Reduced levels of miRNAs 449 and 34 in sperm of mice and men exposed to early life stress.

Exposure of male mice to early life stress alters the levels of specific sperm miRNAs that promote stress-associated behaviors in their offspring. To begin to evaluate whether similar phenomena occur in men, we searched for sperm miRNA changes that occur in both mice and men exposed to early life stressors that have long-lasting effects. For men, we used the Adverse Childhood Experience (ACE) questionnaire. It reveals the degree of abusive and/or dysfunctional family experiences when young, which increases risks of developing future psychological and physical disorders. For male mice, we used adolescent chronic social instability (CSI) stress, which not only enhances sociability defects for >1 year, but also anxiety and defective sociability in female offspring for multiple generations through the male lineage. Here we found a statistically significant inverse correlation between levels of multiple miRNAs of the miR-449/34 family and ACE scores of Caucasian males. Remarkably, we found members of the same sperm miRNA family are also reduced in mice exposed to CSI stress. Thus, future studies should be designed to directly test whether reduced levels of these miRNAs could be used as unbiased indicators of current and/or early life exposure to severe stress. Moreover, after mating stressed male mice, these sperm miRNA reductions persist in both early embryos through at least the morula stage and in sperm of males derived from them, suggesting these miRNA changes contribute to transmission of stress phenotypes across generations. Since offspring of men exposed to early life trauma have elevated risks for psychological disorders, these findings raise the possibility that a portion of this risk may be derived from epigenetic regulation of these sperm miRNAs.

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 vision's 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.

CCN5 Expression in mammals. III. Early embryonic mouse development.

CCN proteins play crucial roles in development, angiogenesis, cell motility, matrix turnover, proliferation, and other fundamental cell processes. Early embryonic lethality in CCN5 knockout and over-expressing mice led us to characterize CCN5 distribution in early development. Previous papers in this series showed that CCN5 is expressed widely in mice from E9.5 to adult; however, its distribution before E9.5 has not been studied. To fill this gap in our knowledge of CCN5 expression in mammals, RT-PCR was performed on preimplantation murine embryos: 1 cell, 2 cell, 4 cell, early morula, late morula, and blastocyst. CCN5 mRNA was not detected in 1, 2, or 4 cell embryos. It was first detected at the early morula stage and persisted to the preimplantation blastocyst stage. Immunohistochemical staining showed widespread CCN5 expression in post-implantation blastocysts (E4.5), E5.5, E6.5, and E7.5 stage embryos. Consistent with our previous study on E9.5 embryos, this expression was not limited to a particular germ layer or cell type. The widespread distribution of CCN5 in early embryos suggests a crucial role in development.

Rhodopsin expression level affects rod outer segment morphology and photoresponse kinetics.

BACKGROUND: The retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly. METHODOLOGY/PRINCIPAL FINDINGS: The structure and function of rods from hemizygous rhodopsin knockout (R+/-) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/- rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics. CONCLUSIONS/SIGNIFICANCE: Rhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/- rods to light.

Palmitoylation stabilizes unliganded rod opsin.

S-palmitoylation is a conserved feature in many G protein-coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm(-/-)) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm(-/-) mice were crossed with Lrat(-/-) mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm(-/-)Lrat(-/-) mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm(-/-)Gnat1(-/-) mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.

Direct rod input to cone BCs and direct cone input to rod BCs challenge the traditional view of mammalian BC circuitry.

Bipolar cells are the central neurons of the retina that transmit visual signals from rod and cone photoreceptors to third-order neurons in the inner retina and the brain. A dogma set forth by early anatomical studies is that bipolar cells in mammalian retinas receive segregated rod/cone synaptic inputs (either from rods or from cones), and here, we present evidence that challenges this traditional view. By analyzing light-evoked cation currents from morphologically identified depolarizing bipolar cells (DBCs) in the wild-type and three pathway-specific knockout mice (rod transducin knockout [Tralpha(-/-)], connexin36 knockout [Cx36(-/-)], and transcription factor beta4 knockout [Bhlhb4(-/-)]), we show that a subpopulation of rod DBCs (DBC(R2)s) receives substantial input directly from cones and a subpopulation of cone DBCs (DBC(C1)s) receives substantial input directly from rods. These results provide evidence of the existence of functional rod-DBC(C) and cone-DBC(R) synaptic pathways in the mouse retina as well as the previously proposed rod hyperpolarizing bipolar-cells pathway. This is grounds for revising the mammalian rod/cone bipolar cell dogma.

Genetic dissection of rod and cone pathways in the dark-adapted mouse retina.

A monumental task of the mammalian retina is to encode an enormous range (>10(9)-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod-bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [WT, Cx36(-/-), Bhlhb4(-/-), and Tralpha(-/-)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod-cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina.

