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FATP1 is involved in lipid transport into cells and in intracellular lipid metabolism. We showed previously that this protein interacts with and inhibits the limiting-step isomerase of the visual cycle RPE65. Here, we aimed to analyze the effect of Fatp1-deficiency in vivo on the visual cycle, structure and function, and on retinal aging. Among the Fatp family members, we observed that only Fatp1 and 4 are expressed in the control retina, in both the neuroretina and the retinal pigment epithelium. In the neuroretina, Fatp1 is mostly expressed in photoreceptors. In young adult Fatp1(-/-) mice, Fatp4 expression was unchanged in retinal pigment epithelium and reduced two-fold in the neuroretina as compared to Fatp1(+/+) mice. The Fatp1(-/-) mice had a preserved retinal structure but a decreased electroretinogram response to light. These mice also displayed a delayed recovery of the b-wave amplitude after bleaching, however, visual cycle speed was unchanged, and both retinal pigment epithelium and photoreceptors presented the same fatty acid pattern compared to controls. In 2 year-old Fatp1(-/-) mice, transmission electron microscopy studies showed specific abnormalities in the retinas comprising choroid vascularization anomalies and thickening of the Bruch membrane with material deposits, and sometimes local disorganization of the photoreceptor outer segments. These anomalies lead us to speculate that the absence of FATP1 accelerates the aging process.

Autophosphorylation of αCaMKII is regarded as a 'molecular memory' for Ca(2+) transients and a crucial mechanism in aversely, but less so in appetitively, motivated learning and memory. While there is a growing body of research implicating αCaMKII in general in behavioral responses to threat or fearful stimuli, little is known about the contribution of the autophosphorylation. The present study asked how αCaMKII autophosphorylation controls anxiety-like behavioral responses toward novel, potentially threatening stimuli. We tested homozygous and heterozygous T286A αCaMKII autophosphorylation deficient mice and wild types in a systematic series of behavioral tests. Homozygous mutants were more active in the open field test and showed reduced anxiety-related behavior in the light/dark test, but these findings were confounded by a hyperlocomotor phenotype. The analysis of elevated plus maze showed significantly reduced anxiety-related behavior in the αCaMKII autophosphorylation-deficient mice which appeared to mediate a hyperlocomotor response. An analysis of home cage behavior, where neither novel nor threatening stimuli were present, showed no differences in locomotor activity between genotypes. Increased locomotion was not observed in the novel object exploration test in the αCaMKII autophosphorylation-deficient mice, implying that hyperactivity does not occur in response to discrete novel stimuli. The present data suggest that the behavior of αCaMKII autophosphorylation-deficient mice cannot simply be described as a low anxiety phenotype. Instead it is suggested that αCaMKII autophosphorylation influences locomotor reactivity to novel environments that are potentially, but not necessarily threatening., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Semaphorin 5A (Sema5A) expression is reduced in the brain of individuals with autism, thus mice with reduced Sema5A levels may serve as a model of this neurodevelopmental disorder. We tested male and female Sema5a knockout mice (B6.129P2SEMA5A(<)™(1DGEN>)/J) and C57BL/6J controls for emotionality, visual ability, prepulse inhibition, motor learning and cognition. Overall, there were only two genotype differences in emotionality: Sema5a mutant mice had more stretch-attend postures in the elevated plus-maze and more defecations in the open field. All mice could see, but Sema5a mice had better visual ability than C57BL/6J mice. There were no genotype differences in sensory-motor gating. Sema5a mice showed higher levels of activity in the elevated plus-maze and light/dark transition box, and there were sex by genotype differences in the Rotarod, suggesting a sex difference in balance and coordination differentially affected by Sema5a. There were no genotype effects on cognition: Sema5a mice did not differ from C57BL/6J in the Morris water maze, set-shifting or cued and contextual fear conditioning. In the social recognition test, all mice preferred social stimuli, but there was no preference for social novelty, thus the Sema5A mice do not have a deficit in social behavior. Overall, there were a number of sex differences, with females showing greater activity and males performing better in tests of spatial learning and memory, but no deficits in the behavior of Sema5A mice. We conclude that the Sema5a mice do not meet the behavioral criteria for a mouse model of autism., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Glutamate and N-methyl-d-aspartate receptor (NMDAR) dysfunction is strongly implicated in the pathophysiology of mood and anxiety disorders. Treatment with NMDAR antagonists has antidepressant efficacy in treatment-resistant depressives. In preclinical rodent models, NMDAR antagonist administration reduces anxiety- and stress-related behaviors in concert with increases in prefrontal cortical (PFC) dendritic spinogenesis and synaptic proteins. While these effects have been attributed to actions at the NMDAR GluN2B subunit, the precise role of cortical GluN2B in mediating emotional behaviors and stress-responsivity is not fully understood. Here, we employed a novel mutant model in which the GluN2B subunit is postnatally deleted in principal neurons in the cortex and the dorsal CA1 subregion of the hippocampus. GluN2BKO mice were phenotyped on a battery of tests for anxiety-related (light/dark exploration, stress-induced hyperthermia) and antidepressant-sensitive (sucrose preference, novelty-induced hypophagia, single-trial forced swim) behaviors. A novel repeated inescapable forced swim paradigm (riFS) was developed to assess behavioral responses to repeated stress in the GluN2BKO mice. For comparison, non-mutant C57BL/6J mice were tested for single-trial forced swim behavior after systemic Ro 25-6981 treatment and for riFS behavior after lesions of the ventromedial prefrontal cortex. riFS-induced alterations in corticolimbic GluN2B expression were also examined in C57BL/6J mice. We found that GluN2BKO mice reduced "despair-like" behavior in the riFS procedure, as compared to GluN2BFLOX controls. By contrast, GluN2BKO mice showed minimal alterations on anxiety-like or antidepressant-sensitive assays, including the single-trial forced swim test. In C57BL/6J mice, induction of "despair-like" responses in the riFS test was attenuated by vmPFC lesions, and was associated with changes in limbic GluN2B expression. Collectively, these data suggest that cortical GluN2B plays a major role in modulating adaptive responses to stress. Current findings provide further support for GluN2B as a key mechanism underlying stress responsivity, and a novel pharmacotherapeutic target for stress-related neuropsychiatric disorders., (Published by Elsevier Ltd.)

