Aqp9 Gene Deletion Enhances Retinal Ganglion Cell (RGC) Death and Dysfunction Induced by Optic Nerve Crush: Evidence that Aquaporin 9 Acts as an Astrocyte-to-Neuron Lactate Shuttle in Concert with Monocarboxylate Transporters To Support RGC Function and Survival.

Aquaporin 9 (AQP9) is an aquaglyceroporin that can transport lactate. Accumulating evidence suggests that astrocyte-to-neuron lactate shuttle (ANLS) plays a critical role in energy metabolism in neurons, including retinal ganglion cells (RGCs). To test the hypothesis that AQP9, in concert with monocarboxylate transporters (MCTs), participates in ANLS to maintain function and survival of RGCs, Aqp9-null mice and wild-type (WT) littermates were subjected to optic nerve crush (ONC) with or without intravitreal injection of an MCT2 inhibitor. RGC density was similar between the Aqp9-null mice and WT mice without ONC, while ONC resulted in significantly more RGC density reduction in the Aqp9-null mice than in the WT mice at day 7. Positive scotopic threshold response (pSTR) amplitude values were similar between the two groups without ONC, but were significantly more reduced in the Aqp9-null mice than in the WT mice 7days after ONC. MCT2 inhibitor injection accelerated RGC death and pSTR amplitude reduction only in the WT mice with ONC. Immunolabeling revealed that both RGCs and astrocytes expressed AQP9, that ONC predominantly reduced astrocytic AQP9 expression, and that MCTs 1, 2, and 4 were co-localized with AQP9 at the ganglion cell layer. These retinal MCTs were also co-immunoprecipitated with AQP9 in the WT mice. ONC decreased the co-immunoprecipitation of MCTs 1 and 4, but did not impact co-immunoprecipitation of MCT2. Retinal glucose transporter 1 expression was increased in Aqp9-null mice. Aqp9 gene deletion reduced and increased the intraretinal L-lactate and D-glucose concentrations, respectively. Results suggest that AQP9 acts as the ANLS to maintain function and survival of RGCs.

Electroretinographic Assessment of Inner Retinal Signaling in the Isolated and Superfused Murine Retina.

PURPOSE: Longer-lasting electroretinographic recordings of the isolated murine retina were initially achieved by modification of a phosphate-buffered nutrient solution originally developed for the bovine retina. During experiments with a more sensitive mouse retina, apparent model-specific limitations were addressed and improvements were analyzed for their contribution to an optimized full electroretinogram (ERG). MATERIAL AND METHODS: Retinas were isolated from dark-adapted mice, transferred to a recording chamber and superfused with different solutions. Scotopic and photopic ERGs were recorded with white flashes every 3 minutes. The phosphate buffer (Sickel-medium) originally used was replaced by a carbonate-based system (Ames-medium), the pH of which was adjusted to 7.7-7.8. Moreover, addition of 0.1 mM BaCl2 was investigated to reduce b-wave contamination by the slow PIII component typically present in the murine ERG. RESULTS: B-wave amplitudes were increased by the pH-shift (pH 7.4 to pH 7.7) from 22.9 ± 1.9 µV to 37.5 ± 2.5 µV. Improved b-wave responses were also achieved by adding small amounts of Ba2+ (100 µM), which selectively suppressed slow PIII components, thereby unmasking more of the true b-wave amplitude (100.0% with vs. 22.2 ± 10.7% without Ba2+). Ames medium lacking amino acids and vitamins was unable to maintain retinal signaling, as evident in a reversible decrease of the b-wave to 31.8 ± 3.9% of its amplitude in complete Ames medium. CONCLUSIONS: Our findings provide optimized conditions for ex vivo ERGs from the murine retina and suggest that careful application of Ba2+ supports reliable isolation of b-wave responses in mice. Under our recording conditions, murine retinas show reproducible ERGs for up to six hours.

Light and dark adaptation mechanisms in the compound eyes of Myrmecia ants that occupy discrete temporal niches.

Ants of the Australian genus Myrmecia partition their foraging niche temporally, allowing them to be sympatric with overlapping foraging requirements. We used histological techniques to study the light and dark adaptation mechanisms in the compound eyes of diurnal (Myrmecia croslandi), crepuscular (M. tarsata, M. nigriceps) and nocturnal ants (M. pyriformis). We found that, except in the day-active species, all ants have a variable primary pigment cell pupil that constricts the crystalline cone in bright light to control for light flux. We show for the nocturnal M. pyriformis that the constriction of the crystalline cone by the primary pigment cells is light dependent whereas the opening of the aperture is regulated by an endogenous rhythm. In addition, in the light-adapted eyes of all species, the retinular cell pigment granules radially migrate towards the rhabdom, a process that in both the day-active M. croslandi and the night-active M. pyriformis is driven by ambient light intensity. Visual system properties thus do not restrict crepuscular and night-active ants to their temporal foraging niche, while day-active ants require high light intensities to operate. We discuss the ecological significance of these adaptation mechanisms and their role in temporal niche partitioning.

