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Electrical coupling, mediated by gap junctions, contributes to signal averaging, synchronization, and noise reduction in neuronal circuits. In addition, gap junctions may also provide alternative neuronal pathways. However, because they are small and especially difficult to image, gap junctions are often ignored in large-scale 3D reconstructions. Here, we reconstruct gap junctions between photoreceptors in the mouse retina using serial blockface-scanning electron microscopy, focused ion beam-scanning electron microscopy, and confocal microscopy for the gap junction protein Cx36. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules at sites of Cx36 labeling, with approximately 50 Cx36 clusters per cone pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. We estimate a mean of 86 Cx36 channels per rod/cone pair, which may provide a maximum conductance of ~1200 pS, if all gap junction channels were open. This is comparable to the maximum conductance previously measured between rod/cone pairs in the presence of a dopamine antagonist to activate Cx36, suggesting that the open probability of gap junction channels can approach 100% under certain conditions.Neurons can talk to each other in two ways: they can send chemical messengers across specialized junctions between two cells, or they can directly pass electrical signals to one another. This latter process is made possible by gap junctions, a system of channel-like structures which connect neighbouring cells and let ions move between them. In most neurons, gap junction channels are made from a specialized protein called connexin 36. Gap junctions are small, difficult to observe, and therefore often ignored by researchers studying neural circuits. In response, Ishibashi et al. focused on nerve cells in the mouse retina, in particular the cones (which detect color during the day) and the rods (which are essential for night vision). Gap junctions between rods and cones allow them to communicate; for example, they enable rod signals to directly activate cones. This provides an alternative route for rod signaling known as the ‘secondary rod pathway’, which seems to be open at night and switches to closed around dawn. Both rods and cones only produce connexin 36, so Ishibashi et al. labeled these proteins with fluorescent tags to pinpoint gap junctions. This showed that each cone makes around 50 gap junctions with nearby rods; however, gap junctions were not detected between cells of the same type. In addition, 3D reconstruction helped to establish the length of each gap junction. Further experiments showed that a typical rod was connected to a cone by about 80 connexin 36 channels. Finally, calculations revealed that the gap junction channels would all need to open to account for the level of electrical activity required for the secondary rod pathway. This suggests that gap junctions may be much more active and important than previously thought. The work by Ishibashi et al. provides a new understanding of the number, size and activity of gap junctions in the retina, potentially paving the way to prevent diseases where light-sensing cells degenerate and cause blindness.

In the retina, signals originating from rod and cone photoreceptors can reach retinal ganglion cells (RGCs)—the output neurons—through different pathways. However, little is known about the exact sensitivities and operating ranges of these pathways. Previously, we created rod- or cone-specificCx36knockout (KO) mouse lines. Both lines are deficient in rod/cone electrical coupling and therefore provide a way to selectively remove the secondary rod pathway. We measured the threshold of the primary rod pathway in RGCs of wild-type mice. Under pharmacological blockade of the primary rod pathway, the threshold was elevated. This secondary component was removed in theCx36KOs to unmask the threshold of the third rod pathway, still below cone threshold. In turn, the cone threshold was estimated by several independent methods. Our work defines the functionality of the secondary rod pathway and describes an additive contribution of the different pathways to the retinal output.

Rhodopsin and cone opsins are essential for light detection in vertebrate rods and cones, respectively. It is well established that rhodopsin is required for rod phototransduction, outer segment disk morphogenesis, and rod viability. However, the roles of cone opsins are less well understood. In this study, we adopted a loss-of-function approach to investigate the physiological roles of cone opsins in mice. We showed that cones lacking cone opsins do not form normal outer segments due to the lack of disk morphogenesis. Surprisingly, cone opsin-deficient cones survive for at least 12 mo, which is in stark contrast to the rapid rod degeneration observed in rhodopsin-deficient mice, suggesting that cone opsins are dispensable for cone viability. Although the mutant cones do not respond to light directly, they maintain a normal dark current and continue to mediate visual signaling by relaying the rod signal through rod-cone gap junctions. Our work reveals a striking difference between the role of rhodopsin and cone opsins in photoreceptor viability.

Adenosine, a major neuromodulator in the central nervous system (CNS), is involved in a variety of regulatory functions such as the sleep/wake cycle. Because exogenous adenosine displays dark- and night-mimicking effects in the vertebrate retina, we tested the hypothesis that a circadian (24 h) clock in the retina uses adenosine to control neuronal light responses and information processing. Using a variety of techniques in the intact goldfish retina including measurements of adenosine overflow and content, tracer labeling, and electrical recording of the light responses of cone photoreceptor cells and cone horizontal cells (cHCs), which are post-synaptic to cones, we demonstrate that a circadian clock in the retina itself—but not activation of melatonin or dopamine receptors—controls extracellular and intracellular adenosine levels so that they are highest during the subjective night. Moreover, the results show that the clock increases extracellular adenosine at night by enhancing adenosine content so that inward adenosine transport ceases. Also, we report that circadian clock control of endogenous cone adenosine A2A receptor activation increases rod-cone gap junction coupling and rod input to cones and cHCs at night. These results demonstrate that adenosine and A2A receptor activity are controlled by a circadian clock in the retina, and are used by the clock to modulate rod-cone electrical synapses and the sensitivity of cones and cHCs to very dim light stimuli. Moreover, the adenosine system represents a separate circadian-controlled pathway in the retina that is independent of the melatonin/dopamine pathway but which nevertheless acts in concert to enhance the day/night difference in rod-cone coupling.

