IV. RESULTADOS Y DISCUSIÓN
4.2. Análisis inferencial y contrastación de hipótesis
4.2.3. Análisis inferencial del modelo 3 obtenido
Luminance detection is the catch-all term for processes which sense light but are non- visual, meaning they simply detect how much light is in the environment at any one time without any detailed spatiotemporal information. The majority of research into luminance detection has focused on its role in setting the so called ‘biological clock’ and how circadian cycles within animals are generated and maintained. This work is of particular interest in a medical context, since, due in large part to artificial light, humans are fast breaking away from the natural cycle of light and dark which has honed our physiology for millennia: the concepts of jet lag, shift work and electric light are all very recent additions to planet Earth (Foster & Wulff 2005).
The circadian regulation of physiology and behaviour is hardwired into all animals on the planet. By having the power to predict the coming of night and day the body is able to work optimally over the course of 24 hours and avoids the highly inefficient method of responding to the changes in the environment as they happen (Roenneberg & Foster, 1997; Foster & Kreitzman, 2004). An internal representation of the Earth day is present in all species. This has been corroborated by the observation that, devoid of external cues, the daily rhythms will continue regardless (Bunning, 1973). This, so called, “free- running” rhythm is found to be close to but never exactly 24 hours (Fig. 1.9).
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11-cis retinal all-transretinal
retinal binding site
A
Bi
ii
43 Figure 1.8 Classic photoreception for vision.
A, In the vertebrate retina there are two types of ciliary photoreceptor cell, called rods and
cones. The outer segment of the photoreceptors contain the photoreceptive machinery while the inner segment synapses onto other cells within the retina for processing of the visual
information. Bi, The first step during phototransduction is the absorption of a photon of light
(hv) by 11-cis retinal. This reaction converts the vitamin A-derived molecule into an all-trans
confirmation. Bii,The retinal is bound to a membrane-bound opsin protein and the
conformational change induced by light activates the opsin. C, Opsins are found in both
vertebrate and invertebrate phototransduction although their activation by light has differential downstream effects: vertebrate phototransduction involves the activation of the G-protein transducin and subsequent activation of PDE resulting in reduced cGMP levels and
hyperpolarisation via the closing of CNG ion channels; invertebrate phototransduction, on the
other hand, involves Gq G-proteins, PLC activation and depolarisation via the opening of TRP
channels. Figures adapted from Peirson & Foster (2006; A & Bii) and Peirson et al (2009; Bi & C).
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Of course, in reality, the internal ‘free-running’ representation of time would be useless if it was not synchronised to the natural day-night cycle. To achieve this, a timing cue from the external environment or “zeitgeber” (time giver) is required to reset or entrain the internal clock (Aschoff, 1984). For most species the primary zeitgeber is the reliable change in the lighting conditions at dawn and dusk and so the resetting of the biological clock by zeitgebers is termed photoentrainment (Roenneberg & Foster, 1997).
Figure 1.9 Free-running of circadian rhythms under constant light conditions.
Ai, Wheel-running activity in a mouse plotted over 17 days. For the first 7 days the mouse was
exposed to a light regime of 12 hours light, 12 hours dark (LD 12:12) and displayed normal
nocturnal activity. On the 8th day the mouse does not experience ‘dawn’ and the lights are kept
off for the remaining 10 days (DD). Wheel-running activity continues to show circadian rhythmicity but gradually advances in time due to running at 23.5 hours rather than exactly 24
hours. Aii, A different mouse initially exposed to normal LD 12:12 conditions is subsequently
exposed to constant light (LL). Under these conditions the wheel-running activity advanced displaying a period of approximately 25 hours. In both conditions the endogenous nature of the circadian rhythm as well as the importance of a light dark cycle to maintain 24 hour periodicity is evident. Figures adapted from Roenneburg & Foster (1997).
While the intrinsic circadian cycles within animals are seemingly well conserved across species, the mechanisms for receiving the photic cue to entrain the rhythm is relatively
Ai
ii
Days o f ex p eri me n t Days o f ex p eri me n t LD 12:12 DD LD 12:12 LL Light regime45
diverse. The pineal organ (or commonly called “pineal gland”) is found in all vertebrates, although it has taken on slightly different forms and functions across species (Peirson et al, 2009). The pineal structures, like the eyes, arose from an invagination of the diencephalon and are the remnants of two dorsally located ‘eyes’ thought to exist in an early ancestor in the vertebrate lineage (Vigh et al, 2002). To this day, some reptiles (Rhynchocephalia and Squamata) and amphibians (Anura) retain an extra-cranial ‘third eye’ in addition to intracranial pineal glands (Pierson et al, 2009). In the case of the reptiles this parietal eye is structurally very similar to the lateral eyes containing both a lens and a cornea (Shand & Foster, 1999). In lamprey and teleosts the pineal complex is also composed of two structures, the pineal and parapineal, although both are located intracranially (Vigh et al, 2002).
The primary role of the pineal is to synthesis and secrete the neurohormone melatonin (Arendt 1998; Korf et al, 1998). Melatonin is released during the dark phase of the light/dark cycle and its levels fluctuate across the course of the day, helping to synchronise many physiological processes to a circadian rhythm. In non-mammalian vertebrates, the pineal is directly light sensitive and located near the surface of the brain. Moreover, the majority of pinealocytes resemble photoreceptor cells in appearance and indeed a mixture of cone-like and rod-like cells are found (Vigh et al, 2002).