Role of extrinsic noise in the sensitivity of the rod pathway: rapid dark adaptation of nocturnal vision in humans.

UNLABELLED: Rod-mediated 500 nm test spots were flashed in Maxwellian view at 5 deg eccentricity, both on steady 10.4 deg fields of intensities (I) from 0.00001 to 1.0 scotopic troland (sc td) and from 0.2 s to 1 s after extinguishing the field. On dim fields, thresholds of tiny (5') tests were proportional to √I (Rose-DeVries law), while thresholds after extinction fell within 0.6 s to the fully dark-adapted absolute threshold. Thresholds of large (1.3 deg) tests were proportional to I (Weber law) and extinction thresholds, to √I. CONCLUSIONS: rod thresholds are elevated by photon-driven noise from dim fields that disappears at field extinction; large spot thresholds are additionally elevated by neural light adaptation proportional to √I. At night, recovery from dimly lit fields is fast, not slow.

Opsin1-2, G(q)α and arrestin levels at Limulus rhabdoms are controlled by diurnal light and a circadian clock.

Dark and light adaptation in photoreceptors involve multiple processes including those that change protein concentrations at photosensitive membranes. Light- and dark-adaptive changes in protein levels at rhabdoms have been described in detail in white-eyed Drosophila maintained under artificial light. Here we tested whether protein levels at rhabdoms change significantly in the highly pigmented lateral eyes of wild-caught Limulus polyphemus maintained in natural diurnal illumination and whether these changes are under circadian control. We found that rhabdomeral levels of opsins (Ops1-2), the G protein activated by rhodopsin (G(q)α) and arrestin change significantly from day to night and that nighttime levels of each protein at rhabdoms are significantly influenced by signals from the animal's central circadian clock. Clock input at night increases Ops1-2 and G(q)α and decreases arrestin levels at rhabdoms. Clock input is also required for a rapid decrease in rhabdomeral Ops1-2 beginning at sunrise. We found further that dark adaptation during the day and the night are not equivalent. During daytime dark adaptation, when clock input is silent, the increase of Ops1-2 at rhabdoms is small and G(q)α levels do not increase. However, increases in Ops1-2 and G(q)α at rhabdoms are enhanced during daytime dark adaptation by treatments that elevate cAMP in photoreceptors, suggesting that the clock influences dark-adaptive increases in Ops1-2 and G(q)α at Limulus rhabdoms by activating cAMP-dependent processes. The circadian regulation of Ops1-2 and G(q)α levels at rhabdoms probably has a dual role: to increase retinal sensitivity at night and to protect photoreceptors from light damage during the day.

Light and vision in the deep-sea benthos: II. Vision in deep-sea crustaceans.

Using new collecting techniques with the Johnson-Sea-Link submersible, eight species of deep-sea benthic crustaceans were collected with intact visual systems. Their spectral sensitivities and temporal resolutions were determined shipboard using electroretinography. Useable spectral sensitivity data were obtained from seven species, and in the dark-adapted eyes, the spectral sensitivity peaks were in the blue region of the visible spectrum, ranging from 470 to 497 nm. Under blue chromatic adaptation, a secondary sensitivity peak in the UV portion of the spectrum appeared for two species of anomuran crabs: Eumunida picta (λ(max)363 nm) and Gastroptychus spinifer (λ(max)383 nm). Wavelength-specific differences in response waveforms under blue chromatic adaptation in these two species suggest that two populations of photoreceptor cells are present. Temporal resolution was determined in all eight species using the maximum critical flicker frequency (CFF(max)). The CFF(max) for the isopod Booralana tricarinata of 4 Hz proved to be the lowest ever measured using this technique, and suggests that this species is not able to track even slow-moving prey. Both the putative dual visual pigment system in the crabs and the extremely slow eye of the isopod may be adaptations for seeing bioluminescence in the benthic environment.

Regression of early diabetic macular oedema is associated with prevention of dark adaptation.

