The circadian response of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGC) signal environmental light level to the central circadian clock and contribute to the pupil light reflex. It is unknown if ipRGC activity is subject to extrinsic (central) or intrinsic (retinal) network-mediated circadian modulation during light entrainment and phase shifting. Eleven younger persons (18–30 years) with no ophthalmological, medical or sleep disorders participated. The activity of the inner (ipRGC) and outer retina (cone photoreceptors) was assessed hourly using the pupil light reflex during a 24 h period of constant environmental illumination (10 lux). Exogenous circadian cues of activity, sleep, posture, caffeine, ambient temperature, caloric intake and ambient illumination were controlled. Dim-light melatonin onset (DLMO) was determined from salivary melatonin assay at hourly intervals, and participant melatonin onset values were set to 14 h to adjust clock time to circadian time. Here we demonstrate in humans that the ipRGC controlled post-illumination pupil response has a circadian rhythm independent of external light cues. This circadian variation precedes melatonin onset and the minimum ipRGC driven pupil response occurs post melatonin onset. Outer retinal photoreceptor contributions to the inner retinal ipRGC driven post-illumination pupil response also show circadian variation whereas direct outer retinal cone inputs to the pupil light reflex do not, indicating that intrinsically photosensitive (melanopsin) retinal ganglion cells mediate this circadian variation.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Dynamics of rod and cone photoreceptor interactions under mesopic light levels

Visual adaptation regulates contrast sensitivity during dynamically changing light conditions (Crawford, 1947; Hecht, Haig & Chase, 1937). These adaptation dynamics are unknown under dim (mesopic) light levels when the rod (R) and long (L), medium (M) and short (S) wavelength cone photoreceptor classes contribute to vision via interactions in shared non-opponent Magnocellular (MC), chromatically opponent Parvocellular (PC) and Koniocellular (KC) visual pathways (Dacey, 2000). This study investigated the time-course of adaptation and post-receptoral pathways mediating receptor specific rod and cone interactions under mesopic illumination. A four-primary photostimulator (Pokorny, Smithson & Quinlan, 2004) was used to independently control the activity of the four photoreceptor classes and their post-receptoral visual athways in human observers. In the first experiment, the contrast sensitivity and time-course of visual adaptation under mesopic illumination were measured for receptoral (L, S, R) and post-receptoral (LMS, LMSR, L-M) stimuli. An incremental (Rapid-ON) sawtooth conditioning pulse biased detection to ON-cells within the visual pathways and sensitivity was assayed relative to pulse onset using a briefly presented incremental probe that did not alter adaptation. Cone.Cone interactions with luminance stimuli (L cone, LMS, LMSR) reduced sensitivity by 15% and the time course of recovery was 25± 5ms-1 (μ ± SEM). PC mediated (+L-M) chromatic stimuli sensitivity loss was less (8%) than for luminance and recovery was slower (μ = 2.95 ± 0.05 ms-1), with KC mediated (S cone) chromatic stimuli showing a high sensitivity loss (38%) and the slowest recovery time (1.6 ± 0.2 ms-1). Rod-Rod interactions increased sensitivity by 20% and the time course of recovery was 0.7 ± 0.2 ms-1 (μ ± SD). Compared to these interaction types, Rod-Cone interactions reduced sensitivity to a lesser degree (5%) and showed the fastest recovery (μ = 43 ± 7 ms-1). In the second experiment, rod contribution to the magnocellular, parvocellular and koniocellular post-receptoral pathways under mesopic illumination was determined as a function of incremental stimulus duration and waveform (rectangular; sawtooth) using a rod colour match procedure (Cao, Pokorny & Smith, 2005; Cao, Pokorny, Smith & Zele, 2008a). For a 30% rod increment, a cone match required a decrease in [L/(L+M)] and an increase in [L+M] and [S/(L+M)], giving a greenish-blue and brighter appearance for probe durations of 75 ms or longer. Probe durations less than 75 ms showed an increase in [L+M] and no change in chromaticity [L/(L+M) or S/(L+M)], uggesting mediation by the MC pathway only for short duration rod stimuli. s We advance previous studies by determining the time-course and nature of photoreceptor specific retinal interactions in the three post-receptoral pathways under mesopic illumination. In the first experiment, the time-course of adaptation for ON cell processing was determined, revealing opponent cell facilitation in chromatic PC and KC pathways. The Rod-Rod and Rod-Cone data identify previously unknown interaction types that act to maintain contrast sensitivity during dynamically changing light conditions and improve the speed of light adaptation under mesopic light levels. The second experiment determined the degree of rod contribution to the inferred post-eceptoral pathways as a function of the temporal properties of the rod signal. r The understanding of the mechanisms underlying interactions between photoreceptors under mesopic illumination has implications for the study of retinal disease. Visual function has been shown to be reduced in persons with age-related maculopathy (ARM) risk genotypes prior to clinical signs of the disease (Feigl, Cao, Morris & Zele, 2011) and disturbances in rod-mediated adaptation have been shown in early phases of ARM (Dimitrov, Guymer, Zele, Anderson & Vingrys, 2008; Feigl, Brown, Lovie-Kitchin & Swann, 2005). Also, the understanding of retinal networks controlling vision enables the development of international lighting standards to optimise visual performance nder dim light levels (e.g. work-place environments, transportation).