The members of the R7 regulator of G-protein signaling (RGS) protein subfamily are versatile regulators of G-protein signaling throughout the nervous system. Recent studies indicate that they are often found in complexes with membrane anchor proteins that serve as versatile modulators of their activity, intracellular targeting, and stability. One striking example is the interplay between the membrane anchor R9AP and the RGS9-1 · Gβ5 GTPase-activating complex responsible for the rapid inactivation of the G-protein transducin in vertebrate photoreceptor cells during their recovery from light excitation. The amount of this complex in photoreceptors sets their temporal resolution and is precisely regulated by the expression level of R9AP, which serves to protect the RGS9-1 and Gβ5 subunits from intracellular proteolysis. In this study, we investigated the mechanism by which R9AP performs its protective function in mouse rods and found that it is entirely confined to recruiting RGS9-1 · Gβ5 to cellular membranes. Furthermore, membrane attachment of RGS9-1 · Gβ5 is sufficient for its stable expression in rods even in the absence of R9AP. Our second finding is that RGS9-1 · Gβ5 possesses targeting information that specifies its exclusion from the outer segment and that this information is neutralized by association with R9AP to allow outer segment targeting. Finally, we demonstrate that the ability of R9AP · RGS9-1 · Gβ5 to accelerate GTP hydrolysis on transducin is independent of its means of membrane attachment, since replacing the transmembrane domain of R9AP with a site for lipid modification did not impair the catalytic activity of this complex.

BDNF regulates components of cognitive processes and has been implicated in psychiatric disorders. Here we report that genetic overexpression of the BDNF mature isoform (BDNF-tg) in female mice impaired working memory functions while sparing components of fear conditioning. BDNF-tg mice also displayed reduced breeding efficiency, higher anxiety-like scores, high self-grooming, impaired prepulse inhibition, and higher susceptibility to seizures when placed in a new empty cage, as compared with wild-type (WT) littermate controls. Control measures of general health, locomotor activity, motor coordination, depression-related behaviors, and sociability did not differ between genotypes. The present findings, indicating detrimental effects of life-long increased BDNF in mice, may inform human studies evaluating the role of BDNF functional genetic variations on cognitive abilities and vulnerability to psychiatric disorders.

Several reports have implicated a role for the histidine triad nucleotide-binding protein-1 (Hint1) in psychiatric disorders. We have studied the emotional behaviour of male Hint1 knockout (Hint1 KO) mice in a battery of tests and performed biochemical analyses on brain tissue. The behavioural analysis revealed that Hint1 KO mice exhibit an increased emotionality phenotype compared to wildtype (WT) mice, while no significant differences in locomotion or general exploratory activity were noted. In the elevated plus-maze (EPM) test, the Hint1 KO animals entered the open arms of the apparatus less often than WT littermates. Similarly, in the dark-light box test, Hint1 KO mice spent less time in the lit compartment and the number of entries were reduced, which further confirmed an increased anxiety-related behaviour. Moreover, the Hint1 KO animals showed significantly more struggling and less floating behaviour in the forced swim test (FST), indicating an increased emotional arousal in aversive situations. Hint1 is known as a protein kinase C (PKC) interacting protein. Western blot analysis showed that PKCγ expression was elevated in Hint1 KO compared to WT mice. Interestingly, PKCγ mRNA levels of the two groups did not show a significant difference, implying a post-transcriptional PKCγ regulation. In addition, PKC enzymatic activity was increased in Hint1 KO compared to WT mice. In summary, our results indicate a role for Hint1 and PKCγ in modulating anxiety-related and stress-coping behaviour in mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.

The possible role of the CB(2) receptor (CB(2)r) in psychiatric disorders has been considered. Several animal models use knockout (KO) mice that display schizophrenia-like behaviors and this study evaluated the role of CB(2)r in the regulation of such behaviors. Mice lacking the CB(2)r (CB(2)KO) were challenged in open field, light-dark box, elevated plus-maze, tail suspension, step down inhibitory avoidance, and pre-pulse inhibition tests (PPI). Furthermore, the effects of treatment with cocaine and risperidone were evaluated using the OF and the PPI test. Gene expression of dopamine D(2) (D(2)r), adrenergic-α(2C) (α(2C)r), serotonergic 5-HT(2A) and 5-HT(2C) receptors (5-HT(2A)r and 5-HT(2C)r) were studied by RT-PCR in brain regions related to schizophrenia. Deletion of CB(2)r decreased motor activity in the OF test, but enhanced response to acute cocaine and produced mood-related alterations, PPI deficit, and cognitive impairment. Chronic treatment with risperidone tended to impair PPI in WT mice, whereas it 'normalized' the PPI deficit in CB(2)KO mice. CB(2)KO mice presented increased D(2)r and α(2C)r gene expressions in the prefrontal cortex (PFC) and locus coeruleus (LC), decreased 5-HT(2C)r gene expression in the dorsal raphe (DR), and 5-HT(2A)r gene expression in the PFC. Chronic risperidone treatment in WT mice left α(2C)r gene expression unchanged, decreased D(2)r gene expression (15 μg/kg), and decreased 5-HT(2C)r and 5-HT(2A)r in PFC and DR. In CB(2)KO, the gene expression of D(2)r in the PFC, of α(2C)r in the LC, and of 5-HT(2C)r and 5-HT(2A)r in PFC was reduced; 5-HT(2C)r and 5-HT(2A)r gene expressions in DR were increased after treatment with risperidone. These results suggest that deletion of CB(2)r has a relation with schizophrenia-like behaviors. Pharmacological manipulation of CB(2)r may merit further study as a potential therapeutic target for the treatment of schizophrenia-related disorders.

Lithium has been used extensively for mood stabilization, and it is particularly efficacious in the treatment of bipolar mania. Like other drugs used in the treatment of psychiatric diseases, it has little effect on the mood of healthy individuals. Our previous studies found that mice with a mutation in the Clock gene (ClockΔ19) have a complete behavioral profile that is very similar to human mania, which can be reversed with chronic lithium treatment. However, the cellular and physiological effects that underlie its targeted therapeutic efficacy remain unknown. Here we find that ClockΔ19 mice have an increase in dopaminergic activity in the ventral tegmental area (VTA), and that lithium treatment selectively reduces the firing rate in the mutant mice with no effect on activity in wild-type mice. Furthermore, lithium treatment reduces nucleus accumbens (NAc) dopamine levels selectively in the mutant mice. The increased dopaminergic activity in the Clock mutants is associated with cell volume changes in dopamine neurons, which are also rescued by lithium treatment. To determine the role of dopaminergic activity and morphological changes in dopamine neurons in manic-like behavior, we manipulated the excitability of these neurons by overexpressing an inwardly rectifying potassium channel subunit (Kir2.1) selectively in the VTA of ClockΔ19 mice and wild-type mice using viral-mediated gene transfer. Introduction of this channel mimics the effects of lithium treatment on the firing rate of dopamine neurons in ClockΔ19 mice and leads to a similar change in dopamine cell volume. Furthermore, reduction of dopaminergic firing rates in ClockΔ19 animals results in a normalization of locomotor- and anxiety-related behavior that is very similar to lithium treatment; however, it is not sufficient to reverse depression-related behavior. These results suggest that abnormalities in dopamine cell firing and associated morphology underlie alterations in anxiety-related behavior in bipolar mania, and that the therapeutic effects of lithium come from a reversal of these abnormal phenotypes.