HYPOTHESIS: Dark-adapted rods consume oxygen at high rates and light adaptation decreases this oxygen burden and can have therapeutic effects on diabetic macular oedema (DMO). METHODS: Patients with mild non-proliferative diabetic retinopathy (DR) and early, untreated non-sight-threatening DMO slept for 6 months wearing masks that illuminated the eyelid of one closed eye by 505 nm light. Exclusion criteria were any concomitant eye disease, DR >ETDRS grade 35, and other systemic diseases. PRIMARY OUTCOME: change of OCT retinal thickness in the local region where oedema was present. RESULTS: A total of 34 out of 40 patients completed the study. Mean baseline OCT macular cube thickness was equivalent for study and fellow eyes. But study eyes had a greater mean thickness in the central subfield zone 1 (282±53 µm) vs (256±19 µm) the fellow eyes. Twenty-eight study eyes showed intraretinal cysts compared with nine in the fellow eyes. At 6 months, only 19 study eyes had cysts while cysts were seen in 20 fellow eyes. After 6 months, the worst affected ETDRS zone and the central subfield zone 1 reduced in thickness in study eyes only by 12 µm (95% CI 20 to -7, P=0.01). The secondary outcomes of change in visual acuity, achromatic contrast sensitivity, and microperimetric thresholds improved significantly in study eyes and deteriorated in fellow eyes. CONCLUSIONS: Sleeping in dim light that can keep rods light adapted may reverse the changes of DMO.

Phosphorylation of G protein-coupled receptor kinase 1 (GRK1) is regulated by light but independent of phototransduction in rod photoreceptors.

Phosphorylation of rhodopsin by G protein-coupled receptor kinase 1 (GRK1, or rhodopsin kinase) is critical for the deactivation of the phototransduction cascade in vertebrate photoreceptors. Based on our previous studies in vitro, we predicted that Ser(21) in GRK1 would be phosphorylated by cAMP-dependent protein kinase (PKA) in vivo. Here, we report that dark-adapted, wild-type mice demonstrate significantly elevated levels of phosphorylated GRK1 compared with light-adapted animals. Based on comparatively slow half-times for phosphorylation and dephosphorylation, phosphorylation of GRK1 by PKA is likely to be involved in light and dark adaptation. In mice missing the gene for adenylyl cyclase type 1, levels of phosphorylated GRK1 were low in retinas from both dark- and light-adapted animals. These data are consistent with reports that cAMP levels are high in the dark and low in the light and also indicate that cAMP generated by adenylyl cyclase type 1 is required for phosphorylation of GRK1 on Ser(21). Surprisingly, dephosphorylation was induced by light in mice missing the rod transducin α-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.

Conical tomography of a ribbon synapse: structural evidence for vesicle fusion.

To characterize the sites of synaptic vesicle fusion in photoreceptors, we evaluated the three-dimensional structure of rod spherules from mice exposed to steady bright light or dark-adapted for periods ranging from 3 to 180 minutes using conical electron tomography. Conical tilt series from mice retinas were reconstructed using the weighted back projection algorithm, refined by projection matching and analyzed using semiautomatic density segmentation. In the light, rod spherules contained ∼470 vesicles that were hemi-fused and ∼187 vesicles that were fully fused (omega figures) with the plasma membrane. Active zones, defined by the presence of fully fused vesicles, extended along the entire area of contact between the rod spherule and the horizontal cell ending, and included the base of the ribbon, the slope of the synaptic ridge and ribbon-free regions apposed to horizontal cell axonal endings. There were transient changes of the rod spherules during dark adaptation. At early periods in the dark (3-15 minutes), there was a) an increase in the number of fully fused synaptic vesicles, b) a decrease in rod spherule volume, and c) an increase in the surface area of the contact between the rod spherule and horizontal cell endings. These changes partially compensate for the increase in the rod spherule plasma membrane following vesicle fusion. After 30 minutes of dark-adaptation, the rod spherules returned to dimensions similar to those measured in the light. These findings show that vesicle fusion occurs at both ribbon-associated and ribbon-free regions, and that transient changes in rod spherules and horizontal cell endings occur shortly after dark onset.

Phosducin regulates transmission at the photoreceptor-to-ON-bipolar cell synapse.

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 preliminary trial to determine whether prevention of dark adaptation affects the course of early diabetic retinopathy.