Image- and non-image-forming vision are essential for animal behavior. Here we use genetically modified mouse lines to examine retinal circuits driving image- and non-image-functions. We describe the outer retinal circuits underlying the pupillary light response (PLR) and circadian photoentrainment, two non-image-forming behaviors. Rods and cones signal light increments and decrements through the ON and OFF pathways, respectively. We find that the OFF pathway drives image-forming vision but cannot drive circadian photoentrainment or the PLR. Cone light responses drive image formation but fail to drive the PLR. At photopic levels, rods use the primary and secondary rod pathways to drive the PLR, whereas at the scotopic and mesopic levels, rods use the primary pathway to drive the PLR, and the secondary pathway is insufficient. Circuit dynamics allow rod ON pathways to drive two non-image-forming behaviors across a wide range of light intensities, whereas the OFF pathway is potentially restricted to image formation.

Outer retinal circuits that drive non-image forming vision in mammals are unknown. Rods and cones signal light increments and decrements to the brain through the ON and OFF pathways, respectively. Although their contribution to image-forming vision is known, the contributions of the ON and OFF pathway to the pupillary light response (PLR), a non-image forming behavior, are unexplored. Here we use genetically modified mouse lines, to comprehensively define the outer retinal circuits driving the PLR. The OFF pathway, which mirrors the ON pathway in image-forming vision, plays no role in the PLR. We found that rods use the primary rod pathway to drive the PLR at scotopic light levels. At photopic light levels, the primary and secondary rod pathways drive normal PLR. Importantly, we find that cones are unable to compensate for rods. Thus, retinal circuit dynamics allow rods to drive the PLR across a wide range of light intensities.

Purpose The present study tested the hypothesis that connexin-36 (Cx36) and gap junctions between photoreceptor cells contribute to the circadian rhythm of the photopic electroretinogram (ERG) b-wave amplitude. Methods Cone-specific disruption of Cx36 was obtained in mice with a floxed Gjd2 gene and human red/green pigment promoter (HRGP)-driven Cre recombinase. Standard ERG, spectral-domain optical coherence tomography (SD-OCT) and histochemical methods were used. Results HRGPcreGjd2fl/fl mice had a selective reduction in Cx36 protein in the outer plexiform layer; no reduction in Cx36 was observed in the inner plexiform layer. Cx36 disruption had no effect on the number of cones, the thickness of the photoreceptor layer, or the scotopic ERG responses. However, there was a reduction of the photopic ERG circadian rhythm, with b-wave amplitudes in the day and the night locked in the daytime, light-adapted state. In HRGPcreGjd2+/+and Gjd2fl/fl controls, the circadian rhythm of light-adapted ERG persisted, similar to that in wild type mice. Conclusions Cx36 regulation contributes to the circadian rhythm of light-adapted ERG; in the absence of photoreceptor gap junctions, mice appear to be in a fully light-adapted state regardless of the time of day. The higher amplitudes and reduced circadian regulation of the b-wave of HRGPcreGjd2fl/fl mice may be due to increased synaptic strength at the cone to ON bipolar cell synapse due to electrotonic isolation of the terminals lacking gap junctions.

Every day and night, the retina undergoes dramatic changes in its physiology and function. The prevailing view is that these daily changes affect the retinal output and thereby visual perception. Recent evidence suggests that modifications in higher-order processing centers, and not in retinal computations, account for variations in visual sensitivity.

Gap-junction-mediated electrical coupling between retinal photoreceptors is an important determinant of photoreceptor function. Yet, quantitative measurements of the junctional conductance between coupled photoreceptors are required to fully assess the effects of coupling on visual performance. Such measurements have been obtained in Salamander and other lower vertebrate retinas but are difficult to acquire in mammalian retinas, in part because of the much smaller size of photoreceptors in mammals. Here, we describe in detail a dual whole-cell patch-clamp technique we recently developed to measure the junctional conductance between photoreceptor pairs in the mouse retina. With this method, electrical coupling strength between mouse photoreceptors can be estimated with high accuracy, and its impact on retinal processing of visual information further evaluated.