Although rolling mouse Nagoya, a Ca(v)2.1α(1) mutant, exhibits ataxia and elevated serotonin concentrations, heterozygous mice have not been examined in detail. Patients with heterozygous mutations in this orthologous gene exhibit neurological disorders. To examine the emotional behavior of heterozygous mice, we used behavioral tasks and examined Ca(v)2.1α(1) message levels, tryptophan hydroxylase expression patterns, and monoamine concentrations in 2- and 22-month-old mice. Reduced anxiety in the elevated plus maze, light-dark exploration, and marble-burying behavioral tests and reduced depression in the forced swimming and tail suspension tests were observed in 22-month-old heterozygous mice compared to aged-matched wild-type mice. The levels of mutant-type Ca(v)2.1α(1) message, phosphorylation of tryptophan hydroxylase, and serotonin increased in the brainstems of 22-month-old heterozygous mice. No difference was observed between 2-month-old heterozygous and wild-type mice in these analyses. These findings suggest that heterozygous mice show age-related emotional changes due to alterations in the serotonin system associated with mutant-type Ca(v)2.1α(1), and that heterozygous mice may represent a novel model to delineate the interaction between Ca(v)2.1 function and synaptic transmission., (Copyright © 2009 Elsevier Inc. All rights reserved.)

Physical exercise is considered to exert a positive neurophysiological effect that helps to maintain normal brain activity in the elderly. Expectations that it could help to fight Alzheimer's disease (AD) were recently raised. This study analyzed the effects of different patterns of physical exercise on the 3xTg-AD mouse. Male and female 3xTg-AD mice at an early pathological stage (4-month-old) have had free access to a running wheel for 1 month, whereas mice at a moderate pathological stage(7-month-old) have had access either during 1 or 6 months. The non-transgenic mouse strain was used as a control. Parallel animal groups were housed in conventional conditions. Cognitive loss and behavioral and psychological symptoms of dementia (BPSD)-like behaviors were present in the 3xTg-AD mice along with alteration in synaptic function and ong-term potentiation impairment in vivo. Brain tissue showed AD-pathology and oxidative-related changes. Disturbances were more severe at the older age tested. Oxidative stress was higher in males but other changes were similar or higher in females. Exercise treatment ameliorated cognitive deterioration and BPSD-like behaviors such as anxiety and the startle response. Synaptic changes were partially protected by exercise. Oxidative stress was reduced. The best neuroprotection was generally obtained after 6 months of exercise in 7-month-old 3xTg-AD mice. Improved sensorimotor function and brain tissue antioxidant defence were induced in both 3xTg-AD and NonTg mice. Therefore, the benefits of aerobic physical exercise on synapse, redox homeostasis, and general brain function demonstrated in the 3xTg-AD mouse further support the value of this healthy life-style against neurodegeneration.

Upon illumination, visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments, vertebrate outer segments or invertebrate rhabdomeres, where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes. Immunocytochemistry and live imaging of GFP-tagged Arr2 demonstrate rapid reversible translocation to stimulated rhabdomeres in stoichiometric proportion to rhodopsin photoisomerization. Translocation is very rapid in normal photoreceptors (time constant <10 s) and can also be resolved in the time course of electroretinogram recordings. Genetic elimination of key phototransduction proteins, including phospholipase C (PLC), Gq, and the light-sensitive Ca2+-permeable TRP channels, slows translocation by 10- to 100-fold. Our results indicate that Arr2 translocation in Drosophila photoreceptors is driven by diffusion, but profoundly accelerated by phototransduction and Ca2+ influx., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

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.

We investigated the gender effects on transient pupillary light reflex (PLR) in healthy young adults between 18 and 22 years old. Both dark-adapted and light-adapted PLRs were measured using green and red stimuli of different intensities. The results indicate that females had significantly larger relative constriction amplitudes than males in a dark-adapted condition. This gender effect depends on the stimulus intensities. The relative constriction amplitude in female subjects increased faster than it did in the males with the stimulus intensity. We did not observe any significant gender differences in the other PLR parameters, including latency, constriction speed, and recovery speed.

The G90D rhodopsin mutation is known to produce congenital night blindness in humans. This mutation produces a similar condition in mice, because rods of animals heterozygous (D+) or homozygous (D+/+) for this mutation have decreased dark current and sensitivity, reduced Ca(2+), and accelerated values of tau(REC) and tau(D), similar to light-adapted wild-type (WT) rods. Our experiments indicate that G90D pigment activates the cascade, producing an equivalent background light of approximately 130 Rh* rod(-1) for D+ and 890 Rh* rod(-1) for D+/+. The active species of the G90D pigment could be unregenerated G90D opsin or G90D rhodopsin, either spontaneously activated (as Rh*) or in some other form. Addition of 11-cis-retinal in lipid vesicles, which produces regeneration of both WT and G90D opsin in intact rods and ROS membranes, had no effect on the waveform or sensitivity of dark-adapted G90D responses, indicating that the active species is not G90D opsin. The noise spectra of dark-adapted G90D and WT rods are similar, and the G90D noise variance is much less than of a WT rod exposed to background light of about the same intensity as the G90D equivalent light, indicating that Rh* is not the active species. We hypothesize that G90D rhodopsin undergoes spontaneous changes in molecular conformation which activate the transduction cascade with low gain. Our experiments provide the first indication that a mutant form of the rhodopsin molecule bound to its 11-cis-chromophore can stimulate the visual cascade spontaneously at a rate large enough to produce visual dysfunction.

The retinal pigment epithelium (RPE) plays an essential role in maintaining the health of the retina. The RPE is also the site of pathologic processes in a wide variety of retinal disorders including monogenic retinal dystrophies, age-related macular degeneration, and retinal detachment. Despite intense interest in the RPE, little is known about its molecular response to ocular damage or disease. We have conducted a comprehensive analysis of changes in transcript abundance (the "genomic response") in the murine RPE after light damage. Several dozen transcripts, many related to cell-cell signaling, show significant increases in abundance in response to bright light; transcripts encoding visual cycle proteins show a decrease in abundance. Similar changes are induced by retinal detachment. Environmental and genetic perturbations that modulate the RPE response to bright light suggest that this response is controlled by the retina. In contrast to the response to bright light, the RPE response to retinal detachment overrides these modulatory affects.

