Christophe Ribelayga

The Circadian Clock in the Retina Controls Rod-Cone Coupling

Although rod and cone photoreceptor cells in the vertebrate retina are anatomically connected or coupled by gap junctions, a type of electrical synapse, rod-cone electrical coupling is thought to be weak. Using tracer labeling and electrical recording in the goldfish retina and tracer labeling in the mouse retina, we show that the retinal circadian clock, and not the retinal response to the visual environment, controls the extent and strength of rod-cone coupling by activating dopamine D(2)-like receptors in the day, so that rod-cone coupling is weak during the day but remarkably robust at night. The results demonstrate that circadian control of rod-cone electrical coupling serves as a synaptic switch that allows cones to receive very dim light signals from rods at night, but not in the day. The increase in the strength and extent of rod-cone coupling at night may facilitate the detection of large dim objects.

A circadian clock in the fish retina regulates dopamine release via activation of melatonin receptors

Although many biochemical, morphological and physiological processes in the vertebrate retina are controlled by a circadian (24 h) clock, the location of the clock and how the clock alters retinal function are unclear. For instance, several observations have suggested that dopamine, a retinal neuromodulator, may play an important role in retinal rhythmicity but the link between dopamine and a clock located within or outside the retina remains to be established. We found that endogenous dopamine release from isolated goldfish retinae cultured in continuous darkness for 56 h clearly exhibited a circadian rhythm with high values during the subjective day. The continuous presence of melatonin (1 nm) in the culture medium abolished the circadian rhythm of dopamine release and kept values constantly low and equal to the night‐time values. The selective melatonin antagonist luzindole (1  μm) also abolished the dopamine rhythm but the values were high and equal to the daytime values. Melatonin application during the late subjective day introduced rod input and reduced cone input to fish cone horizontal cells, a state usually observed during the subjective night. In contrast, luzindole application during the subjective night decreased rod input and increased cone input. Prior application of dopamine or spiperone, a selective dopamine D2‐like antagonist, blocked the above effects of melatonin and luzindole, respectively. These findings indicate that a circadian clock in the vertebrate retina regulates dopamine release by the activation of melatonin receptors and that endogenous melatonin modulates rod and cone pathways through dopamine‐mediated D2‐like receptor activation.

Dopamine mediates circadian clock regulation of rod and cone input to fish retinal horizontal cells

A circadian (24‐hour) clock regulates the light responses of fish cone horizontal cells, second order neurones in the retina that receive synaptic contact from cones and not from rods. Due to the action of the clock, cone horizontal cells are driven by cones in the day, but primarily driven by rods at night. We show here that dopamine, a retinal neurotransmitter, acts as a clock signal for the day by increasing cone input and decreasing rod input to cone horizontal cells. The amount of endogenous dopamine released from in vitro retinae was greater during the subjective day than the subjective night. Application of dopamine or quinpirole, a dopamine D2‐like agonist, during the subjective night increased cone input and eliminated rod input to the cells, a state usually observed during the subjective day. In contrast, application of spiperone, a D2‐like antagonist, or forskolin, an activator of adenylyl cyclase, during the subjective day reduced cone input and increased rod input. SCH23390, a D1 antagonist, had no effect. Application of Rp‐cAMPS, an inhibitor of cAMP‐dependent protein kinase, or octanol, an alcohol that uncouples gap junctions, during the night increased cone input and decreased rod input. Because D2‐like receptors are on photoreceptor cells, but not horizontal cells, the results suggest that the clock‐induced increase in dopamine release during the day activates D2‐like receptors on photoreceptor cells. The resultant decrease in intracellular cyclic AMP and protein kinase A activation then mediates the increase in cone input and decrease in rod input.

Adenosine and Dopamine Receptors Coregulate Photoreceptor Coupling via Gap Junction Phosphorylation in Mouse Retina

Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell–cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibit adenylyl cyclase (AC) and reduce PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to coregulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod–cone gap junctions but possibly including some rod–rod and cone–cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately downregulated expression of D4R and AC type 1 mRNA. We conclude that adenosine and dopamine coregulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function.

Adrenergic and peptidergic regulations of hydroxyindole-O-methyltransferase activity in rat pineal gland.

Mechanisms involved in the regulation of hydroxyindole-O-methyltransferase (HIOMT) activity were investigated in the rat pineal. Isoproterenol, db-cAMP, PACAP or VIP had no acute (6 h) effect whereas NPY, thapsigargin and a PKC activator stimulated HIOMT activity by 30-40%. Chronic stimulation (6 days) with isoproterenol, db-cAMP, or each peptide prevented the long-term decrease of HIOMT activity. Phenylephrine had neither short- nor long-term effect on enzyme activity. These results indicate that HIOMT activity is long- and short-term regulated by various neurotransmitters.