UNLABELLED: This study was designed to determine whether a new form of treatment of diabetic retinopathy (DR) was acceptable to patients and whether reduction in the maximal activity of rods in diabetes could affect the progress of DR. METHODS: In 12 patients, trans-lid retinal illumination of one eye was employed during sleep to prevent the depolarisation of rods and thus reduce their metabolic activity. TECHNIQUES: A headband was used to place a source of chemical light over one eye, with its fellow as a control. MEASUREMENTS: Colour contrast thresholds were measured before and after a period of treatment in treated eyes, and the changes were compared to those in untreated fellow eyes, and areas of 'dark retinal anomalies' (microaneurysms, dot haemorrhages) were measured at the same time points. RESULTS: Patients found this intervention to be acceptable, and no adverse effects were noted. In the majority of cases, and for each outcome measure, the treated eyes improved relative to their fellows. The intervention significantly reduced the tritan thresholds in treated eyes relative to their fellows (P=0.03), and the area of dark retinal anomalies decreased in treated eyes and increased in untreated eyes, with a similar probability. CONCLUSIONS: The study showed that this intervention is safe. Although the study was not powered to study efficacy, the results are promising and consistent with other reports that indicate the retina in DR is suffering from hypoxia; however, further trials should be undertaken.

Relationships among visual cycle retinoids, rhodopsin phosphorylation, and phototransduction in mouse eyes during light and dark adaptation.

Phosphorylation and regeneration of rhodopsin, the prototypical G-protein-coupled receptor, each can influence light and dark adaptation. To evaluate their relative contributions, we quantified rhodopsin, retinoids, phosphorylation, and photosensitivity in mice during a 90 min illumination followed by dark adaptation. During illumination, all-trans-retinyl esters and, to a lesser extent, all-trans-retinal accumulate and reach the steady state in <1 h. Each major phosphorylation site on rhodopsin reaches a steady state level of phosphorylation at a different time during illumination. The dominant factor that limits dark adaptation is isomerization of retinal. During dark adaptation, dephosphorylation of rhodopsin occurs in two phases. The faster phase corresponds to rapid dephosphorylation of regenerated rhodopsin present at the end of the illumination period. The slower phase corresponds to dephosphorylation of rhodopsin as it forms by regeneration. We conclude that rhodopsin phosphorylation has three physiological functions: it quenches phototransduction, reduces sensitivity during light adaptation, and suppresses bleached rhodopsin activity during dark adaptation.

Retinophilin is a light-regulated phosphoprotein required to suppress photoreceptor dark noise in Drosophila.

Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.

Influence of the rod photoresponse on light adaptation and circadian rhythmicity in the cone ERG.

PURPOSE: In the mammalian retina, rod and cone pathways are fundamentally intertwined, with signals from both converging on cone bipolar cells to reach retinal ganglion cells. Psychophysical and electrophysiological data suggests that, as a consequence, rod signal transduction has a suppressive effect on the activity of cone pathways. It therefore might be assumed that the balance between rod and cone input to cone bipolar cells would be subject to dynamic regulation. There is evidence of light and time-of-day dependent alterations in this parameter. Here we set out to determine the extent to which such changes in rod-cone pathway convergence explain alterations in cone pathway function associated with light adaptation and circadian phase by recording cone electroretinograms (ERGs) in mice deficient in rod phototransduction. METHODS: Cone-isolated ERGs elicited by bright flashes superimposed on a rod saturating background light were recorded from wild-type and rod transducin deficient (Gnat1(-/-)) mice. The process of light adaptation was observed by tracing changes in the ERG waveform over 20 min exposure to the background light in these genotypes, and circadian control by comparing responses at subjective midday and midnight. RESULTS: The cone ERG b-wave exhibited significantly enhanced amplitude and reduced latency (implicit time) in Gnat1(-/-) mice under all conditions. Light adaptation was associated with a robust increase in b-wave amplitude in Gnat1(-/-) mice but, in contrast to wild types, almost no change in implicit time. Gnat1(-/-) mice retained circadian rhythms in the cone ERG with b-wave amplitudes larger and latencies reduced during the subjective day. CONCLUSIONS: Rod phototransduction has a strong suppressive effect on the cone ERG. Light adaptation in cone pathways relies in part on reductions in this effect, although mechanisms intrinsic to cone pathways also play an important role. Similarly, while changes in coupling between rod and cone pathways over the course of the day may contribute to circadian regulation of the cone pathway they are not sufficient to explain circadian rhythms in the wild-type cone ERG.

c-Fos expression in the visual system of the tree shrew (Tupaia belangeri).