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker in mammals. A salient feature of the SCN is that cells of a particular phenotype are topographically organized; this organization defines functionally distinct subregions that interact to generate coherent rhythmicity. In Syrian hamsters (Mesocricetus auratus), a dense population of directly retinorecipient calbindin D(28K) (CalB) neurons in the caudal SCN marks a subregion critical for circadian rhythmicity. In mouse SCN, a dense cluster of CalB neurons occurs during early postnatal development, but in the adult CalB neurons are dispersed through the SCN. In the adult retina CalB colocalizes with melanopsin-expressing ganglion cells. In the present study, we explored the role of CalB in modulating circadian function and photic entrainment by investigating mice with a targeted mutation of the CalB gene (CalB-/- mice). In constant darkness (DD), CalB-/- animals either become arrhythmic (40%) or exhibit low-amplitude locomotor rhythms with marked activity during subjective day (60%). Rhythmic clock gene expression is blunted in these latter animals. Importantly, CalB-/- mice exhibit anomalies in entrainment revealed following transfer from a light : dark cycle to DD. Paradoxically, responses to acute light pulses measured by behavioral phase shifts, SCN FOS protein and Period1 mRNA expression are normal. Together, the developmental pattern of CalB expression in mouse SCN, the presence of CalB in photoresponsive ganglion cells and the abnormalities seen in CalB-/- mice suggest an important role for CalB in mouse circadian function.

Many aspects of photoreceptor metabolism are regulated as diurnal or circadian rhythms. The nature of the signals that drive rhythms in mouse photoreceptors is unknown. Dopamine amacrine cells in mouse retina express core circadian clock genes, leading us to test the hypothesis that dopamine regulates rhythms of protein phosphorylation in photoreceptor cells. To this end we investigated the phosphorylation of phosducin, an abundant photoreceptor-specific phosphoprotein. In mice exposed to a daily light-dark cycle, robust daily rhythms of phosducin phosphorylation and retinal dopamine metabolism were observed. Phospho-phosducin levels were low during the daytime and high at night, and correlated negatively with levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid. The effect of light on phospho-phosducin levels was mimicked by pharmacological activation of dopamine D4 receptors. The amplitude of the diurnal rhythm of phospho-phosducin was reduced by > 50% in D4 receptor-knockout mice, due to higher daytime levels of phospho-phosducin. In addition, the daytime level of phospho-phosducin was significantly elevated by L-745,870, a dopamine D4 receptor antagonist. These data indicate that dopamine and other light-dependent processes cooperatively regulate the diurnal rhythm of phosducin phosphorylation. Under conditions of constant darkness a circadian rhythm of phosducin phosphorylation was observed, which correlated negatively with the circadian rhythm of 3,4-dihydroxyphenylacetic acid levels. The circadian fluctuation of phospho-phosducin was completely abolished by constant infusion of L-745,870, indicating that the rhythm of phospho-phosducin level is driven by dopamine. Thus, dopamine release in response to light and circadian clocks drives daily rhythms of protein phosphorylation in photoreceptor cells.

The rod pigment, rhodopsin, shows spontaneous isomerization activity. This quantal noise produces a dark light of approximately 0.01 photons s(-1) rod(-1) in human, setting the threshold for rod vision. The spontaneous isomerization activity of human cone pigments has long remained a mystery because the effect of a single isomerized pigment molecule in cones, unlike that in rods, is small and beyond measurement. We have now overcome this problem by expressing human red cone pigment transgenically in mouse rods in order to exploit their large single-photon response, especially after genetic removal of a key negative-feedback regulation. Extrapolating the measured quantal noise of transgenic cone pigment to native human red cones, we obtained a dark rate of approximately 10 false events s(-1) cone(-1), almost 10(3)-fold lower than the overall dark transduction noise previously reported in primate cones. Our measurements provide a rationale for why mammalian red, green and blue cones have comparable sensitivities, unlike their amphibian counterparts.

Evolutionary and other functional accounts of the retina and its normal development highlight different aspects of control of its growth and form than genomic and mechanistic accounts. Discussing examples from opsin expression, developmental regulation of the eye's size and optical quality, regulation of eye size with respect to brain and body size, and the development of the fovea, these different aspects of control are contrasted. Contributions of mouse models, particularly with regard to relative timing of events in different species are reviewed, introducing a Web-based utility for exploration of timing issues (www.translatingtime.net). Variation at the individual level, in early experience, and also across species is an essential source of information to understand normal development and its pathologies.

We present the case report of two brothers, PF-21 years old and PN-19 years old, to whom the fundus examination, perimetry and dark adaptation established the diagnosis of Retinitis Pigmentosa. The otorhinolaryngology exam and the audiogram revealed, in both cases, bilateral sensorineural deafness. The simultaneous presence of these two conditions completes the clinical findings of Usher syndrome. The common ectodermic origin of the retina and the inner ear could explain this pathological association.

Gbeta5 exists as two splice variants, Gbeta5-S and Gbeta5-L, which interact with and stabilize the R7 members of the regulators of G-protein signaling (RGSs): RGS6, RGS7, RGS9, and RGS11. Although the role of Gbeta5-L and RGS9-1 is established in photoreceptors, the physiological functions of Gbeta5-S and other R7 RGS proteins remain unclear. We found that the electroretinogram of Gbeta5-/- mice lacks the b-wave component and that Gbeta5-S and RGS11 colocalize with Go alpha at the tips of the ON-bipolar cell dendrites. Unexpectedly, we found a significant reduction in the number of synaptic triads in the outer plexiform layer (OPL) of the Gbeta5-/- mice, which is evident at postnatal day 14. Transgenic expression of Gbeta5-L in rods failed to rescue the b-wave or the OPL defects. These results indicate that Gbeta5-S is indispensable for OPL integrity and normal light responses of the retina.