In several species, including the lamprey, zebrafish and chicken, the pineal is also capable of generating endogenous circadian rhythms, meaning the whole
photoneuroendocrine system is housed in one structure (Korf et al, 1998). In fact, in house sparrows that have become arrhythmic after pinealectomy, a transplanted pineal will restore robust rhythmic activity within just a few days (Zimmerman & Menaker, 1979). This work neatly demonstrates the hormonal nature of the signal, since the donor pineal has no chance to make neural connections but can still entrain the physiology of the recipient bird. Although the mammalian pineal contains many constituents of the phototransduction cascade (Korf et al, 1985; Foster et al, 1989), crucially, it contains no chromophore and has lost its direct sensitivity to light (Vollrath, 1981; Foster et al, 1989; Korf et al, 1998; Arendt, 1998). Moreover, circadian rhythms in mammals are set within the suprachiasmatic nucleus (SCN) of the hypothalamus and the pineal itself is purely a secretory structure.
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The mammalian SCN is thought to be the master clock of the body. The first studies on the SCN showed that this tiny cluster of approximately 20,000 cells in the rat, were necessary to maintain regular patterns of hormone secretion and behaviours including feeding and locomotion (Moore & Eichler, 1972). By recording directly from the SCN it has been shown to follow a rhythmic pattern of neural activity, with more activity during the day and less at night (Inouye & Kawamura 1979). This is true both in intact animals, the isolated SCN and individual SCN neurons (Welsh et al. 1995). The SCN communicates with the rest of the brain via both direct neural connections and as yet unknown chemical messengers and controls processes all over the body via
neuroendocrine signalling (for a review see Kriegsfeld & Silver 2006). Despite this view, it cannot be ignored that almost all peripheral tissues are capable of circadian oscillation in gene expression (Foster & Kreitzman, 2004). The story is clearly very complicated but, whether or not other cells and tissues are hardwired to oscillate over the 24 hour day, it is the SCN and the SCN alone that can restore rhythmic activity once it is lost (Ralph et al, 1990; Foster & Kreitzman, 2004).
All photic information from the retina leaves via the optic nerve which is composed of the axons of retinal ganglion cells (RGCs). As well the carrying visual information to the lateral geniculate nucleus and midbrain, en route to the primary visual cortex, the optic nerve contains the retinohypothalamic tract (RHT). The RHT, as its name suggests, links a proportion of RGCs to the hypothalamus and allows external photic information to reach the SCN. In mammals, therefore, photoreceptors for both vision and luminance detection are found in the retina. Blind or enucleated mammals cannot entrain to a light dark cycle (Nelson & Zucker 1981) (Foster et al, 1991); however, mutant mice, with intact retinas but without rod or cone photorecpetors (rd/rd cl), retain this ability (Freedman et al, 1999). Furthermore, these mice showed normal pineal melatonin regulation suggesting the link between the retina and the circadian machinery of the hypothalamus is independent of rods and cones (Lucas et al, 1999).
Electrophysiological recordings from retinal neurons labelled via retrograde tracing from the SCN in rats highlighted a population of RGCs that were directly photosensitive (Berson et al. 2002). These results were backed up by calcium imaging from the rd/rd cl
mice which again highlighted a population of intrinsically photosensitive sensitive RGCs (ipRGC; Sekaran et al, 2003). Moreover, this sub-set of RGCs, which make up
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about 1% of the total population, were found to express melanopsin, a novel photopigment in the mammalian retina (Provencio et al. 2000; Hattar et al. 2002).
Melanopsin has since been shown to be crucial for the phototransduction in vertebrate luminance detection. In studies using melanopsin knockout mice (Opn4-/-), the animals displayed disrupted entrainment to light (Panda et al, 2002; Ruby et al, 2002).
Furthermore, knockout of melanopsin as well as rods and cones resulted in mice that were completely unresponsive to light (Hattar et al, 2003). Surprisingly, the mechanism for melanopsin-mediated phototransduction seems to be more like that found in
invertebrate systems than a typical vertebrate signalling cascade (Peirson & Foster 2006). Briefly, ipRGCs and cells transfected with melanopsin depolarise in response to light; signalling seems to be dependent on Gq/G11-type G-proteins and not Gi/Go-type
and PLC and TRP channels are also implicated in the signalling cascade (for review see Peirson et al. 2009).
The input from ipRGCs converges on the SCN from sites all over the retina and, unlike the topographic arrangement of RGC inputs to the visual cortex (Rodieck, 1998), is unpatterned (Cooper et al, 1993). By averaging irradiance from the entire visible environment, and not the radiance from a single point, the SCN can then get a more accurate ‘picture’ of the true light level. Experimental evidence corroborates this since the system for photoentrainment is less sensitive overall than the visual system and also requires longer integration times (Nelson & Takahashi 1991). The slower integration time helps to avoid errors caused by momentarily encountering a particularly dim or bright photic environment, while the high threshold for activation serves to reduce the chance of saturation over the several minutes needed for accurate irradiance detection.