assigned to differences in laminar patterning observed in different models, genotypes or across developmental time. CONCLUSION: IPLaminator is an easy to use software application that will greatly speed and standardize quantification of neuron organization.