Highly abundant short-chain alcohol dehydrogenases (RDHs) in the retina were assumed to be involved in the recycling of 11-cis-retinal chromophore in the visual cycle. Mutations in human RDH genes are associated with Fundus albipunctatus, a mild form of night blindness (RDH5) and an autosomal recessive, childhood-onset severe retinal dystrophy (RDH12). Rdh12 knockout mice were found to be susceptible to light-induced photoreceptor apoptosis, whereas Rdh5 and Rdh8 knockout mice displayed only delayed dark adaptation. However, each knockout mouse eventually regenerated normal levels of visual pigments, suggesting that RDHs compensate for each other in the visual cycle. Here, we established RDH double knockout (Rdh8(-/-)Rdh12(-/-)) and triple knockout (Rdh5(-/-)Rdh8(-/-)Rdh12(-/-)) mice generated on various genetic backgrounds including a rod alpha-transducin knockout to test cone function. RDH activity was severely reduced in Rdh8(-/-)Rdh12(-/-) retina extracts, whereas Rdh8(-/-) RDH activity was intermediate and Rdh12(-/-) RDH activity was reduced only slightly. Surprisingly, all multiple knockout mice produced sufficient amounts of the chromophore to regenerate rhodopsin and cone pigments in vivo. Three-month-old Rdh8(-/-)Rdh12(-/-) mice characteristically displayed a slowly progressing rod-cone dystrophy accompanied by accumulation of N-retinylidene-N-retinylethanolamine (A2E), a toxic substance known to contribute to retinal degeneration. A2E accumulation and retinal degeneration were prevented by application of retinylamine, a potent retinoid cycle inhibitor. The results suggest that RDH8 and RDH12 are dispensable in support of the visual cycle but appear to be key components in clearance of free all-trans-retinal, thereby preventing A2E accumulation and photoreceptor cell death.

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Phosphorylation of rhodopsin by rhodopsin kinase GRK1 is an important desensitization mechanism in scotopic vision. For cone vision GRK1 is not essential. However, cone opsin is phosphorylated following light stimulation. In cone-dominant animals as well as in humans, but not in rodents, GRK7, a cone-specific homolog of GRK1, has been identified in cone outer segments. To investigate the function of GRK7 in vivo, we cloned two orthologs of grk7 in zebrafish and knocked down gene expression of grk7a in zebrafish larvae by morpholino antisense nucleotides. Photoresponse recovery in Grk7a-deficient larvae was delayed in electroretinographic measurements, and temporal contrast sensitivity was reduced, particularly under bright-light conditions. These results show that function of a cone-specific kinase is essential for cone vision in the zebrafish retina and argue that pigment bleaching and spontaneous decay alone are not sufficient for light adaptation and rapid cone response inactivation.

Nanophthalmos is a rare disorder of eye development characterized by extreme hyperopia (farsightedness), with refractive error in the range of +8.00 to +25.00 diopters. Because the cornea and lens are normal in size and shape, hyperopia occurs because insufficient growth along the visual axis places these lensing components too close to the retina. Nanophthalmic eyes show considerable thickening of both the choroidal vascular bed and scleral coat, which provide nutritive and structural support for the retina. Thickening of these tissues is a general feature of axial hyperopia, whereas the opposite occurs in myopia. We have mapped recessive nanophthalmos to a unique locus at 11q23.3 and identified four independent mutations in MFRP, a gene that is selectively expressed in the eye and encodes a protein with homology to Tolloid proteases and the Wnt-binding domain of the Frizzled transmembrane receptors. This gene is not critical for retinal function, as patients entirely lacking MFRP can still have good refraction-corrected vision, produce clinically normal electro-retinograms, and show only modest anomalies in the dark adaptation of photoreceptors. MFRP appears primarily devoted to regulating axial length of the eye. It remains to be determined whether natural variation in its activity plays a role in common refractive errors.

Peripherin/rds (P/rds) is a membrane glycoprotein essential for the photoreceptor outer segment disc morphogenesis and maintenance. More than half of the disease-causing mutations in P/rds have been linked to different forms of macular dystrophy; the most common one is substitution of tryptophan for arginine at position 172 (R172W). Here we confirm the patient phenotype associated with the expression of R172W mutation in transgenic mice. Functional, structural and biochemical analyses showed that, while R172W P/rds is appropriately localized, a direct correlation exists between transgene expression levels and the onset/severity of the phenotype. In the wild-type background, both cone and rod photoreceptors' structure and function were significantly diminished, which indicates a dominant-negative, cone-rod defect. Whereas rds(+/-) mice maintained the normal cone function at early ages, cone responses in R172W/rds(+/-) mice were diminished to 41% of the wild-type level signifying a preferential damaging effect of the mutation on cones. Conversely, R172W/rds(+/-) mice showed a significant rescue of rod function and improvement of rod outer segment structure. Although rds(-/-) mice have no detectable rod or cone responses, R172W/rds(-/-) animals retained 30% of wild-type structure and rod function, but no significant rescue of cone function was detected at 1 month of age. No biochemical abnormalities were observed in complex formation and association with Rom-1; however, R172W protein was more sensitive to tryptic digestion, indicative of a change in protein conformation, possibly contributing to the cone-dominated phenotype. As the first animal model for P/rds-associated cone-rod dystrophy, R172W mice provide a valuable tool for studying the pathophysiology of P/rds-associated human retinal dystrophies and the development of therapeutic strategies to intervene in these diseases.

Guanylyl cyclase-activating proteins (GCAPs) are Ca2+-binding proteins that activate guanylyl cyclase when free Ca2+ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca2+ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca2+ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nM free Ca2+, the normal resting Ca2+ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca2+ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca2+-dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca2+ in the outer segment, which may trigger the apoptotic process.

Dopamine is an important neurotransmitter regulating light-dark adaptation in the retina. The effect of dopamine is widespread and dependent on the localization of its receptors. We performed single cell RT-PCR on A-type horizontal cells visually selected from dissociated rabbit retinal neurons. Dopamine D1-receptor mRNA was positively identified. Subsequent sequencing of the fragment showed 82% homology with rat D1 receptor and 87% homology with human D1 receptor. This study supported previous observations that dopamine regulated A-type horizontal cell coupling via D1 receptors at the level of transcription.

Sleep enhances plasticity in neocortex, and thereby improves sensory learning. Here we show that sleep itself undergoes changes as a consequence of waking experience during a late critical period in cats and mice. Dark-rearing produced a robust and reversible decrement of slow-wave electrical activity during sleep that was restricted to visual cortex and impaired by gene-targeted reduction of NMDA receptor function.