Modulation of molecular interactions and function by rhodopsin palmitylation.

Rhodopsin is palmitylated at two cysteine residues in its carboxyl terminal region. We have looked at the effects of palmitylation on the molecular interactions formed by rhodopsin using single-molecule force spectroscopy and the function of rhodopsin using both in vitro and in vivo approaches. A knockin mouse model expressing palmitate-deficient rhodopsin was used for live animal in vivo studies and to obtain native tissue samples for in vitro assays. We specifically looked at the effects of palmitylation on the chromophore-binding pocket, interactions of rhodopsin with transducin, and molecular interactions stabilizing the receptor structure. The structure of rhodopsin is largely unperturbed by the absence of palmitate linkage. The binding pocket for the chromophore 11-cis-retinal is minimally altered as palmitate-deficient rhodopsin exhibited the same absorbance spectrum as wild-type rhodopsin. Similarly, the rate of release of all-trans-retinal after light activation was the same both in the presence and absence of palmitylation. Significant differences were observed in the rate of transducin activation by rhodopsin and in the force required to unfold the last stable structural segment in rhodopsin at its carboxyl terminal end. A 1.3-fold reduction in the rate of transducin activation by rhodopsin was observed in the absence of palmitylation. Single-molecule force spectroscopy revealed a 2.1-fold reduction in the normalized force required to unfold the carboxyl terminal end of rhodopsin. The absence of palmitylation in rhodopsin therefore destabilizes the molecular interactions formed in the carboxyl terminal end of the receptor, which appears to hinder the activation of transducin by light-activated rhodopsin.

Overexpression of rhodopsin alters the structure and photoresponse of rod photoreceptors.

Rhodopsins are densely packed in rod outer-segment membranes to maximize photon absorption, but this arrangement interferes with transducin activation by restricting the mobility of both proteins. We attempted to explore this phenomenon in transgenic mice that overexpressed rhodopsin in their rods. Photon capture was improved, and, for a given number of photoisomerizations, bright-flash responses rose more gradually with a reduction in amplification--but not because rhodopsins were more tightly packed in the membrane. Instead, rods increased their outer-segment diameters, accommodating the extra rhodopsins without changing the rhodopsin packing density. Because the expression of other phototransduction proteins did not increase, transducin and its effector phosphodiesterase were distributed over a larger surface area. That feature, as well as an increase in cytosolic volume, was responsible for delaying the onset of the photoresponse and for attenuating its amplification.

Rhodopsin-mediated retinitis pigmentosa.

Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous group of diseases that cause blindness. Mutations within the rhodopsin gene account for approximately 25% of autosomal dominantly inherited RP cases. Therefore, understanding the mechanisms causing rhodopsin-mediated RP has a significant health impact. To date, results from multiple labs indicate that rhodopsin-mediated RP pathogenesis does not share a common mechanism of degeneration. There is strong evidence that multiple mechanisms are involved, including protein misfolding, mislocalization, release of toxic products, and aberrant signaling. Development of effective treatments requires investigation of the mechanism involved in the different rhodopsin mutations. This chapter focuses on the mechanisms by which rhodopsin mutations cause retinal degeneration, as well as potential therapeutic strategies to treat the disease.

A novel form of transducin-dependent retinal degeneration: accelerated retinal degeneration in the absence of rod transducin.

PURPOSE: Rhodopsin mutations account for approximately 25% of human autosomal dominant retinal degenerations. However, the molecular mechanisms by which rhodopsin mutations cause photoreceptor cell death are unclear. Mutations in genes involved in the termination of rhodopsin signaling activity have been shown to cause degeneration by persistent activation of the phototransduction cascade. This study examined whether three disease-associated rhodopsin substitutions Pro347Ser, Lys296Glu, and the triple mutant Val20Gly, Pro23His, Pro27Leu (VPP) caused degeneration by persistent transducin-mediated signaling activity. METHODS: Transgenic mice expressing each of the rhodopsin mutants were crossed onto a transducin alpha-subunit null (Tr(alpha)(-/-)) background, and the rates of photoreceptor degeneration were compared with those of transgenic mice on a wild-type background. RESULTS: Mice expressing VPP-substituted rhodopsin had the same severity of degeneration in the presence or absence of Tr(alpha). Unexpectedly, mice expressing Pro347Ser- or Lys296Glu-substituted rhodopsins exhibited faster degeneration on a Tr(alpha)(-/-) background. To test whether the absence of alpha-transducin contributed to degeneration by favoring the formation of stable rhodopsin/arrestin complexes, mutant Pro347Ser(+), Tr(alpha)(-/-) mice lacking arrestin (Arr(-/-)) were analyzed. Rhodopsin/arrestin complexes were found not to contribute to degeneration. CONCLUSIONS: The authors hypothesized that the decay of metarhodopsin to apo-opsin and free all-trans-retinaldehyde is faster with Pro347Ser-substituted rhodopsin than it is with wild-type rhodopsin. Consistent with this, the lipofuscin fluorophores A2PE, A2E, and A2PE-H(2), which form from retinaldehyde, were elevated in Pro347Ser transgenic mice.

Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins.

Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments.

G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor.

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.

Noninvasive volumetric imaging and morphometry of the rodent retina with high-speed, ultrahigh-resolution optical coherence tomography.

PURPOSE: To demonstrate high-speed, ultrahigh-resolution optical coherence tomography (OCT) for noninvasive, in vivo, three-dimensional imaging of the retina in rat and mouse models. METHODS: A high-speed, ultrahigh-resolution OCT system using spectral, or Fourier domain, detection has been developed for small animal retinal imaging. Imaging is performed with a contact lens and postobjective scanning. An axial image resolution of 2.8 mum is achieved with a spectrally broadband superluminescent diode light source with a bandwidth of approximately 150 nm at approximately 900-nm center wavelength. Imaging can be performed at 24,000 axial scans per second, which is approximately 100 times faster than previous ultrahigh-resolution OCT systems. High-definition and three-dimensional retinal imaging is performed in vivo in mouse and rat models. RESULTS: High-speed, ultrahigh-resolution OCT enabled high-definition, high transverse pixel density imaging of the murine retina and visualization of all major intraretinal layers. Raster scan protocols enabled three-dimensional volumetric imagingand comprehensive retinal segmentation algorithms allowed measurement of retinal layers. An OCT fundus image, akin to a fundus photograph was generated by axial summation of three-dimensional OCT data, thus enabling precise registration of OCT measurements to retinal fundus features. CONCLUSIONS: High-speed, ultrahigh-resolution OCT enables imaging of retinal architectural morphology in small animal models. OCT fundus images allow precise registration of OCT images and repeated measurements with respect to retinal fundus features. Three-dimensional OCT imaging enables visualization and quantification of retinal structure, which promises to allow repeated, noninvasive measurements to track disease progression, thereby reducing the need for killing the animal for histology. This capability can accelerate basic research studies in rats and mice and their translation into clinical patient care.

Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis.

BACKGROUND: RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. METHODS AND FINDINGS: Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. CONCLUSIONS: By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65(-/-) mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.

Phototransduction in a transgenic mouse model of Nougaret night blindness.

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.

Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.

Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl(-/-) mice, using suction pipette recordings from their "inner segments" (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the alpha-subunit of the G-protein transducin (G(t)alpha(-/-)), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (lambda(max) = 360 nm) and M- (lambda(max) = 508 nm) cone opsins and to be maximally sensitive at 360 nm ("S-cones"); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm ("M-cones"). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was approximately 70 ms, and approximately 0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A approximately 4 s(-2)) was found to be about twofold lower than in rods (A approximately 8 s(-2)). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate "dark light."

A critical role of CaBP4 in the cone synapse.

PURPOSE: CaBP4, a photoreceptor-specific protein of the rods and cones, is essential for the development and maintenance of the mouse photoreceptor synapse. In this study, double CaBP4/rod alpha-transducin knockout (Cabp4(-/-)Gnat1(-/-)) mice lacking the rod-mediated component of electrophysiologic responses were generated and analyzed to investigate the role of CaBP4 in cones. METHODS: The retinal morphology and physiologic function of 2-month-old Cabp4(-/-)Gnat1(-/-) mice were analyzed using immunocytochemistry, electron microscopy, and single-flash and flicker electroretinography (ERG). RESULTS: The thickness of the outer plexiform layer and the number of photoreceptor terminals in Cabp4(-/-)Gnat1(-/-) mice were reduced to levels similar to those of Cabp4(-/-) mice. Single-flash and flicker ERG showed that the amplitude and sensitivity of the b-wave in the Cabp4(-/-)Gnat1(-/-) mice were severely attenuated compared with those in wild-type and Gnat1(-/-) mice. CONCLUSIONS: Results indicate that the cone synaptic function in Cabp4(-/-)Gnat1(-/-) mice was severely disrupted, whereas the morphologic defects observed in Cabp4(-/-)Gnat1(-/-) mice were similar to those of single Cabp4(-/-) knockout mice. This and a previous study reveal that CaBP4 is critical for signal transmission from rods and cones to second-order neurons.

Enhanced shutoff of phototransduction in transgenic mice expressing palmitoylation-deficient rhodopsin.

Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.