Cameron S. Cowan

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone.

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy. In rat first molar organs, the NH2-terminal fragment localized to predentin, whereas the COOH-terminal fragment was mainly restricted to mineralized dentin. In the growth plate of bone, the NH2-terminal fragment appeared in the proliferation and hypertrophic zones, whereas the COOH-terminal fragment occupied the ossification zone. Forster resonance energy transfer analysis showed colocalization of both fragments of DMP1 in odontoblasts and predentin, as well as hypertrophic chondrocytes within the growth plates of bone. The biochemical analysis of bovine teeth showed that predentin is rich in DMP1-PG, whereas mineralized dentin primarily contains the COOH-terminal fragment. We conclude that the differential patterns of expression of NH2-terminal and COOH-terminal fragments of DMP1 reflect their potentially distinct roles in the biomineralization of dentin and bone matrices.

Connexin 36 and rod bipolar cell independent rod pathways drive retinal ganglion cells and optokinetic reflexes

Rod pathways are a parallel set of synaptic connections which enable night vision by relaying and processing rod photoreceptor light responses. We use dim light stimuli to isolate rod pathway contributions to downstream light responses then characterize these contributions in knockout mice lacking rod transducin-α (Trα), or certain pathway components associated with subsets of rod pathways. These comparisons reveal that rod pathway driven light sensitivity in retinal ganglion cells (RGCs) is entirely dependent on Trα, but partially independent of connexin 36 (Cx36) and rod bipolar cells. Pharmacological experiments show that rod pathway-driven and Cx36-independent RGC ON responses are also metabotropic glutamate receptor 6-dependent. To validate the RGC findings in awake, behaving animals we measured optokinetic reflexes (OKRs), which are sensitive to changes in ON pathways. Scotopic OKR contrast sensitivity was lost in Trα(-/-) mice, but indistinguishable from controls in Cx36(-/-) and rod bipolar cell knockout mice. Mesopic OKRs were also altered in mutant mice: Trα(-/-) mice had decreased spatial acuity, rod BC knockouts had decreased sensitivity, and Cx36(-/-) mice had increased sensitivity. These results provide compelling evidence against the complete Cx36 or rod BC dependence of night visions ON component. Further, the findings suggest the parallel nature of rod pathways provides considerable redundancy to scotopic light sensitivity but distinct contributions to mesopic responses through complicated interactions with cone pathways.

Space–time codependence of retinal ganglion cells can be explained by novel and separable components of their receptive fields

Reverse correlation methods such as spike‐triggered averaging consistently identify the spatial center in the linear receptive fields (RFs) of retinal ganglion cells (GCs). However, the spatial antagonistic surround observed in classical experiments has proven more elusive. Tests for the antagonistic surround have heretofore relied on models that make questionable simplifying assumptions such as space–time separability and radial homogeneity/symmetry. We circumvented these, along with other common assumptions, and observed a linear antagonistic surround in 754 of 805 mouse GCs. By characterizing the RFs space–time structure, we found the overall linear RFs inseparability could be accounted for both by tuning differences between the center and surround and differences within the surround. Finally, we applied this approach to characterize spatial asymmetry in the RF surround. These results shed new light on the spatiotemporal organization of GC linear RFs and highlight a major contributor to its inseparability.

Effects of Chronic and Acute Intraocular Pressure Elevation on Scotopic and Photopic Contrast Sensitivity in Mice

Purpose To compare the impact of intraocular pressure (IOP) elevation on scotopic and photopic contrast sensitivity in mice. Methods We chronically elevated the IOP of wild-type mice via injection of polystyrene beads or acutely via injection of highly cohesive sodium hyaluronate. Some eyes with chronically elevated IOP were treated with either topical brimonidine tartrate 0.1% or brinzolamide 1%. Scotopic and photopic contrast sensitivity was assessed at peak spatiotemporal frequencies at multiple time points, with an established optokinetic technique. Retinal ganglion cell (RGC) counts were determined with an antibody to class III beta-tubulin. Correlations among IOP level, RGC count, and scotopic or photopic contrast sensitivity were performed. Results Six weeks of IOP elevation caused a generalized reduction of photopic contrast sensitivity and a preferential reduction of scotopic contrast sensitivity at peak spatiotemporal frequencies. The administration of brinzolamide but not brimonidine caused a significant reduction in cumulative IOP, whereas brimonidine, but not brinzolamide, prevented RGC loss. Both brimonidine and brinzolamide prevented contrast sensitivity loss, but brimonidine did so at earlier time points and across a wider range of lighting conditions. Following either chronic or acute IOP elevation, scotopic contrast sensitivity was impacted most prominently by IOP level and not by RGC count, while photopic contrast sensitivity was impacted by a combination of factors. Conclusions It is possible that scotopic-specific retinal circuitry is altered preferentially by IOP elevation, and that changes in scotopic contrast sensitivity will assist with glaucoma detection. Brimonidine appears to prevent RGC loss via an IOP-independent mechanism.

