Search

Prolonged exposure to motion in one direction often leads to the illusion of motion in the opposite direction for stationary objects. This motion aftereffect likely arises across several visual areas from adaptive changes in the balance of activity and competitive interactions. We examined whether or not the mouse was susceptible to this same illusion to determine whether it would be a suitable model for learning about the neural representation of the motion aftereffect. Under a classical conditioning paradigm, mice learned to lick when presented with motion in one direction and not the opposite direction. When the mice were adapted to motion preceding this test, their lick behavior for zero coherence motion was biased for motion in the opposite direction of the adapting stimulus. Overall, lick count versus motion coherence shifted in the opposite direction of the adapting stimulus. This suggests that although the mouse has a simpler visual system compared with primates, it still is subject to the motion aftereffect and may elucidate the underlying circuitry.

Unlabelled: Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs, with five subtypes named M1-M5) are a unique subclass of RGCs with axons that project directly to many brain nuclei involved in non-image-forming functions such as circadian photoentrainment and the pupillary light reflex. Recent evidence suggests that melanopsin-based signals also influence image-forming visual function, including light adaptation, but the mechanisms involved are unclear. Intriguingly, a small population of M1 ipRGCs have intraretinal axon collaterals that project toward the outer retina. Using genetic mouse models, we provide three lines of evidence showing that these axon collaterals make connections with upstream dopaminergic amacrine cells (DACs): (1) ipRGC signaling to DACs is blocked by tetrodotoxin both in vitro and in vivo, indicating that ipRGC-to-DAC transmission requires voltage-gated Na(+) channels; (2) this transmission is partly dependent on N-type Ca(2+) channels, which are possibly expressed in the axon collateral terminals of ipRGCs; and (3) fluorescence microscopy reveals that ipRGC axon collaterals make putative presynaptic contact with DACs. We further demonstrate that elimination of M1 ipRGCs attenuates light adaptation, as evidenced by an impaired electroretinogram b-wave from cones, whereas a dopamine receptor agonist can potentiate the cone-driven b-wave of retinas lacking M1 ipRGCs. Together, the results strongly suggest that ipRGCs transmit luminance signals retrogradely to the outer retina through the dopaminergic system and in turn influence retinal light adaptation., Significance Statement: Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a third class of retinal photoreceptors that are known to mediate physiological responses such as circadian photoentrainment. However, investigation into whether and how ipRGCs contribute to vision has just begun. Here, we provide convergent anatomical and physiological evidence that axon collaterals of ipRGCs constitute a centrifugal pathway to DACs, conveying melanopsin-based signals from the innermost retina to the outer retina. We further demonstrate that retrograde signals likely influence visual processing because elimination of axon collateral-bearing ipRGCs impairs light adaptation by limiting dopamine-dependent facilitation of the cone pathway. Our findings strongly support the hypothesis that retrograde melanopsin-based signaling influences visual function locally within the retina, a notion that refutes the dogma that RGCs only provide physiological signals to the brain., (Copyright © 2016 the authors 0270-6474/16/367184-14$15.00/0.)

The extracellular potential of excitable and nonexcitable cells with respect to ground is ∼0 mV. One of the known exceptions in mammals is the cochlear duct, where the potential is ∼80-100 mV, called the endocochlear potential (EP). The EP serves as the "battery" for transduction of sound, contributing toward the sensitivity of the auditory system. The stria vascularis (StV) of the cochlear duct is the station where the EP is generated, but the cell-specific roles in the StV are ill defined. Using the intermediate cell (IC)-specific tyrosinase promoter, under the control of diphtheria toxin (DT), we eliminated and/or halted differentiation of neural crest melanocytes after migration to the StV. The ensuing adult transgenic mice are profoundly deaf. Additionally, the EP was abolished. Expression of melanocyte early marker and Kir4.1 in ICs precedes the onset of pigment synthesis. Activation of DT leads to loss of ICs. Finally, in accord with the distinct embryology of retinal pigmented cells, transgenic mice with toxigenic ablation of neural crest-derived melanocytes have intact visual responses. We assert that the tyrosinase promoter is the distinct target for genetic manipulation of IC-specific genes.

Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors.

Memory-retrieval processing of cross-modal sensory preconditioning is vital for understanding the plasticity underlying the interactions between modalities. As part of the sensory preconditioning paradigm, it has been hypothesized that the conditioned response to an unreinforced cue depends on the memory of the reinforced cue via a sensory link between the two cues. To test this hypothesis, we studied cross-modal memory-retrieval processing in a genetically tractable model organism, Drosophila melanogaster. By expressing the dominant temperature-sensitive shibire(ts1) (shi(ts1)) transgene, which blocks synaptic vesicle recycling of specific neural subsets with the Gal4/UAS system at the restrictive temperature, we specifically blocked visual and olfactory memory retrieval, either alone or in combination; memory acquisition remained intact for these modalities. Blocking the memory retrieval of the reinforced olfactory cues did not impair the conditioned response to the unreinforced visual cues or vice versa, in contrast to the canonical memory-retrieval processing of sensory preconditioning. In addition, these conditioned responses can be abolished by blocking the memory retrieval of the two modalities simultaneously. In sum, our results indicated that a conditioned response to an unreinforced cue in cross-modal sensory preconditioning can be recalled through parallel pathways.

A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. Although much is now known about the role of the α-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary βγ-complex in this process remains a mystery. Here, we show that elimination of the transducin γ-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin βγ-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions.

In rod photoreceptors, deactivation of the light-activated G-protein-coupled receptor rhodopsin (R*) is initiated by phosphorylation and completed through subsequent binding of visual arrestin (Arr1). The in vivo kinetics of these individual interactions have proven difficult to determine with precision since R* lifetime is much shorter than the lifetimes of downstream G-protein and effector molecules. Here, we have used a transgenic mouse line with accelerated downstream deactivation kinetics to reveal the contribution of Arr1 binding to the overall time course of rhodopsin deactivation. Photoresponses revealed that the lifetime of R* is significantly increased in rods that express half of the normal amount of Arr1, in a manner consistent with a twofold decrease in the rate of Arr1 binding across a wide range of flash strengths. A basic model of photoresponse deactivation consistent with established photoreceptor biochemistry shows that R* phosphorylation and Arr1 binding occur with a time constant of approximately 40 ms in wild-type mouse rods, much faster than previous estimates.

The rate of synaptic transmission between photoreceptors and bipolar cells has been long known to depend on conditions of ambient illumination. However, the molecular mechanisms that mediate and regulate transmission at this ribbon synapse are poorly understood. We conducted electroretinographic recordings from dark- and light-adapted mice lacking the abundant photoreceptor-specific protein phosducin and found that the ON-bipolar cell responses in these animals have a reduced light sensitivity in the dark-adapted state. Additional desensitization of their responses, normally caused by steady background illumination, was also diminished compared with wild-type animals. This effect was observed in both rod- and cone-driven pathways, with the latter affected to a larger degree. The underlying mechanism is likely to be photoreceptor specific because phosducin is not expressed in other retina neurons and transgenic expression of phosducin in rods of phosducin knock-out mice rescued the rod-specific phenotype. The underlying mechanism functions downstream from the phototransduction cascade, as evident from the sensitivity of phototransduction in phosducin knock-out rods being affected to a much lesser degree than b-wave responses. These data indicate that a major regulatory component responsible for setting the sensitivity of signal transmission between photoreceptors and ON-bipolar cells is confined to photoreceptors and that phosducin participates in the underlying molecular mechanism.

Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme. Type 3 deiodinase, encoded by Dio3, is expressed in the immature mouse retina. In Dio3(-/-) mice, approximately 80% of cones are lost through neonatal cell death. Cones that express opsin photopigments for response to both short (S) and medium-long (M) wavelength light are lost. Rod photoreceptors, which mediate dim light vision, remain essentially intact. Excessive thyroid hormone in wild-type pups also eliminates cones. Cone loss is mediated by cone-specific thyroid hormone receptor beta2 (TRbeta2) as deletion of TRbeta2 rescues cones in Dio3(-/-) mice. However, rescued cones respond to short but not longer wavelength light because TRbeta2 under moderate hormonal stimulation normally induces M opsin and controls the patterning of M and S opsins over the retina. The results suggest that type 3 deiodinase limits hormonal exposure of the cone to levels that safeguard both cone survival and the patterning of opsins that is required for cone function.