Vision is a highly rhythmic function adapted to the extensive changes in light intensity occurring over the 24-hour day. This adaptation relies on rhythms in cellular and molecular processes, which are orchestrated by a network of circadian clocks located within the retina and in the eye, synchronized to the day/night cycle and which, together, fine-tune detection and processing of light information over the 24-hour period and ensure retinal homeostasis. Systematic or high throughput studies revealed a series of genes rhythmically expressed in the retina, pointing at specific functions or pathways under circadian control. Conversely, knockout studies demonstrated that the circadian clock regulates retinal processing of light information. In addition, recent data revealed that it also plays a role in development as well as in aging of the retina. Regarding synchronization by the light/dark cycle, the retina displays the unique property of bringing together light sensitivity, clock machinery, and a wide range of rhythmic outputs. Melatonin and dopamine play a particular role in this system, being both outputs and inputs for clocks. The retinal cellular complexity suggests that mechanisms of regulation by light are diverse and intricate. In the context of the whole eye, the retina looks like a major determinant of phase resetting for other tissues such as the retinal pigmented epithelium or cornea. Understanding the pathways linking the cell-specific molecular machineries to their cognate outputs will be one of the major challenges for the future.

Although it is well established that the vertebrate retina contains endogenous circadian clocks that regulate retinal physiology and function during day and night, the processes that the clocks affect and the means by which the clocks control these processes remain unresolved. We previously demonstrated that a circadian clock in the goldfish retina regulates rod-cone electrical coupling so that coupling is weak during the day and robust at night. The increase in rod-cone coupling at night introduces rod signals into cones so that the light responses of both cones and cone horizontal cells, which are post-synaptic to cones, become dominated by rod input. By comparing the light responses of cones, cone horizontal cells and rod horizontal cells, which are post-synaptic to rods, under dark-adapted conditions during day and night, we determined whether the daily changes in the strength of rod-cone coupling could account entirely for rhythmic changes in the light response properties of cones and cone horizontal cells. We report that although some aspects of the day/night changes in cone and cone horizontal cell light responses, such as response threshold and spectral tuning, are consistent with modulation of rod-cone coupling, other properties cannot be solely explained by this phenomenon. Specifically, we found that at night compared to the day the time course of spectrally-isolated cone photoresponses was slower, cone-to-cone horizontal cell synaptic transfer was highly non-linear and of lower gain, and the delay in cone-to-cone horizontal cell synaptic transmission was longer. However, under bright light-adapted conditions in both day and night, cone-to-cone horizontal cell synaptic transfer was linear and of high gain, and no additional delay was observed at the cone-to-cone horizontal cell synapse. These findings suggest that in addition to controlling rod-cone coupling, retinal clocks shape the light responses of cone horizontal cells by modulating cone-to-cone horizontal cell synaptic transmission.

Rod photoreceptors are electrically coupled through gap junctions. Coupling is a key determinant of their light response properties, but whether rod electrical coupling is dynamically regulated remains elusive and controversial. Here, we have obtained direct measurements of the conductance between adjacent rods in mouse retina and present evidence that rod electrical coupling strength is dependent on the time of day, the lighting conditions, and the mouse strain. Specifically, we show in CBA/Ca mice that under circadian conditions, the rod junctional conductance has a median value of 98 pS during the subjective day and of 493 pS during the subjective night. In C57BL/6 mice, the median junctional conductance between dark-adapted rods is ∼140 pS, regardless of the time in the circadian cycle. Adaptation to bright light decreases the rod junctional conductance to ∼0 pS, regardless of the time of day or the mouse strain. Together, these results establish the high degree of plasticity of rod electrical coupling over the course of the day. Estimates of the rod coupling strength will provide a foundation for further investigations of rod interactions and the role of rod coupling in the ability of the visual system to anticipate, assimilate, and respond to the daily changes in ambient light intensity.SIGNIFICANCE STATEMENTMany cells in the CNS communicate via gap junctions, or electrical synapses, the regulation of which remains largely unknown. Here, we show that the strength of electrical coupling between rod photoreceptors of the retina is regulated by the time of day and the lighting conditions. This mechanism may help us understand some key aspects of day and night vision as well as some visual malfunctions.

Circadian rhythms are present in most leaving organisms and these rhythms are not just a consequence of the day/night fluctuation, but rather they are generated by endogenous biological clocks with a periodicity of about 24 hrs. In mammals, the master pacemaker of circadian rhythms is localized in the suprachiasmatic nuclei of the hypothalamus (SCN). The SCN controls circadian rhythms in peripheral organs. The retina also contains circadian clocks which regulate many aspects of retinal physiology, independently of the SCN. Emerging experimental evidence indicates that the retinal circadian clocks also affect ocular health and a few studies have now demonstrated that disruption of retinal clocks may contribute to the development of retinal diseases. Our study indicates that in mice lacking the clock gene Bmal1 photoreceptor viability during aging is significantly reduced. Bmal1 knock-out mice at 8-9 months of age have 20-30% less nuclei in the outer nuclear layer. No differences were observed in the other retinal layers. Our study suggests that the retinal circadian clock is an important modulator of photoreceptor health.

