Daily Organization of Retinal Function
“The only reason for time is so that everything doesn’t happen at once.”
We use a combination of anatomical, cellular, molecular, biochemical, electrophysiological, pharmacological, genetic, behavioral, and computational modeling approaches to fully understand the biological basis and functional importance of circadian clocks in the mammalian retina.
Current projects include the following:
- The biological basis of circadian clocks in the retina
The core mechanism of circadian clocks is contained within single cells and relies on a specific set of genes (the clock genes) and their protein products interlocked in translational-transcriptional feedback loops that self-regenerate with a period close to 24 h. Our recently published data, together with others, reveal an important disparity in the expression of the core clock components among retinal layers and cell types. The core components of the mammalian circadian clock are expressed in most retinal cell types, and their patterns of expression show great disparity in both amplitude and phase among retinal layers and cell types. Globally, these findings suggest that the retinal circadian system is built upon an array of clocks, each present in different cell types. Our central hypothesis is that the neural retina is a heterogeneous tissue in terms of clock activity: circadian clocks are present in most retinal cell types, and each clock cell type controls specific aspects of retinal function through a restricted clock pathway. Therefore, knocking out clock function in specific retinal cell types will help establish the specific contribution of each clock to the overall temporal organization of the tissue’s function. We have developed several mouse models that are retinal cell type-specific and clock-deficient and are actively pursuing this research avenue.
Techniques/tools: mouse genetics, retinal cell type-specific clock-deficient mouse models, immunohistochemistry, confocal microscopy
- The nature of the effectors/outputs of the retinal clocks
There is a variety of ways through which a clock mechanism can modulate neuronal activity, such as controlling gene expression in the clock cell itself. To identify candidate genes and signaling pathways controlled by a circadian clock in photoreceptors, namely rods and cones, we have developed an experimental approach based on conditional knockout strategies, cell sorting, and RNA sequencing and analysis.
We are also interested in the control of the synthesis/release of retinal neurohormones by the circadian clocks, because neurohormones diffuse through retinal layers and influence large populations of neurons. Thereby, circadian clocks can affect entire functional pathways and/or set the gain of the retina. Our work and that of others has essentially focused on dopamine, adenosine, and melatonin. Yet, the signaling pathways through which these neurohormones control retinal function remain poorly understood, and many aspects of their control by circadian clocks remain to be clarified. Using our clock-deficient mouse models, we are currently investigating the retinal clocks responsible for the control of these neurohormones. This approach is complemented with precise electrophysiology experiments (see below).
Techniques/tools: mouse genetics, retina or retinal cell type-specific clock-deficient mouse models, immunohistochemistry, confocal microscopy, High Performance Liquid Chromatography (HPLC, electrochemical detection, UV detection), Fluorescence-Assisted Cell Sorting (FACS), RNAseq and analysis
- The impact of the clocks on the activity of specific retinal neurons and functional pathways
Knowledge of the circadian clock regulation of retinal function in mammals is essentially based on mass (electroretinographic) recordings, a technique that provides limited insight on the nature of the cellular/subcellular processes underlying the circadian-induced changes in retinal function. Few studies have focused on the control of clocks on the light responses of single retinal cell types. Yet, identification of the neuronal targets of the clocks is key to our understanding of the clock control mechanisms of retinal physiology and function. We have developed a novel technique, voltage clamp of single or pairs of photoreceptors in mouse retinas maintained by superfusion and found a circadian component in the light response kinetics and the state of electrical coupling that is sensitive to the melatonin and dopamine. We are dissecting this clock pathway that includes a clock within the photoreceptor layer, melatonin, dopamine, and gap junctions between photoreceptors and are building a realistic computational model of the mouse photoreceptor network.
We are pursuing the understanding of the clock pathways that control the daily change in photoreceptor electrical coupling and the consequences of such regulation for downstream circuits. Our approach here includes single cell and paired recordings of mouse photoreceptors and of second-order neurons in a variety of genetically modified animals including photoreceptor type-specific clock- or gap junction-deficient animals.
Techniques/tools: mouse genetics, retina- or retinal cell type-specific clock-deficient mouse models, photoreceptor type-specific gap-junction-deficient mouse models, single-cell (path-clamp) recording on mouse intact retina or retinal slice, simultaneous (patch-clamp) recording of pairs of adjacent photoreceptors in retinal slice, tracer injection, confocal microscopy, computational modeling
- The aging-dependent decline in retinal function and the role of circadian clocks in this process
An increasing body of evidence indicates that circadian clocks and their outputs influence functional and trophic processes in the retina. For instance, retina-specific ablation of the clock mechanism leads to abnormal retinal transcriptional activity and defective inner retinal electrical responses to light. In addition, a decrease in the efficacy or of the rhythm of some circadian factors, such as dopamine, adenosine, and melatonin, may affect photoreceptor survival. For instance, impairment of melatonin signaling affects retinal cell viability, and dopamine is necessary for the circadian nature of light-adapted vision as well as optimal contrast detection and acuity. Understanding the basis of circadian clocks in the retina is therefore key to our understanding of retina normal function and dysfunction. We are currently examining our retina- or retinal cell type-specific clock knock-out mice for morphological and visual defects. Our hope is to identify specific retinal clock cell types (and their associated clock pathways) that are critical for the maintenance of retinal tissue and normal retinal function.
Techniques/tools: mouse genetics, retinal cell type-specific clock-deficient mouse models, retina-specific clock-deficient mouse models, immunohistochemistry, confocal microscopy, assessment of the pupillary light reflex, optomotor reflex-based visual test, wheel-based locomotor assay, electroretinogram