UNLABELLED: c-Fos is a nuclear phosphoprotein coded by the proto-oncogen c-fos which can be detected immunohistochemically after both physiological and pathological stimuli. This property is of great importance, because it offers a valuable tool for morphofunctional identification of activated neurons. We have studied the neuronal activity in the visual pathway of Tupaia belangeri within the following anatomical structures: retina, superior colliculus (SC), dorsal lateral geniculate nucleus (dLGN), pulvinar (Pu), parabigeminal (PBG) nucleus and primary visual cortex (V1) analyzing the c-Fos expression after exposing the tree shrews to different light stimuli (white light -control positive group-, green light, blue light and darkness conditions -control negative group-). Our findings suggest that in the retina, the ganglion cells and the cells of the inner nuclear layer respond better to blue and green light stimuli, when comparing the c-Fos expression between white, green, blue lights and darkness conditions. However, in the SC, dLGN, Pu, PBG nucleus and V1 another pattern of c-Fos expression is observed: a maximum expression for the control positive group, a minimum expression for the control negative group and intermediate expressions within the blue and green light groups. CONCLUSION: the expression levels of c-Fos protein are able to show significant differences between distinct light stimuli in all anatomical structures studied (retina, SC, dLGN, Pu, PBG and V1) of T. belangeri.

Diacylglyceride lipase activity in rod outer segments depends on the illumination state of the retina.

We have demonstrated that the competition between phosphatidic acid (PA) and lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) for lipid phosphate phosphatases (LPP) generates different levels of diacylglycerol (DAG) depending on the illumination state of the retina. The aim of the present research was to determine the diacylglyceride lipase (DAGL) activity in purified rod outer segments (ROS) obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. [2-(3)H]monoacylglycerol (MAG), the product of DAGL, was evaluated from [2-(3)H]DAG generated by LPP action on [2-(3)H]PA in the presence of either LPA, S1P or C1P. MAG production was inhibited by 55% in BLROS and by 25% when the enzymatic assay was carried out in ROS obtained from dark-adapted retinas and incubated under room light (LROS). The most important events occurred in DROS where co-incubation of [2-(3)H]PA with LPA, S1P or C1P diminished MAG production. A higher level of DAGL activity was observed in LROS than in BLROS, though this difference was not apparent in the presence of LPA, S1P or C1P. DAGL activity in depleted DROS was diminished with respect to that in entire DROS. LPA, S1P and C1P produced a similar decrease in MAG production in depleted DROS whereas only C1P significantly diminished MAG generation in depleted BLROS. Sphingosine and ceramide inhibited MAG production in entire DROS and stimulated its generation in BLROS. Sphingosine and ceramide stimulated MAG generation in both depleted DROS and BLROS. Under our experimental conditions the degree of MAG production depended on the illumination state of the retina. We therefore suggest that proteins related to phototransduction phenomena are involved in the effects observed in the presence of S1P/sphingosine or C1P/ceramide.

Dark adaptation recovery of human rod bipolar cell response kinetics estimated from scotopic b-wave measurements.

We recorded ganzfeld scotopic ERGs to examine the responses of human rod bipolar cells in vivo, during dark adaptation recovery following bleaching exposures, as well as during adaptation to steady background lights. In order to be able to record responses at relatively early times in recovery, we utilized a 'criterion response amplitude' protocol in which the test flash strength was adjusted to elicit responses of nearly constant amplitude. In order to provide accurate and unbiased measures of response kinetics, we utilized a curve-fitting procedure to fit a smooth function to the measured responses in the vicinity of the peak, thereby extracting both the time-to-peak and the amplitude of the responses. Following bleaching exposures, the responses exhibited both desensitization and accelerated kinetics. During early post-bleach recovery, the flash sensitivity and time-to-peak varied according to a power-law expression (with an exponent of 6), as found in the presence of steady background light. This light-like phenomenon, however, appeared to be set against the backdrop of a second, more slowly recovering 'pure' desensitization, most clearly evident at late post-bleach times. The post-bleach 'equivalent background intensity' derived from measurements of flash sensitivity faded initially with an S2 slope of approximately 0.24 decades min(-1), and later as a gentle S3 tail. When calculated from kinetics, the results displayed only the S2 slope. While the recovery of rod bipolar cell response kinetics can be described accurately by a declining level of opsin in the rods, the sensitivity of these cells is reduced further than expected by this mechanism alone.

Dopamine modulates diurnal and circadian rhythms of protein phosphorylation in photoreceptor cells of mouse retina.

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.

PPARalpha is involved in photoentrainment of the circadian clock.

We found that bezafibrate, a ligand of peroxisome proliferator-activated receptor alpha (PPARalpha), advances the active phase of mice under light-dark (LD) conditions in a photoperiod-dependent manner. Bezafibrate gradually advanced the activity onset that consequently almost completely reversed the active phase from the dark to the light period under a long photoperiod (18 h of light and 6 h of darkness: LD 18 : 6). The activity onset was not changed under a short photoperiod (LD 8 : 16) or under constant illumination. These observations suggest that PPARalpha is involved in entrainment of the circadian clock to environmental LD conditions.