Key points Rod photoreceptors play a key role in vision in dim light; in the mammalian retina, although rods are anatomically connected or coupled by gap junctions, a type of electrical synapse, the functional importance and regulation of rod coupling has remained elusive. We have developed a new technique in the mouse: perforated patch-clamp recording of rod inner segments in isolated intact retinae maintained by superfusion. We find that rod electrical coupling is controlled by a circadian clock and dopamine, and is weak during the day and stronger at night. The results also indicate that the signal-to-noise ratio for a dim light response is increased at night because of coupling. Our observations will provide a framework for understanding the daily variations in human vision as well as the basis of specific retinal malfunctions. Abstract Rod single-photon responses are critical for vision in dim light. Electrical coupling via gap junction channels shapes the light response properties of vertebrate photoreceptors, but the regulation of rod coupling and its impact on the single-photon response have remained unclear. To directly address these questions, we developed a perforated patch-clamp recording technique and recorded from single rod inner segments in isolated intact neural mouse retinae, maintained by superfusion. Experiments were conducted at different times of the day or under constant environmental conditions, at different times across the circadian cycle. We show that rod electrical coupling is regulated by a circadian clock and dopamine, so that coupling is weak during the day and strong at night. Altogether, patch-clamp recordings of single-photon responses in mouse rods, tracer coupling, receptive field measurements and pharmacological manipulations of gap junction and dopamine receptor activity provide compelling evidence that rod coupling is modulated in a circadian manner. These data are consistent with computer modelling. At night, single-photon responses are smaller due to coupling, but the signal-to-noise ratio for a dim (multiphoton) light response is increased at night because of signal averaging between coupled rods.

In the retina, electrical coupling through gap junctions is present in all major classes of neurons. Electrical coupling in the retina was first reported more than four decades ago and great strides have been made in understanding its properties and roles. It has become clear that dynamic modulation of electrical coupling is a critical element of circadian and light-adaptive changes that optimize retinal function for vastly different light conditions. This review summarizes current knowledge of the mechanisms regulating electrical coupling between photoreceptors, horizontal cells and amacrine cells, which play prominent roles in adaptation. In each case we review what is known of the pathways involved in plasticity and the interpretations and hypotheses regarding its functional effects. We assert that dynamic regulation of electrical coupling is extremely diverse and likely plays an integral if not a central role in normal retinal function and physiology by fine-tuning neuronal circuit activity.

Opsin 4 (Opn4)/melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) play a major role in non-image-forming visual system. Although advances have been made in understanding their morphological features and functions, the molecular mechanisms that regulate their formation and survival remain unknown. Previously, we found that mouse T-box brain 2 (Tbr2) (also known as Eomes), a T-box-containing transcription factor, was expressed in a subset of newborn RGCs, suggesting that it is involved in the formation of specific RGC subtypes. In thisin vivostudy, we used complex mouse genetics, single-cell dye tracing, and behavioral analyses to determine whether Tbr2 regulates ipRGC formation and survival. Our results show the following: (1) Opn4 is expressed exclusively in Tbr2-positive RGCs; (2) no ipRGCs are detected whenTbr2is genetically ablated before RGC specification; and (3) most ipRGCs are eliminated whenTbr2is deleted in established ipRGCs. The few remaining ipRGCs display abnormal dendritic morphological features and functions. In addition, some Tbr2-expressing RGCs can activateOpn4expression on the loss of native ipRGCs, suggesting that Tbr2-expressing RGCs may serve as a reservoir of ipRGCs to regulate the number of ipRGCs and the expression levels of Opn4.

Dopamine is a key neurotransmitter in the retina and plays a central role in the light adaptive processes of the visual system. The sole source of retinal dopamine is dopaminergic amacrine cells (DACs). We and others have previously demonstrated that DACs are activated by rods, cones and intrinsically photosensitive retinal ganglion cells (ipRGCs) upon illumination. However, it is still not clear how each class of photosensitive cells generates light responses in DACs. We genetically isolated cone function in mice to specifically examine the cone-mediated responses of DACs and their neural pathways. In addition to the reported excitatory input to DACs from light-increment (ON) bipolar cells, we found that cones alternatively signal to DACs via a retrograde signalling pathway from ipRGCs. Cones also produce ON and light-decrement (OFF) inhibitory responses in DACs, which are mediated by other amacrine cells, likely driven by type 1 and type 2/3a OFF bipolar cells, respectively. Dye injections indicated that DACs had similar morphological profiles with or without ON/OFF inhibition. Our data demonstrate that cones utilize specific parallel excitatory and inhibitory circuits to modulate DAC activity and efficiently regulate dopamine release and the light-adaptive state of the retina.