A butterfly eye's view of birds

The striking color patterns of butterflies and birds have long interested biologists. But how these animals see color is less well understood. Opsins are the protein components of the visual pigments of the eye. Color vision has evolved in butterflies through opsin gene duplications, through positive selection at individual opsin loci, and by the use of filtering pigments. By contrast, birds have retained the same opsin complement present in early-jawed vertebrates, and their visual system has diversified primarily through tuning of the short-wavelength-sensitive photoreceptors, rather than by opsin duplication or the use of filtering elements. Butterflies and birds have evolved photoreceptors that might use some of the same amino acid sites for generating similar spectral phenotypes across approximately 540 million years of evolution, when rhabdomeric and ciliary-type opsins radiated during the early Cambrian period. Considering the similarities between the two taxa, it is surprising that the eyes of birds are not more diverse. Additional taxonomic sampling of birds may help clarify this mystery.

Mesopic rod and S-cone interactions revealed by modulation thresholds

We analyzed mesopic rod and S-cone interactions in terms of their contributions to the blue-yellow opponent pathway. Stimuli were generated using a 4-primary colorimeter. Mixed rod and S-cone modulation thresholds (constant L-, M-cone excitation) were measured as a function of their phase difference. Modulation amplitude was equated using threshold units and contrast ratios. This study identified three interaction types: (1) A linear and antagonistic rod:S-cone interaction, (2) probability summation (3) and a previously unidentified mutual nonlinear reinforcement. Linear rod:S-cone interactions occur within the blue-yellow opponent pathway. Probability summation involves signaling by different post-receptoral pathways. The origin of the nonlinear reinforcement is possibly at the photoreceptors.

Melanopsin expressing intrinsically photosensitive Retinal Ganglion cells in retinal disease

Melanopsin containing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) are a class of photoreceptors with established roles in non-image forming processes. Their contributions to image forming vision may include the estimation of brightness. Animal models have been central for understanding the physiological mechanisms of ipRGC function and there is evidence of conservation of function across species. ipRGCs can be divided into 5 ganglion cell subtypes that show morphological and functional diversity. Research in humans has established that ipRGCs signal environmental irradiance to entrain the central body clock to the solar day for regulating circadian processes and sleep. In addition, ipRGCs mediate the pupil light reflex (PLR), making the PLR a readily accessible behavioural marker of ipRGC activity. Less is known about ipRGC function in retinal and optic nerve disease, with emerging research providing insight into their function in diabetes, retinitis pigmentosa, glaucoma and hereditary optic neuropathy. We briefly review the anatomical distributions, projections and basic physiological mechanisms of ipRGCs, their proposed and known functions in animals and humans with and without eye disease. We introduce a paradigm for differentiating inner and outer retinal inputs to the pupillary control pathway in retinal disease and apply this paradigm to patients with age-related macular degeneration (AMD). In these cases of patients with AMD, we provide the initial evidence that ipRGC function is altered, and that the dysfunction is more pronounced in advanced disease. Our perspective is that with refined pupillometry paradigms, the pupil light reflex can be extended to AMD assessment as a tool for the measurement of inner and outer retinal dysfunction.

Retinal anatomy of the New Zealand kiwi: Structural traits consistent with their nocturnal behavior

Kiwi (Apteryx spp.) have a visual system unlike that of other nocturnal birds, and have specializations to their auditory, olfactory and tactile systems. Eye size, binocular visual fields and visual brain centers in kiwi are proportionally the smallest yet recorded among birds. Given the many unique features of the kiwi visual system, we examined the laminar organization of the kiwi retina to determine if they evolved increased light sensitivity with a shift to a nocturnal niche or if they retained features of their diurnal ancestor. The laminar organization of the kiwi retina was consistent with an ability to detect low light levels similar to that of other nocturnal species. In particular, the retina appeared to have a high proportion of rod photoreceptors compared to diurnal species, as evidenced by a thick outer nuclear layer, and also numerous thin photoreceptor segments intercalated among the conical shaped cone photoreceptor inner segments. Therefore, the retinal structure of kiwi was consistent with increased light sensitivity, although other features of the visual system, such as eye size, suggest a reduced reliance on vision. The unique combination of a nocturnal retina and smaller than expected eye size, binocular visual fields and brain regions make the kiwi visual system unlike that of any bird examined to date. Whether these features of their visual system are an evolutionary design that meets their specific visual needs or are a remnant of a kiwi ancestor that relied more heavily on vision is yet to be determined.