A Circadian Clock and Light/Dark Adaptation Differentially Regulate Adenosine in the Mammalian Retina

Although the purine adenosine acts as an extracellular neuromodulator in the mammalian CNS in both normal and pathological conditions and regulates sleep, the regulation of extracellular adenosine in the day and night is incompletely understood. To determine how extracellular adenosine is regulated, rabbit neural retinas were maintained by superfusion at different times of the regular light/dark and circadian cycles. The adenosine level in the superfusate, representing adenosine overflow from the retinas, and the adenosine level in retinal homogenates, representing adenosine content, were measured using HPLC with fluorescence detection in the absence or presence of blockers of adenosine transport and/or extracellular adenosine synthesis. We report that darkness, compared with illumination, increases the level of extracellular adenosine, and that a circadian clock also increases extracellular adenosine at night. In addition, we show that the darkness-evoked increase in the level of extracellular adenosine results primarily from an increase in the conversion of extracellular ATP into adenosine, but that the clock-induced increase at night results primarily from an increase in the accumulation of intracellular adenosine. We also show that a slightly hypoxic state increases adenosine content and overflow to an extent similar to that of the clock. Our findings demonstrate that the extracellular level of adenosine in the mammalian retina is differentially regulated by a circadian clock and the lighting conditions and is maximal at night under dark-adapted conditions. We conclude that adenosine is a neuromodulator involved in both circadian clock and dark-adaptive processes in the vertebrate retina.

Photoneural regulation of rat pineal hydroxyindole-O-methyltransferase (HIOMT) messenger ribonucleic acid expression: an analysis of its complex relationship with HIOMT activity.

In the pineal gland, synthesis of melatonin requires O-methylation catalyzed by hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4). We investigated in vivo the molecular mechanisms involved in the regulation of rat pineal HIOMT messenger RNA (mRNA) expression and activity using in situ hybridization and radioenzymatic assay. HIOMT mRNA levels and activity are both detectable during the daytime and display nocturnal increases of 100% and 30%, respectively. These variations are controlled by the endogenous clock, as they persist in constant darkness. The nocturnal increase in HIOMT mRNA mainly results from a b1-adrenergic stimulation of HIOMT gene expression without requiring de novo synthesis of a transcription factor. In contrast, the nocturnal increase in HIOMT activity appears independent of b1/a1-adrenergic stimulation. A light pulse at night abolishes the nighttime increase in HIOMT mRNA, but not HIOMT activity. Constant light application for up to 11 days does not depress HIOMT mRNA levels lower than the daytime levels, but decreases enzyme activity down to 50% of the daytime level. This finding indicates that the nocturnal stimulation of HIOMT gene expression is required for sustaining a basal level of activity over a few days. Our data suggest 1) that HIOMT gene expression is partly regulated by b1-stimulation; and 2) that HIOMT activity is regulated over the short term by a nonnoradrenergic stimulus and over the long term by noradrenergic stimulation. (Endocrinology 140: 1375‐1384, 1999)

Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue.

Photoperiodic Control of the Rat Pineal Arylalkylamine-N-Acetyltransferase and Hydroxyindole-O-Methyltransferase Gene Expression and Its Effect on Melatonin Synthesis

Photoperiodic changes of pineal melatonin (MEL) profile are accompanied by parallel changes of arylalkylamine-N-acetyltransferase (AA-NAT) activity. In the present study, the authors investigated, for the first time, whether two other important variables of pineal metabolism, AA-NAT and hydroxyindole-O-methyltransferase (HIOMT) gene expression, also may be affected by the photoperiod. Evening rises in AA-NAT and HIOMT mRNA and in circulating MEL occurred concomitantly with an increased delay from dark onset as scotophase shortened. On the opposite, the morning declines of all three variables occurred with different kinetics but were locked to light onset. These observations demonstrate that the daily rhythms in AA-NAT and HIOMT gene expression are modulated by the photoperiod and bring further evidence in favor of nor adrenaline as the possible link between the endogenous clock and MEL. Interestingly, the duration of the nocturnal peak in HIOMT mRNA was positively correlated with HIOMT activity. In conclusion, this study adds two important links to the chain of mechanisms involved in the photoperiodic control of pineal metabolism. First, photoperiodic modulation of the MEL rhythm primarily results from changes in the AA-NAT gene expression. Second, the photoperiodic regulation of HIOMT activity occurs at the transcriptional level.

Identification of a circadian clock-controlled neural pathway in the rabbit retina.

Background Although the circadian clock in the mammalian retina regulates many physiological processes in the retina, it is not known whether and how the clock controls the neuronal pathways involved in visual processing. Methodology/Principal Findings By recording the light responses of rabbit axonless (A-type) horizontal cells under dark-adapted conditions in both the day and night, we found that rod input to these cells was substantially increased at night under control conditions and following selective blockade of dopamine D2, but not D1, receptors during the day, so that the horizontal cells responded to very dim light at night but not in the day. Using neurobiotin tracer labeling, we also found that the extent of tracer coupling between rabbit rods and cones was more extensive during the night, compared to the day, and more extensive in the day following D2 receptor blockade. Because A-type horizontal cells make synaptic contact exclusively with cones, these observations indicate that the circadian clock in the mammalian retina substantially increases rod input to A-type horizontal cells at night by enhancing rod-cone coupling. Moreover, the clock-induced increase in D2 receptor activation during the day decreases rod-cone coupling so that rod input to A-type horizontal cells is minimal. Conclusions/Significance Considered together, these results identify the rod-cone gap junction as a key site in mammals through which the retinal clock, using dopamine activation of D2 receptors, controls signal flow in the day and night from rods into the cone system.