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master circadian clock in mammals. Nocturnal light pulses that reset the circadian clock also lead to rapid increases in levels of Per1 and Per2 mRNA in the SCN, suggesting that these genes are involved in the synchronization to light. During the day, when light has no phase-shifting effects in nocturnal rodents, the consequences of light exposure for Per expression have been less thoroughly studied. Therefore, the effects of light exposure during the day were assessed on Per1 and Per2 mRNA in the SCN of mice. Expression of Per1 and Per2 was generally increased by 30-min light pulses during the subjective day, with more pronounced effects in the morning. One exception was noted for a transient decrease in Per2 expression after a short light pulse applied at midday. Prolonged light exposure (up to 3 hr) starting at midday markedly increased Per2 expression but not that of Per1. Moreover, the amplitude of the daily variations of both Per and the duration of Per1 peak was increased in mice exposed to a light-dark cycle compared with those transferred to constant darkness. Finally, the amplitude of the daily variations of both Per and the basal level of Per1 were increased in mice under a light-dark cycle compared with animals synchronized to a skeleton photoperiod (i.e., with daily dawn and dusk 1-hr exposures to light). Taken together, the results indicate that prolonged light exposure during daytime positively modulates daily levels of Per1 and Per2 mRNA in the SCN of mice., (Copyright 2003 Wiley-Liss, Inc.)

We have previously reported that clock mutant mice on Jcl:ICR background show about 2-h delayed circadian profiles in body temperature, spontaneous activity and sleep-wake rhythm, and that they appear to be useful as a model of evening-type of individual. Hippocampal acetylcholine (ACh) release which is positively correlated with attention, learning and memory shows a circadian variation. In this study, changes in hippocampal ACh release in transitional phase from light (rest) to dark (active) period in clock mutant mice were monitored using an in vivo microdialysis method. Compared with wild mice, the increase in hippocampal ACh in the first 2 h of the active period in the mutant mice was suppressed in parallel with peak frequency in electroencephalogram theta rhythm. The molecular basis of the circadian system appears to have a strong effect on hippocampal cholinergic function, and is probably associating with individual temporal differences in voluntary behavior, cognition, learning and/or memory performance.

The localization and regulation of chicken cryptochrome 1 (cCry1) mRNA expression in retina was investigated by laser capture microdissection and quantitative real-time RT-PCR. Laser capture microdissection (LCM) of retinal cell layers showed the highest level of cCry1 expression in the ganglion cell and photoreceptor layers. In both layers, expression was high during the daytime and low at night in subjects exposed to a 12:12 h light:dark cycle. Robust circadian oscillations of cCry1 mRNA levels were observed in constant (24 h day) light, but not in constant darkness, with the highest expression during daytime at zeitgeber time (ZT) 8. Unlike cCry1, circadian rhythms of the melatonin-synthesizing enzyme, arylalkylamine N-acetyltransferase, persisted in constant darkness, suggesting that rhythmic cCry1 expression is not essential for circadian clock function or output. On the second day of constant darkness, when cCry1 expression is arrhythmic, light exposure for 2 h significantly increased retinal cCry1 mRNA levels at ZT 4 and 8, times that cCry1 expression is induced in LD and LL. Similar light exposure ending at ZT 20 had no significant effect. Thus, expression of cCry1 mRNA is regulated dually by light and circadian clocks.

In Drosophila photoreceptors, the amplification responsible for generating quantum bumps in response to photoisomerization of single rhodopsin molecules has been thought to be mediated downstream of phospholipase C (PLC), since bump amplitudes were reportedly unaffected in mutants with greatly reduced levels of either G protein or PLC. We now find that quantum bumps in such mutants are reduced approximately 3- to 5-fold but are restored to near wild-type values by mutations in the rdgA gene encoding diacylglycerol kinase (DGK) and also by depleting intracellular ATP. The results demonstrate that amplification requires activation of multiple G protein and PLC molecules, identify DGK as a key enzyme regulating amplification, and implicate diacylglycerol as a messenger of excitation in Drosophila phototransduction.

The mechanism whereby brief light exposure resets the mammalian circadian clock in a phase dependent manner is not known, but is thought to involve Per gene expression. At the behavioural level, a light pulse produces phase delays in early subjective night, phase advances in late subjective night, and no phase shifts in mid-subjective night or subjective day. To understand the relationship between Per gene activity and behavioural phase shifts, we examined light-induced mPer1 and mPer2 expression in the suprachiasmatic nucleus (SCN) of the mouse, in the subjective night, with a view to understanding SCN heterogeneity. In the VIP-containing region of the SCN (termed 'core'), light-induced mPer1 expression occurs at all times of the subjective night, while mPer2 induction is seen only in early subjective night. In the remaining regions of the SCN (termed 'shell'), a phase delaying light pulse produces no mPer1 but significant mPer2 expression, while a phase advancing light pulse produces no mPer2 but substantial mPer1 induction. Moreover, following a light pulse during mid-subjective night, neither mPer1 nor mPer2 are induced in the shell. The results reveal that behavioural phase shifts occur only when light-induced Per gene expression spreads from the core to the shell SCN, with mPer1 expression in shell corresponding to phase advances, and mPer2 corresponding to phase delays. The results indicate that the time course and the localization of light-induced Per gene expression in SCN reveals important aspects of intra-SCN communication.

Temporal changes of mRNA expression of three clock genes, qPer2, qPer3 and qClock, were studied in the suprachiasmatic nucleus (SCN) of Japanese quail under different light conditions, as well as under the condition of continuous melatonin. In addition, the expression of melatonin receptor genes, Mel1a and Mel1c, in the SCN were also examined. The expression of qPer2 mRNA showed robust oscillation during both light and dark (LD) 12:12 cycles and under constant dark conditions (DD), but did not exhibit circadian rhythmicity in constant light conditions (LL), instead being expressed at a consistently high level. Expression of qPer3 also showed robust oscillation under both LD and DD conditions. Unlike qPer2 however, qPer3 mRNA expression remained rhythmic under LL conditions. Contrary to the findings on the other clock genes, no remarkable rhythmicity was detectable in either light condition. Both Mel1a and Mel1c mRNAs were detected in the SCN, however, Mel1a mRNA levels were higher than Mel1c and showed daily rhythmicity. Although implantation of melatonin tubes caused constant high levels of plasma melatonin and consequently masked the endogenous daily melatonin rhythm, no significant differences in the expression pattern of any of the three clock genes were observed between birds with and without constant melatonin. In addition, a single injection of melatonin did not affect mRNA expression of these clock genes. These results suggest that melatonin does not affect transcription of clock genes, but may act on the mechanism of synchronization among SCN oscillatory cells.