Rpe65(-/-) mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10-20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated the Rpe65(-/-)Grk1(-/-) mouse. The retinae of Rpe65(-/-)Grk1(-/-) mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative to Rpe65(-/-) mice. These data show that opsin phosphorylation in the Rpe65(-/-) mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degenerating Rpe65(-/-)Grk1(-/-) mice resulted in lower overall opsin activity and in higher rod sensitivity compared with Rpe65(-/-) mice. In Rpe65(-/-)Grk1(-/-)Gnat1(-/-) mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration in Grk1(-/-) mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin in Grk1(-/-)Gnat1(-/-) mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.

Prominin-1/CD133 (Prom-1) is a commonly used marker of neuronal, vascular, hematopoietic and other stem cells, yet little is known about its biological role and importance in vivo. Here, we show that loss of Prom-1 results in progressive degeneration of mature photoreceptors with complete loss of vision. Despite the expression of Prom-1 on endothelial progenitors, photoreceptor degeneration was not attributable to retinal vessel defects, but caused by intrinsic photoreceptor defects in disk formation, outer segment morphogenesis, and associated with visual pigment sorting and phototransduction abnormalities. These findings shed novel insight on how Prom-1 regulates neural retinal development and phototransduction in vertebrates.

A systematic forward genetic Drosophila screen for electroretinogram mutants lacking synaptic transients identified the fuseless (fusl) gene, which encodes a predicted eight-pass transmembrane protein in the presynaptic membrane. Null fusl mutants display >75% reduction in evoked synaptic transmission but, conversely, an approximately threefold increase in the frequency and amplitude of spontaneous synaptic vesicle fusion events. These neurotransmission defects are rescued by a wild-type fusl transgene targeted only to the presynaptic cell, demonstrating a strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at the synapse show a severely impaired exo-endo synaptic vesicle cycling pool. Consistently, ultrastructural analyses reveal accumulated vesicles arrested in clustered and docked pools at presynaptic active zones. In the absence of Fusl, calcium-dependent neurotransmitter release is dramatically compromised and there is little enhancement of synaptic efficacy with elevated external Ca(2+) concentrations. These defects are causally linked with severe loss of the Cacophony voltage-gated Ca(2+) channels, which fail to localize normally at presynaptic active zone domains in the absence of Fusl. These data indicate that Fusl regulates assembly of the presynaptic active zone Ca(2+) channel domains required for efficient coupling of the Ca(2+) influx and synaptic vesicle exocytosis during neurotransmission.

Rods and cones subserve mouse vision over a 100 million-fold range of light intensity (-6 to 2 log cd m(-2)). Rod pathways tune vision to the temporal frequency of stimuli (peak, 0.75 Hz) and cone pathways to their speed (peak, approximately 12 degrees/s). Both pathways tune vision to the spatial components of stimuli (0.064-0.128 cycles/degree). The specific photoreceptor contributions were determined by two-alternative, forced-choice measures of contrast thresholds for optomotor responses of C57BL/6J mice with normal vision, Gnat2(cpfl3) mice without functional cones, and Gnat1-/- mice without functional rods. Gnat2(cpfl3) mice (threshold, -6.0 log cd m(-2)) cannot see rotating gratings above -2.0 log cd m(-2) (photopic vision), and Gnat1-/- mice (threshold, -4.0 log cd m(-2)) are blind below -4.0 log cd m(-2) (scotopic vision). Both genotypes can see in the transitional mesopic range (-4.0 to -2.0 log cd m(-2)). Mouse rod and cone sensitivities are similar to those of human. This parametric study characterizes the functional properties of the mouse visual system, revealing the rod and cone contributions to contrast sensitivity and to the temporal processing of visual stimuli.