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from non-functional alleles of the genes coding two key enzymes of the melatonin synthesis pathway: arylalkylamine-N-acetyltransferase (Aanat) and N-acetylserotonin-O-methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin-proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin-dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin-dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically-modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin-proficient B6 congenic line will be widely distributed.

Although rod and cone photoreceptor cells in the vertebrate retina are anatomically connected or coupled by gap junctions, rod-cone coupling is thought to be weak. By using a combination of tracer labeling and electrical recording in the goldfish retina and tracer labeling in the mouse retina, we show that the retinal circadian clock, a local endogenous process, and not the retinal response to the visual environment, controls the extent and strength of rod-cone coupling by activating dopamine D2-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 coupling serves as a synaptic switch between the rod and cone pathways, so that cones and neurons postsynaptic to cones 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 enhance the reliability of the rod light response and facilitate the detection of large dim objects.

The gap-junction-forming protein connexin36 (Cx36) represents the anatomical substrate of photoreceptor electrical coupling in mammals. The strength of coupling is directly correlated to the phosphorylation of Cx36 at two regulatory sites: Ser110 and Ser293. Our previous work demonstrated that the extent of biotinylated tracer coupling between photoreceptor cells, which provides an index of the extent of electrical coupling, depends on the mouse strain. In the C57Bl/6J strain, light or dopamine reduces tracer coupling and Cx36 phosphorylation in photoreceptors. Conversely, darkness or a dopaminergic antagonist increases tracer coupling and Cx36 phosphorylation, regardless of the daytime. In the CBA/CaJ strain, photoreceptor tracer coupling is not only regulated by light and dopamine, but also by a circadian clock, a type of oscillator with a period close to 24 h and intrinsic to the retina, so that under prolonged dark-adapted conditions tracer coupling is broader at night compared to daytime. In the current study, we examined whether the modulation of photoreceptor coupling by a circadian clock in the CBA/CaJ mouse photoreceptors reflected a change in Cx36 protein expression and/or phosphorylation. We found no significant change in Cx36 expression or in the number of Cx36 gap junction among the conditions examined. However, we found that Cx36 phosphorylation is higher under dark-adapted conditions at night than in the daytime, and is the lowest under prolonged illumination at any time of the day/night cycle. Our observations are consistent with the view that the circadian clock regulation of photoreceptor electrical coupling is mouse strain-dependent and highlights the critical position of Cx36 phosphorylation in the control of photoreceptor coupling.

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 microM) 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 D(2)-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 D(2)-like receptor activation.

In the Syrian hamster, the role of noradrenaline in the regulation of melatonin synthesis is less clear than in the rat. During pineal ontogenesis in the rat, noradrenaline is the major transmitter involved in the onset of melatonin synthesis and melatonin rhythm. We analysed the involvement of noradrenaline in the ontogenesis of melatonin synthesis in the Syrian hamster and compared it with that of the rat. We followed the developmental profile of melatonin content in parallel with those of mRNA expression and activity of AA-NAT, the melatonin rhythm-generating enzyme. In addition, we tested the effect of noradrenergic drugs at early steps of pineal ontogenesis. In the Syrian hamster, the night-time Aa-nat mRNA, first detected 3 days after birth, increases progressively up to a maximum reached at 30 days of age and then decreases significantly towards adulthood. The daytime level of Aa-nat mRNA remains always low. A significant day/night rhythm appears 10 days after birth, is maximal (200-fold nocturnal increase) 30 days after birth and decreases slowly towards adulthood. Ontogenesis of the AA-NAT activity rhythm is similar, although with a much lower amplitude of day/night variations (four-fold). The developmental pattern of melatonin content is similar to that of AA-NAT and could be correlated with the appearance of sympathetic innervation in the pineal gland. However, neither alpha- nor beta-adrenergic antagonists inhibit the night-time Aa-nat mRNA transcription in the 9-day-old Syrian hamster, in contrast to what is observed in the adult. For comparison, the beta-adrenergic antagonist propranolol inhibits Aa-nat gene expression in 2-day-old rat. These results show that both species are different in the regulation of the appearance of melatonin synthesis and that Syrian hamster is peculiar from birth in term of noradrenaline involvement in the activation of melatonin synthesis.

The time giver hormone melatonin is secreted with both daily and seasonal rhythms in the mammalian pineal gland. We report here that the two main melatoninsynthesizing enzymes N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) are differently involved in the regulation of the daily and photoperiodic rhythms of melatonin synthesis. The activity of the unstable NAT enzyme is stimulated acutely at night by norepinephrine via cAMP-dependent transcriptional mechanisms, indicating that it triggers the nocturnal increase in melatonin synthesis and drives its duration according to the night length. In contrast, the activity of the stable HIOMT enzyme is regulated chronically by a norepinephrine-induced, 2-fold increase in HIOMT mRNA that occurs every night. Such regulation leads to a photoperiodic variation in HIOMT activity which is positively related to duration of the night. Whether in vivo or in vitro, at high NAT activity, HIOMT activity appears to limit the amount of melatonin release. ...