Distinct subcomponents of mouse retinal ganglion cell receptive fields are differentially altered by light adaptation

HighlightsRGCs increase sensitivity under dim light by broadening spatiotemporal integration.We identified an antagonistic surround under scotopic conditions in a subset of RGCs.The center and surround differentially broaden temporal integration under dim light.Spatial alterations under dim light are more heterogeneous than temporal ones.Asymmetries between ON and OFF RGCs are decreased under dim conditions. Abstract The remarkable dynamic range of vision is facilitated by adaptation of retinal sensitivity to ambient lighting conditions. An important mechanism of sensitivity adaptation is control of the spatial and temporal window over which light is integrated. The retina accomplishes this by switching between parallel synaptic pathways with differing kinetics and degrees of synaptic convergence. However, the relative shifts in spatial and temporal integration are not well understood – particularly in the context of the antagonistic spatial surround. Here, we resolve these issues by characterizing the adaptation‐induced changes to spatiotemporal integration in the linear receptive field center and surround of mouse retinal ganglion cells. While most ganglion cells lose their antagonistic spatial surround under scotopic conditions, a strong surround is maintained in a subset. We then applied a novel technique that allowed us to analyze the receptive field as a triphasic temporal filter in the center and a biphasic filter in the surround. The temporal tuning of the surround was relatively maintained across adaptation conditions compared to the center, which greatly increased its temporal integration. Though all phases of the center’s triphasic temporal response slowed, some shifted significantly less. Additionally, adaptation differentially shifted ON and OFF pathway temporal tuning, reducing their asymmetry under scotopic conditions. Finally, spatial integration was significantly increased by dark adaptation in some cells while it decreased it in others. These findings provide novel insight into how adaptation adjusts visual information processing by altering fundamental properties of ganglion cell receptive fields, such as center‐surround antagonism and space‐time integration.

Elevated IOP alters the space–time profiles in the center and surround of both ON and OFF RGCs in mouse

Significance Glaucoma is a leading cause of blindness worldwide and many patients with glaucoma do not realize they have the disease until a significant visual deficit occurs. Here we record from mouse retinal ganglion cells and determine how they properties change in a mouse model of experiment glaucoma. We identify multiple changes to retinal ganglion cell functional properties and potential circuits mediating these changes. The findings from this study will help glaucoma patients in two ways. The novel functional changes we see can help identify new diagnostic tests that could identify patients prior to gross vision loss. Additionally, identification of specific retinal pathways mediating glaucomatous injury will help develop new treatments that extend to retinal cells that are currently being ignored. Glaucoma is a leading cause of blindness worldwide, and is characterized by progressive retinal ganglion cell (RGC) death. An experimental model of glaucoma has been established by elevating the intraocular pressure (IOP) via microbead occlusion of ocular fluid outflow in mice. Studies in this model have found visual dysfunction that varied with adaptational state, occurred before anatomical changes, and affected OFF RGCs more than ON RGCs. These results indicate subtle alterations in the underlying retinal circuitry that could help identify disease before irreversible RGC changes. Therefore, we looked at how RGC function was altered with elevated IOP under both photopic and scotopic conditions. We first found that responses to light offset are diminished with IOP elevation along with a concomitant decrease in receptive field center size for OFF RGCs. In addition, the antagonistic surround strength and size was reduced in ON RGCs. Furthermore, elevation of IOP significantly accelerated the photopic temporal tuning of RGC center responses in both ON and OFF RGCs. We found that some of the IOP-induced functional changes to OFF RGCs relied on ON cross-over pathways, indicating dysfunction in inner retinal circuitry. Overall, these results suggest that IOP alters multiple functions in the retina depending on the adaptational state. They provide a basis for designing multiple functional tests for early detection of glaucoma and for circuit-specific therapeutic targets in treatment of this blinding disease.

The ON Crossover Circuitry Shapes Spatiotemporal Profile in the Center and Surround of Mouse OFF Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are often grouped based on their functional properties. Many of these functional properties, such as receptive field (RF) size, are driven by specific retinal circuits. In this report, we determined the role of the ON bipolar cell (BC) mediated crossover circuitry in shaping the center and surround of OFF RGCs. We recorded from a large population of mouse RGCs using a multielectrode array (MEA) while pharmacologically removing the ON BC-mediated crossover circuit. OFF sustained and transient responses to whole field stimuli are lost under scotopic conditions, but maintained under photopic conditions. Though photopic light responses were grossly maintained, we found that photopic light response properties were altered. Using linear RF mapping, we found a significant reduction in the antagonistic surround and a decrease in size of the RF center. Using a novel approach to separate the distinct temporal filters present in the RF center, we see that the crossover pathway contributes specifically to the sluggish antagonistic filter in the center. These results provide new insight into the role of crossover pathways in driving RGCs and also demonstrate that the distinct inputs driving the RF center can be isolated and assayed by RGC activity.