Whereas the mammalian retina possesses a repertoire of factors known to establish general retinal cell types, these factors alone cannot explain the vast diversity of neuronal subtypes. In other CNS regions, the differentiation of diverse neuronal pools is governed by coordinately acting LIM-homeodomain proteins including the Islet-class factor Islet-1 (Isl1). We report that deletion of Isl1 profoundly disrupts retinal function as assessed by electroretinograms and vision as assessed by optomotor behavior. These deficits are coupled with marked reductions in mature ON- and OFF-bipolar (>76%), cholinergic amacrine (93%), and ganglion (71%) cells. Mosaic deletion of Isl1 permitted a chimeric analysis of "wild-type" cells in a predominantly Isl1-null environment, demonstrating a cell-autonomous role for Isl1 in rod bipolar and cholinergic amacrine development. Furthermore, the effects on bipolar cell development appear to be dissociable from the preceding retinal ganglion cell loss, because Pou4f2-null mice are devoid of similar defects in bipolar cell marker expression. Expression of the ON- and OFF-bipolar cell differentiation factors Bhlhb4 and Vsx1, respectively, requires the presence of Isl1, whereas the early bipolar cell marker Prox1 initially did not. Thus, Isl1 is required for engaging bipolar differentiation pathways but not for general bipolar cell specification. Spatiotemporal expression analysis of additional LIM-homeobox genes identifies a LIM-homeobox gene network during bipolar cell development that includes Lhx3 and Lhx4. We conclude that Isl1 has an indispensable role in retinal neuron differentiation within restricted cell populations and this function may reflect a broader role for other LIM-homeobox genes in retinal development, and perhaps in establishing neuronal subtypes.

CaBP4 is a calmodulin-like neuronal calcium-binding protein that is crucial for the development and/or maintenance of the cone and rod photoreceptor synapse. Previously, we showed that CaBP4 directly regulates Ca(v)1 L-type Ca2+ channels, which are essential for normal photoreceptor synaptic transmission. Here, we show that the function of CaBP4 is regulated by phosphorylation. CaBP4 is phosphorylated by protein kinase C zeta (PKCzeta) at serine 37 both in vitro and in the retina and colocalizes with PKCzeta in photoreceptors. CaBP4 phosphorylation is greater in light-adapted than dark-adapted mouse retinas. In electrophysiological recordings of cells transfected with Ca(v)1.3 and CaBP4, mutation of the serine 37 to alanine abolished the effect of CaBP4 in prolonging the Ca2+ current through Ca(v)1.3 channel, whereas inactivating mutations in the CaBP4 Ca2+-binding sites strengthened Ca(v)1.3 modulation. These findings demonstrate how light-stimulated changes in CaBP4 phosphorylation and Ca2+ binding may regulate presynaptic Ca2+ signals in photoreceptors.

The Nougaret form of dominant stationary night blindness is linked to a G38D mutation in the rod transducin-alpha subunit (Talpha). In this study, we have examined the mechanism of Nougaret night blindness using transgenic mice expressing TalphaG38D. The biochemical, electrophysiological, and vision-dependent behavioral analyses of the mouse model revealed a unique phenotype of reduced rod sensitivity, impaired activation, and slowed recovery of the phototransduction cascade. Two key deficiencies in TalphaG38D function, its poor ability to activate PDE6 (cGMP phosphodiesterase) and decreased GTPase activity, are found to be the major mechanisms altering visual signaling in transgenic mice. Despite these defects, rod-mediated sensitivity in heterozygous mice is not decreased to the extent seen in heterozygous Nougaret patients.