Dopamine (DA) is a light-adaptive factor that plays an important role in daytime vision. DA controls the gain of retinal signal transmission partly by controlling gap junction coupling of retinal n...

SummaryThe recent discovery of the critical role of TRPM1 in retinal function in vertebrates has come as a surprise and has already provided important insights into a common cause of blindness.

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.

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)

We have paid close attention to the elegant work from Dr Cangiano's group, in particular to the pioneer work in obtaining electrical recordings from mouse photoreceptors using patch-clamp electrodes. As with any technique, perforated or whole cell patch-clamp techniques have limitations, due in part to the dialysis of the pipette content into the cell and/or of the cell content into the pipette. These limitations are evident in small cells such as mammalian photoreceptors. Intracellular access through patch electrodes in mouse photoreceptors introduces such artefacts as rundown of the light responses and spontaneous increase in rod–cone coupling. Dr Cangiano's group has reported with great detail these limitations in two recent publications (Cangiano et al. 2012; Asteriti et al. 2014). However, despite all the limitations, intracellular access yields critical information about mammalian photoreceptor physiology that cannot be obtained with other techniques, including important information about the state of electrical coupling between neighbouring photoreceptors. We agree with Asteriti and Cangiano that the kinetics of the rod light responses we recorded in intact mouse retinae during the daytime, as reported in Jin et al. (2015), are at the higher end of the range of published values. These relatively slow kinetics may possibly reflect rundown due to perforated patch recording. As indicated in the Methods section of our paper, we found differences between the protocol used by the Cangiano group and ours. In particular, we used pipettes of relatively high resistance (15–20 MΩ) and found that this was key to obtaining stable recordings and limited rundown. It was not our intention to infer that our recordings were perfectly physiological, but we now feel that a few additional words could have been added to prevent the reader from making the assumption that they were. Specifically, we should have clarified that we had optimized the patch-clamp procedure in order to minimize the rundown and coupling effects that may result from patch clamping. In response to ‘It further surprised us the statement of Jin et al. (2015) that the “kinetics of the current and voltage responses to light compared favourably with those measured with suction and patch electrodes in mouse retinal slices”’, this statement was referring to the overall sensitivity and kinetics, including the polarity of the responses, and was not intended to make a precise comparison. However, we disagree with Asteriti and Cangiano on their claim that the possible rundown/coupling effect of patch clamping significantly complicates the interpretation of our findings. Indeed, electrical recording of single photon responses, tracer coupling and receptor field measurements provide compelling evidence that rod coupling is minimal during the day/subjective day. This demonstrates that, under our conditions, patching of a rod per se has limited effect on the state of rod electrical coupling. It follows that the day/night difference in coupling reflected in the increase in the number of effectively coupled rods (Ne), tracer coupling and receptor field size at night is due to the action of a circadian clock, as there is no reason to believe that the coupling effect of the patch electrode would differ between day and night. Similarly, the rundown effect due to patching should be independent of the time of day, and hence the day/night difference in response kinetics essentially reflects the action of the clock. Finally, the rundown effect has been shown to have no impact on the rod response amplitude (Cangiano et al. 2012) and therefore is probably not responsible for the decrease in response amplitude at night. Thus, as the effects of rundown and coupling due to intracellular access should be the same day and night, and daytime measurements demonstrate that both have limited impact on rod electrical coupling and response kinetics, we conclude that the day/night difference in rod electrical coupling and light response properties we observed is real and if anything may have been slightly underestimated. Finally, we acknowledge Asteriti and Cangiano's last statement: ‘one must realize the limitations of patch recordings of photoreceptors and plan for appropriate controls, timing of the recordings and devise alternative approaches.’ We do realize the limitations of patch recording mouse photoreceptors, but we think that the advantages of the technique greatly outweigh its limitations. The mechanisms through which intracellular access of mouse photoreceptors induces rundown and spontaneous electrical coupling remain unknown, but it is legitimate to think that they will eventually be identified and counteracted. The Jin et al. (2015) paper establishes time of day as a key determinant of the rod light response and electrical coupling. So far, we have used single cell recordings and single photon response statistics, tracer coupling and receptive field size as proxies of rod electrical coupling. We are moving towards simultaneous patch-clamp recordings of pairs of adjacent rods in various mouse models. Direct measurements of the rod junctional conductance and the use of genetic tools will not only provide important clues about the biophysical mechanisms involved in the regulation of rod function but also help in the understanding of the perturbations triggered by accessing mammalian photoreceptors with perforated patch electrodes. This will move the field forward.