Northern blotting indicated that the level of induced fos was higher in normal than dark reared cat visual cortex (VC) at 5 weeks of age, comparable at 10 weeks, and higher in dark reared than normal cat visual cortex at 20 weeks. Fos egr-1, induction was higher in dark reared visual cortex at all ages. Thus, dark rearing has similar effects on fos, but not egr-1, expression as it has on physiological neuronal plasticity during the VC critical period., (Copyright 2002 Elsevier Science B.V.)

The diurnal changes in arginine vasopressin (AVP) mRNA and heteronuclear (hn) RNA, an indicator for gene transcription, were examined in the hypothalamus of Sprague-Dawley rats using in situ hybridization. AVP hnRNA levels in the suprachiasmatic nucleus (SCN) varied during a 24-h cycle and showed a peak at day-time [Zeitgeber time (ZT) 5], which preceded the peak in AVP mRNA levels by 4 h. AVP hnRNA was undetectable at ZT 13 and 17, indicating that the gene transcription was almost shut down at these time points. AVP mRNA levels in the SCN continued to decrease at night (ZT 13, 17 and 21) when there were minimal changes in transcription, suggesting rapid turnover of mRNA. Similar diurnal changes in AVP hnRNA levels were observed without photic cues. On the other hand, AVP hnRNA or mRNA levels in the supraoptic nucleus, where AVP is synthesized in response to plasma osmolarity and/or volume, did not show any circadian rhythm. These data suggest that both dynamic changes in AVP gene transcription and rapid turnover of mRNA contribute to the diurnal variation in AVP mRNA levels in the SCN.

The ability of nursing mothers to entrain the circadian pacemaker of rat pups was examined by measuring the rat Per1 (rPer1) and rPer2 expression levels in the suprachiasmatic nuclei (SCN). Newborn rats from mothers under a light-dark cycle (LD) were blinded immediately after birth and reared by foster mothers under either LD (LD blind pups) or reversed light-dark cycle (DL; DL blind pups). At postnatal day (P)6, small but significant phase differences were observed in the circadian gene expression rhythms of the SCN not only between the blind and sighted pups, but also between the two groups of blind pups, indicating the involvement of both free-running and maternal influence in phase-resetting the circadian rhythms of blind pups. However, from P6 to P13 the circadian rhythms of both LD and DL blind pups showed phase delays of similar extent, which suggests that the influence of nursing mothers was lost. From P13 to P20 (the day of weaning), the rPer1 and rPer2 rhythms phase-shifted in a different manner, the rPer2 rhythm being related more closely to the behavioural rhythm than was the rPer1. This finding suggests a differential influence of mothers on the rPer1 and rPer2 rhythms in the third week of life. It is concluded that the ability of nursing mothers to entrain pup circadian oscillation depends on the developmental stage.

Electrical silencing of Drosophila circadian pacemaker neurons through targeted expression of K+ channels causes severe deficits in free-running circadian locomotor rhythmicity in complete darkness. Pacemaker electrical silencing also stops the free-running oscillation of PERIOD (PER) and TIMELESS (TIM) proteins that constitutes the core of the cell-autonomous molecular clock. In contrast, electrical silencing fails to abolish PER and TIM oscillation in light-dark cycles, although it does impair rhythmic behavior. On the basis of these findings, we propose that electrical activity is an essential element of the free-running molecular clock of pacemaker neurons along with the transcription factors and regulatory enzymes that have been previously identified as required for clock function.

In neonatal retinal explants cultured long-term green cones are missing. Recently it was reported that thyroid hormone beta2 receptors (TR-beta2) are essential for these green cones to differentiate. Therefore transcript level of these receptors was investigated in our mouse retinal explants. However, thyroid receptors function as heterodimers with retinoid receptors (RR); so the fate of selected RRs was similarly analyzed using semi-quantitative RT-PCR. Loss of TR-beta2 and RR (RXR-gamma and ROR-beta2) mRNAs was observed after culturing the neonatal retina for 12 days. This indicates that these proteins are involved in determination of green cone identity. In addition, levels of the selected RR transcripts are differentially affected by short- or long-term culture. In the latter case an attached retinal pigment epithelium seems to play a protective role. Furthermore, divergent diurnal peaks of RR mRNAs are present in young as well as aged mouse retina and neocortex. This data might be relevant in the context of human ageing disorders.

Some isolated populations exhibit an increased prevalence of rare recessive diseases. The island of Newfoundland is a characteristic geographic isolate, settled by a small number of families primarily during the late 1700s and early 1800s. During our studies of this population, we identified a group of families exhibiting a retinal dystrophy reminiscent of retinitis punctata albescens but with a substantially lower age at onset and more-rapid and distinctive progression, a disorder that we termed "Newfoundland rod-cone dystrophy" (NFRCD). The size of one of these families was sufficient to allow us to perform a genomewide screen to map the NFRCD locus. We detected significant linkage to markers on the long arm of chromosome 15, in a region encompassing RLBP1, the gene encoding the cellular retinaldehyde-binding protein. Previously, mutations in RLBP1 have been associated with other retinal dystrophies, leading us to hypothesize that RLBP1 mutations might also cause NFRCD. To test this hypothesis, we sequenced all coding exons and splice junctions of RLBP1. We detected two sequence alterations, each of which is likely to be pathogenic, since each segregates with the disease and is predicted to interfere with mRNA splicing. In contrast to some previously reported RLBP1 mutations, which yield a protein that may retain some residual activity, each NFRCD mutation is likely to give rise to a null allele. This difference may account for the severe phenotype in these families and exemplifies the molecular continuum that underlies clinically distinct but genetically related entities.

It has been proposed that cis-retinol dehydrogenase (cRDH) acts within the body to catalyze the oxidation of 9-cis-retinol, an oxidative step needed for 9-cis-retinoic acid synthesis, the oxidation of 11-cis-retinol [an oxidative step needed for 11-cis-retinal (visual chromophore) synthesis], and 3 alpha-hydroxysteroid transformations. To assess in vivo the physiological importance of each of these proposed actions of cRDH, we generated cRDH-deficient (cRDH-/-) mice. The cRDH-/- mice reproduce normally and appear to be normal. However, the mutant mice do have a mild visual phenotype of impaired dark adaptation. This phenotype is evidenced by electroretinagram analysis of the mice and by biochemical measures of eye levels of retinoid intermediates during recovery from an intense photobleach. Although it is thought that cRDH is expressed in the eye almost solely in retinal pigment epithelial cells, we detected cRDH expression in other retinal cells, including ganglion cells, amacrine cells, horizontal cells, and the inner segments of the rod photoreceptor cells. Aside from the eye, there are no marked differences in retinoid levels in other tissues throughout the body for cRDH-/- compared with cRDH+/+ mice. Moreover, we did not detect any non-visual phenotypic changes for cRDH-/- mice, suggesting that these mice do not have problems in metabolizing 3 alpha-hydroxysteroids.Thus, cRDH may act essentially in the visual cycle but is redundant for catalyzing 9-cis-retinoic acid formation and 3 alpha-hydroxysteroid metabolism.

