Hyperpolarization-activated current (I(h)) in ganglion-cell photoreceptors

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and serve as the primary retinal drivers of non-image-forming visual functions such as circadian photoentrainment, the pupillary light reflex, and suppression of melatonin production in the pineal. Past electrophysiological studies of these cells have focused on their intrinsic photosensitivity and synaptic inputs. Much less is known about their voltage-gated channels and how these might shape their output to non-image-forming visual centers. Here, we show that rat ipRGCs retrolabeled from the suprachiasmatic nucleus (SCN) express a hyperpolarization-activated inwardly-rectifying current (I(h)). This current is blocked by the known I(h) blockers ZD7288 and extracellular cesium. As in other systems, including other retinal ganglion cells, I(h) in ipRGCs is characterized by slow kinetics and a slightly greater permeability for K(+) than for Na(+). Unlike in other systems, however, I(h) in ipRGCs apparently does not actively contribute to resting membrane potential. We also explore non-specific effects of the common I(h) blocker ZD7288 on rebound depolarization and evoked spiking and discuss possible functional roles of I(h) in non-image-forming vision. This study is the first to characterize I(h) in a well-defined population of retinal ganglion cells, namely SCN-projecting ipRGCs.     

Mice Deficient of Glutamatergic Signaling from Intrinsically Photosensitive Retinal Ganglion Cells Exhibit Abnormal Circadian Photoentrainment

Several aspects of behavior and physiology, such as sleep and wakefulness, blood pressure, body temperature, and hormone secretion exhibit daily oscillations known as circadian rhythms. These circadian rhythms are orchestrated by an intrinsic biological clock in the suprachiasmatic nuclei (SCN) of the hypothalamus which is adjusted to the daily environmental cycles of day and night by the process of photoentrainment. In mammals, the neuronal signal for photoentrainment arises from a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that send a direct projection to the SCN. ipRGCs also mediate other non-image-forming (NIF) visual responses such as negative masking of locomotor activity by light, and the pupillary light reflex (PLR) via co-release of neurotransmitters glutamate and pituitary adenylate cyclase-activating peptide (PACAP) from their synaptic terminals. The relative contribution of each neurotransmitter system for the circadian photoentrainment and other NIF visual responses is still unresolved. We investigated the role of glutamatergic neurotransmission for circadian photoentrainment and NIF behaviors by selective ablation of ipRGC glutamatergic synaptic transmission in mice. Mutant mice displayed delayed re-entrainment to a 6 h phase shift (advance or delay) in the light cycle and incomplete photoentrainment in a symmetrical skeleton photoperiod regimen (1 h light pulses between 11 h dark periods). Circadian rhythmicity in constant darkness also was reduced in some mutant mice. Other NIF responses such as the PLR and negative masking responses to light were also partially attenuated. Overall, these results suggest that glutamate from ipRGCs drives circadian photoentrainment and negative masking responses to light.     

Melanopsin regulates visual processing in the mouse retina

The discovery of melanopsin-dependent inner retinal photoreceptors in mammals has precipitated a fundamental reassessment of such non-image forming (NIF) light responses as circadian photoentrainment and the pupil light reflex. By contrast, it remains unclear whether these new photoreceptors also play a role in classical image-forming vision. The retinal ganglion cells that subserve inner retinal photoreception (ipRGCs) project overwhelmingly to brain areas involved in NIF responses, indicating that, in terms of central signaling, their predominant function is non-image forming. However, ipRGCs also exhibit intraretinal communication via gap junction coupling, which could allow them to modulate classical visual pathways within this tissue. Here, we explore this second possibility by using melanopsin knockout (Opn4-/-) mice to examine the role of inner retinal photoreceptors in diurnal regulation of retinal function. By using electroretinography in wild-type mice, we describe diurnal rhythms in both the amplitude and speed of the retinal cone pathway that are a function of both prior light exposure and circadian phase. Unexpectedly, loss of the melanopsin gene abolishes circadian control of these parameters, causing significant attenuation of the diurnal variation in cone vision. Our results demonstrate for the first time a melanopsin-dependent regulation of visual processing within the retina, revealing an important function for inner retinal photoreceptors in optimizing classical visual pathways according to time of day.     

Melanopsin-based brightness discrimination in mice and humans

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a small number of intrinsically photoreceptive retinal ganglion cells (ipRGCs), expressing the photopigment melanopsin. ipRGCs are known to support various accessory visual functions including circadian photoentrainment and pupillary reflexes. However, despite anatomical and physiological evidence that they contribute to the thalamocortical visual projection, no aspect of visual discrimination has been shown to rely upon ipRGCs. Based on their currently known roles, we hypothesized that ipRGCs may contribute to distinguishing brightness. This percept is related to an object's luminance-a photometric measure of light intensity relevant for cone photoreceptors. However, the perceived brightness of different sources is not always predicted by their respective luminance. Here, we used parallel behavioral and electrophysiological experiments to first show that melanopsin contributes to brightness discrimination in both retinally degenerate and fully sighted mice. We continued to use comparable paradigms in psychophysical experiments to provide evidence for a similar role in healthy human subjects. These data represent the first direct evidence that an aspect of visual discrimination in normally sighted subjects can be supported by inner retinal photoreceptors.     

Evidence for mouse sulfhydryl oxidase‐assisted cross‐linking of major seminal vesicle proteins

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high‐molecular‐weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)‐dependent Sox was purified from mouse SVS using ion exchange and high‐performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The K_m and V_max of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross‐linking activity among SVS I–III monomers to form HMWCs in SVS.     

Sox neuro, a new Drosophila Sox gene expressed in the developing central nervous system

We describe the identification and detailed expression pattern of a second Drosophila Sox gene, SoxNeuro (SoxN), highly related to mammalian group B Sox1, 2, 3 genes. SoxN is expressed in a highly dynamic pattern during embyogenesis, being associated with the development of the central nervous system (CNS), from the early steps onwards. We present strong evidence that the early SoxN neuroectoderm expression is controlled by the zygotic dorso-ventral patterning genes (dpp, sog, brk, twi).     

Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells

The retinal ganglion cell layer of the eye comprises a subtype of cells characterized by their intrinsic photosensitivity and expression of melanopsin (ipRGCs). These cells regulate a variety of non-image-forming (NIF) functions such as light entrainment of circadian rhythms, acute suppression of locomotor activity (masking), and pupillary light reflex. Two neurotransmitters have been identified in ipRGCs, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP). To date, little is known about their release and interplay. Here, we describe the presence and co-localization of vesicular glutamate transporter 2 (VGLUT2; a marker of glutamate signaling) and PACAP in ipRGCs and their projections in the brain. Nine adult male Wistar rats were assigned to one of three groups; anterograde tracing (n = 3), eye enucleation (n = 3), and untreated (n = 3). Under anaesthesia, rats were transcardially perfusion-fixated, after which the brains and eyes were removed for double immunohistochemical staining using a polyclonal anti-VGLUT2 antibody and a mouse monoclonal anti-PACAP antibody. Results revealed that VGLUT2- and PACAP-immunoreactivity (-ir) were present in ipRGCs and co-localized in their projections in the suprachiasmatic nucleus, the intergeniculate leaflet, and the olivary pretectal nucleus. We conclude that there is evidence to support the use of glutamate and PACAP as neurotransmitters in NIF photoperception by rat ipRGCs, and that these neurotransmitters are co-stored and probably released from the same nerve terminals. Furthermore, we conclude that VGLUT2 is the preferred subtype of vesicular transporter used by these cells.