Timely deactivation of G-protein signaling is essential for the proper function of many cells, particularly neurons. Termination of the light response of retinal rods requires GTP hydrolysis by the G-protein transducin, which is catalyzed by a protein complex that includes regulator of G-protein signaling RGS9-1 and the G-protein beta subunit Gbeta5-L. Disruption of the Gbeta5 gene in mice (Gbeta5-/-) abolishes the expression of Gbeta5-L in the retina and also greatly reduces the expression level of RGS9-1. We examined transduction in dark- and light-adapted rods from wild-type and Gbeta5-/- mice. Responses of Gbeta5-/- rods were indistinguishable in all respects from those of RGS9-/- rods. Loss of Gbeta5-L (and RGS9-1) had no effect on the activation of the G-protein cascade, but profoundly slowed its deactivation and interfered with the speeding of incremental dim flashes during light adaptation. Both RGS9-/- and Gbeta5-/- responses were consistent with another factor weakly regulating GTP hydrolysis by transducin in a manner proportional to the inward current. Our results indicate that a complex containing RGS9-1-Gbeta5-L is essential for normal G-protein deactivation and rod function. In addition, our light adaptation studies support the notion than an additional weak GTPase-accelerating factor in rods is regulated by intracellular calcium and/or cGMP.

Transducins couple visual pigments to cGMP hydrolysis, the only recognized phototransduction pathway in vertebrate photoreceptors. Here we describe a zebrafish mutant, no optokinetic response f(w21) (nof), with a nonsense mutation in the gene encoding the alpha subunit of cone transducin. Retinal morphology and levels of phototransduction enzymes are normal in nof retinas, but cone transducin is undetectable. Dark current in nof cones is also normal, but it is insensitive to moderate intensity light. The nof cones do respond, however, to bright light. These responses are produced by a light-stimulated, but transducin-independent, release of Ca2+ into the cone cytoplasm. Thus, in addition to stimulating transducin, light also independently induces release of Ca2+ into the photoreceptor cytoplasm.

Inhibition is crucial for normal function in the nervous system. In the CNS, inhibition is mediated primarily by the amino acid GABA via activation of two ionotropic GABA receptors, GABA(A) and GABA(C). GABA(A) receptor composition and function have been well characterized, whereas much less is known about native GABA(C) receptors. Differences in molecular composition, anatomical distributions, and physiological properties strongly suggest that GABA(A) receptors and GABA(C) receptors have distinct functional roles in the CNS. To determine the functional role of GABA(C) receptors, we eliminated their expression in mice using a knock-out strategy. Although native rodent GABA(C) receptors are composed of rho1 and rho2 subunits, we show that after rho1 subunit expression was selectively eliminated there was no GABA(C) receptor expression. We assessed GABA(C) receptor function in the retina because GABA(C) receptors are highly expressed on the axon terminals of rod bipolar cells and because this site modulates the visual signal to amacrine and ganglion cells. In GABA(C)rho1 null mice, GABA-evoked responses, normally mediated by GABA(C) receptors, were eliminated, and signaling from rod bipolar cells to third order cells was altered. These data demonstrate that elimination of the GABA(C)rho1 subunit, via gene targeting, results in the absence of GABA(C) receptors in the retina and selective alterations in normal visual processing.

G-Protein receptor kinase 1 (GRK1) ("rhodopsin kinase") is necessary for the inactivation of photoactivated rhodopsin, the light receptor of the G-protein transduction cascade of rod photoreceptors. GRK1 has also been reported to be present in retinal cones in which its function is unknown. To examine the role of GRK1 in retinal cone signaling pathways, we measured in mice having null mutations of GRK1 (GRK1 -/-) cone-driven electroretinographic (ERG) responses, including an a-wave component identified as the field potential generated by suppression of the circulating current of the cone photoreceptors. Dark-adapted GRK1 -/- animals generated cone-driven ERGs having saturating amplitudes and sensitivities in both visible and UV spectral regions similar to those of wild-type (WT) mice. However, after exposure to a bright conditioning flash, the cone-driven ERGs of GRK1 -/- animals recovered 30-50 times more slowly than those of WT mice and similarly slower than the cone-driven ERGs of mice homozygously null for arrestin (Arrestin -/-), whose cone (but not rod) response recoveries were found to be as rapid as those of WT. Our observations argue that GRK1 is essential for normal deactivation of murine cone phototransduction and provide the first functional evidence for a major role of a specific GRK in the inactivation of vertebrate cone phototransduction.