The abundance of the integral membrane protein synaptophysin in synaptic vesicles and its multiple possible functional contributions to transmitter exocytosis and synaptic vesicle formation stand in sharp contrast to the observed lack of defects in synaptophysin knockout mice. Assuming that deficiencies are compensated by the often coexpressed synaptophysin isoform synaptoporin, we now show that retinal rod photoreceptors, which do not synthesize synaptoporin either in wild-type or in knockout mice, are affected by the loss of synaptophysin. Multiple pale-appearing photoreceptors, as seen by electron microscopy, possess reduced cytoplasmic electron density, swollen mitochondria, an enlarged cell surface area, and, most importantly, a significantly reduced number of synaptic vesicles with an unusually bright interior. Quantification of the number of synaptic vesicles per unit area, not only in these, but also in all other rod terminals of knockout animals, reveals a considerable reduction in vesicles that is even more pronounced during the dark period, i.e., at times of highest synaptic activity. Moreover, activity-dependent reduction in synaptic vesicle diameter, typically occurring in wild-type mice, is not detected in knockout animals. The large number of clathrin-coated pits and vesicles in dark-adapted synaptophysin knockout mice is taken as an indication of compensatory usage of synaptophysin-independent pathway(s), and, conversely, in view of the overall reduction in the number of synaptic vesicles, as an indication for the presence of another synaptophysin-dependent synaptic vesicle recycling pathway. Our results provide in vivo evidence for the importance of the integral membrane protein synaptophysin for synaptic vesicle recycling and formation.

A new assay was designed, named checker, that measures the individual response to light in the fruitfly Drosophila melanogaster larva. In this assay the Drosophila larva apparently modulates its pattern of locomotion when faced with a choice between a dark and lit environment by orienting its movement towards the dark environment. We show that, in this assay, a response to light can be measured as an increase in residence time in the dark versus the lit quadrant. Mutations that disrupt phototransduction in the adult Drosophila abolish the larval response to light, demonstrating that this larval visual function is similar to that of the adult fly. Similarly, no response to light was detected in strains where the larval visual system (photoreceptors and target area) was disrupted by a mutation in the homeobox containing gene sine oculis (so) gene. Ablation of photoreceptors by the targeted expression of the cell death gene hid under the control of the photoreceptor-specific transcription factor glass (gl) abolishes this response entirely. Finally, we demonstrate that this response to light can be mediated by rhodopsins other than the blue absorbing Rh1.

Photoisomerization of 11-cis-retinal to all-trans-retinal and reduction to all-trans-retinol occur in photoreceptor outer segments whereas enzymatic esterification of all-trans-retinol, isomerization to 11-cis-retinol, and oxidation to 11-cis-retinal occur in adjacent cells. The processes are linked into a visual cycle by intercellular diffusion of retinoids. Knowledge of the mechanistic aspects of the visual cycle is very limited. In this study, we utilize chemical analysis of visual cycle retinoids to assess physiological roles for components inferred from in vitro experiments and to understand why excised mouse eyes fail to regenerate their bleached visual pigment. Flash illumination of excised mouse eyes or eyecups, in which regeneration of rhodopsin does not occur, produced a block in the visual cycle after all-trans-retinal formation; constant illumination of eyecups produced a block in the cycle after all-trans-retinol formation; and constant illumination of whole excised eyes resulted in a block of the cycle after formation of all-trans-retinyl ester. These blocks emphasize the role of cellular metabolism in the visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) has been postulated to play a role in intercellular retinoid transfer in the retina; however, the rates of recovery of 11-cis-retinal and of regeneration of rhodopsin in the dark in IRBP-/- mice were very similar to those found with wild-type (wt) mice. Thus, IRBP is necessary for photoreceptor survival but is not essential for a normal rate of visual pigment turnover. Arrestin forms a complex with activated rhodopsin, quenches its activity, and affects the release of all-trans-retinal in vitro. The rate of recovery of 11-cis-retinal in arrestin-/- mice was modestly delayed relative to wt, and the rate of rhodopsin recovery was approximately 80% of that observed with wt mice. Thus, the absence of arrestin appeared to have a minor effect on the kinetics of the visual cycle.

Purpose: The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes contains highly conserved cis-acting elements, which include an AP-1 and/or Nrl response element (NRE: An extended AP-1 like sequence). Transactivation of AP-1 or NRE appears necessary to drive expression of these rod-specific genes during adulthood, however, their role during development is relatively unknown. Therefore, we determined the spatial and temporal relationships between rod morphological and functional development, rod-specific gene expression, and expression of the bZIP transcription factors c-fos, junD and Nrl., Methods: Retinas from 0-45 day old (PN0-45) dark- and light-adapted Long-Evans rats were used. Morphological development was monitored by light and electron microscopy. Whole retinal trypsin-activated cGMP-PDE activity and rhodopsin content were measured biochemically. The expression of opsin, beta-PDE, c-fos, junD and Nrl mRNAs were determined by Northern blot analysis. The cellular localization of Nrl was examined with in situ hybridization., Results: The mRNAs for opsin, beta-PDE and c-fos were observed at PN0-2, while cGMP-PDE activity and rhodopsin were detected first at PN5: coincident with rod outer segment development. The developmental pattern of cGMP-PDE activity and rhodopsin accumulation paralleled the expression of beta-PDE and opsin mRNA and all reached their maximal levels by PN45. Nrl expression, for all three transcripts found in the rat retina, was low on PN2 and reached its maximal level at PN14. The c-fos and Nrl expression preceded beta-PDE and opsin mRNA expression by 1-2 days. Nrl expression was detected first in the distal post-mitotic retina at PN5 and then in all nuclear layers during retinal development. Maximal expression shifted from the ganglion cells to the outer nuclear layer as the neural retina matured. In contrast, junD expression was highest at PN0 and declined to a stable level by PN10., Conclusions: Colocalization of Nrl and c-Fos suggests that expression of rod-specific genes, which utilize AP-1 or NRE sites in their promoter, could be regulated through the formation of Nrl-Fos dimers. We hypothesize that Nrl and c-Fos play a fundamental role in the initiation and regulation of the rod-specific gene expression in developing and adult rod photoreceptors.