Hydroxyindole-O-methyltransferase (HIOMT) is the enzyme involved in the last step of the melatonin synthesis pathway. Recently, a cDNA encoding HIOMT has been isolated from a rat pineal gland library. Using this cDNA, we developed a highly sensitive in situ hybridisation protocol to investigate the distribution of HIOMT mRNA in both the rat brain and dissociated pinealocytes maintained in primary cell culture. In the rat brain, HIOMT mRNA was only detected in the three parts of the pineal complex: the superficial pineal, the stalk and the deep pineal. No extra-pineal hybridisation labelling was observed. These results strongly suggest that melatonin synthesis also occurs in the deep part and the stalk of the pineal gland. HIOMT mRNA was markedly expressed in cultured pinealocytes. No particular subcellular area was observed to express HIOMT mRNA specifically, as the labelling was homogeneously distributed in the cytosol and in the axon-like processes. In conclusion, the use of in situ and in vitro hybridisation with a pineal riboprobe has detected notable HIOMT expression restricted to pinealocytes.

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.

Purpose Retinal dopamine deficiency is a potential cause of myopia and visual deficits in retinopathy of prematurity (ROP). We investigated the cellular mechanisms responsible for lowered levels of retinal dopamine in an oxygen-induced retinopathy (OIR) mouse model of ROP. Methods Retinopathy was induced by exposing mice to 75% oxygen from postnatal day 7 (P7) to P12. Oxygen-induced retinopathy and age-matched control mice were euthanized at P12, P17, P25, or P42 to P50. Immunohistochemistry, electrophysiology, and biochemical approaches were used to determine the effect of OIR on the structure and function of dopaminergic amacrine cells (DACs). Results The total number of DACs was unchanged in OIR retinas at P12 despite significant capillary dropout in the central retina. However, a significant loss of DACs was observed in P17 OIR retinas (in which neovascularization was maximal), with the cell loss being more profound in the central (avascular) than in the peripheral (neovascular) regions. Cell loss was persistent in both regions at P25, at which time retinal neovascularization had regressed. At P42, the percentage of DACs lost (54%) was comparable to the percent decrease in total dopamine content (53%). Additionally, it was found that DACs recorded in OIR retinas at P42 to P50 had a complete dendritic field and exhibited relatively normal spontaneous and light-induced electrical activity. Conclusions The results suggest that remaining DACs are structurally and functionally intact and that loss of DACs is primarily responsible for the decreased levels of retinal dopamine observed after OIR.

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.

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent(1,2). In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue. Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions(3,4). Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling(3-8). For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation(7,8). However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance. Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading(9-12), which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas(7, 8,13), and can be used to study coupling between other retinal neurons, as described here.

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 darkadapted 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.

Most vertebrate retinas have both rod and cone photoreceptor cells. Based primarily on electrical recordings of dissociated rods and cones, it has been accepted that rods operate at low light levels at night and cones operate at high light levels in the day. Recent evidence, however, demonstrates that cones in the intact retina respond to very dim light stimuli at night (though not in the day), because the circadian (24-h) clock in the retina opens an electrical synapse (gap-junctional channel) between rods and cones at night. The increased direct communication between rods and cones at night may facilitate the detection of large dim objects, and influence the health and survival of the photoreceptor cells.

Horizontal cells are second order neurons that receive direct synaptic input from photoreceptors. In teleosts horizontal cells can be divided into two categories, cone-connected and rod-connected. Although the anatomy and physiology of fish cone horizontal cells have been extensively investigated, less is known about rod horizontal cells. This study was undertaken to determine whether light and/or the circadian clock regulate gap junctional coupling between goldfish rod horizontal cells. We used fine-tipped, microelectrode intracellular recording to monitor rod horizontal cells under various visual stimulation conditions, and tracer (biocytin) iontophoresis to visualize their morphology and evaluate the extent of coupling. Under dark-adapted conditions, rod horizontal cells were extensively coupled to cells of like-type (homologous coupling) with an average of ∼120 cells coupled. Under these conditions, no differences were observed between day, night, the subjective day, and subjective night. In addition, under dark-adapted conditions, application of the dopamine D2-like agonist quinpirole (1 μM), the D2-like antagonist spiperone (10 μM), or the D1-like antagonist SCH23390 (10 μM) had no effect on rod horizontal cell tracer coupling. In contrast, the extent of tracer coupling was reduced by ∼90% following repetitive light (photopic range) stimulation of the retina or application of the D1-agonist SKF38393 (10 μM) during the subjective day and night. We conclude that similarly to cone horizontal cells, rod horizontal cells are extensively coupled to one another in darkness and that the extent of coupling is dramatically reduced by bright light stimulation or dopamine D1-receptor activation. However, in contrast to cone horizontal cells whose light responses are under the control of the retinal clock, the light responses of rod horizontal cells under dark-adapted conditions were similar during the day, night, subjective day, and subjective night thus demonstrating that they are not under the influence of the circadian clock.

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.

In fish and other vertebrate retinas, although dopamine release is regulated by both light and an endogenous circadian (24-hour) clock, light increases dopamine release to a greater extent than the clock. The clock increases dopamine release during the subjective day so that D2-like receptors are activated. It is not known, however, whether the retinal clock also activates D1 receptors, which display a much lower sensitivity to dopamine in intact tissue. Because activation of the D1 receptors on fish cone horizontal (H1) cells uncouples the gap junctions between the cells, we studied whether the clock regulates the extent of biocytin tracer coupling in the goldfish retina. Tracer coupling between H1 cells was extensive under dark-adapted conditions (low scotopic range) and similar in the subjective day, subjective night, day, and night. An average of approximately 180 cells were coupled in each dark-adapted condition. However, bright light stimulation or application of the D1 agonist SKF38393 (10 μM) dramatically reduced H1 cell coupling. The D2 agonist quinpirole (1 μM) or application of the D1 antagonist SCH23390 (10 μM) and/or the D2 antagonist spiperone (10 μM) had no effect on H1 cell coupling in dark-adapted retinas. These observations demonstrate that H1 cell gap junctional coupling and thus D1 receptor activity are not affected by endogenous dopamine under dark-adapted conditions. The results suggest that two different dopamine systems are present in the goldfish retina. One system is controlled by an endogenous clock that activates low threshold D2-like receptors in the day, whereas the second system is controlled by light and involves activation of higher threshold D1 receptors. J. Comp. Neurol. 467:243–253, 2003. © 2003 Wiley-Liss, Inc.

Melatonin, the major hormone produced by the pineal gland, displays characteristic daily and seasonal patterns of secretion. These robust and predictable rhythms in circulating melatonin are strong synchronizers for the expression of numerous physiological processes in photoperiodic species. In mammals, the nighttime production of melatonin is mainly driven by the circadian clock, situated in the suprachiasmatic nucleus of the hypothalamus, which controls the release of norepinephrine from the dense pineal sympathetic afferents. The pivotal role of norepinephrine in the nocturnal stimulation of melatonin synthesis has been extensively dissected at the cellular and molecular levels. Besides the noradrenergic input, the presence of numerous other transmitters originating from various sources has been reported in the pineal gland. Many of these are neuropeptides and appear to contribute to the regulation of melatonin synthesis by modulating the effects of norepinephrine on pineal biochemistry. The aim of this review is firstly to update our knowledge of the cellular and molecular events underlying the noradrenergic control of melatonin synthesis; and secondly to gather together early and recent data on the effects of the nonadrenergic transmitters on modulation of melatonin synthesis. This information reveals the variety of inputs that can be integrated by the pineal gland; what elements are crucial to deliver the very precise timing information to the organism. This also clarifies the role of these various inputs in the seasonal variation of melatonin synthesis and their subsequent physiological function.

Zinc is strikingly co-localized with glutamate-containing vesicles in the synaptic terminals of retinal photoreceptors, and it is thought to be co-released with glutamate onto postsynaptic neurons such as horizontal cells and bipolar cells. Here we examined exogenous zinc modulation of glutamate receptors on cultured retinal horizontal cells using patch-clamp recording and endogenous zinc effect on intact horizontal cells using intracellular recording techniques. Application of 3, 30, and 300 μM zinc reduced the whole cell peak current of response to 200 μM glutamate by 2, 30, and 56%, respectively. Zinc suppression of glutamate response persisted in the presence of 10 μM cyclothiazide (CTZ). Glutamate responses of outside-out patches were completely abolished by 30 μM 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), and the receptor desensitization was blocked by 30 μM CTZ, indicating that receptor target for the zinc action on horizontal cells is α-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptors. Zinc decreased the amplitude of outside-out patch peak current without an effect on either its 10–90% rise time or the rate of receptor desensitization. Dose-response curves for glutamate show that zinc reduced the maximal current evoked by glutamate and increased EC50 from 50 ± 3 to 70 ± 6 μM without changing the Hill coefficient. Chelation of endogenous zinc with 1 mM Ca-EDTA depolarized horizontal cells in the intact retina by 3 mV, consistent with relief of the partial glutamate receptor inhibition by zinc. Overall, the results describe a unimodal form of zinc modulation of AMPA-type glutamate receptor responses not previously described in native neuronal preparations and a novel role for endogenous zinc in modulating neurotransmission.

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 D(2)-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 D(2)-like antagonist, or forskolin, an activator of adenylyl cyclase, during the subjective day reduced cone input and increased rod input. SCH23390, a D(1) antagonist, had no effect. Application of R(p)-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 D(2)-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 D(2)-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.