Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

term (i.e., across days), continuous measurement of general activity under controlled lighting conditions is a widely used approach in analyzing the circadian oscillator. Although general activity may be measured by infrared beam-break systems, the wheel-running assay is a robust readout of temporal activity, because rodents limit their wheel-running activity to night and rest during the day. In a typical assay, mice are individually housed in special cages equipped with a running wheel whose rotation is computer monitored (Siepka and Takahashi, 2005). Under a normal 12-hour light, 12-hour dark (LD) lighting regimen, mice of the widely used C57Bl/6J strain start their wheel-running activity at dark onset and are active throughout the night. Such a tight, 24-hour activity rhythm arises from photoentrainment of the circadian clock to the imposed LD cycle and from the lightinduced suppression of activity, or negative masking, that occurs in mice (Mrosovsky, 1999). Because negative masking may be confused with bona fide circadian entrainment, it is important to be able to distinguish between them. This is best accomplished by administering a discrete period of light to the animals during the dark phase. In mice, this action results in the immediate cessation of wheel-running behavior and the onset of an inactive state for the duration of the light pulse. In other words, light negatively masks or inhibits the activity of nocturnal animals, and the phenomenon is called negative masking.

The effect of light in adjusting the phase of the oscillator is best assessed in mice held under constant darkness. Mice are first entrained to a LD cycle for up to 2 weeks and then released into constant darkness (DD). Under DD, the time of activity onset is no longer determined by the time of dark onset but by the phase of the clock. The time difference between activity onset on the first day of DD and dark onset during the prior entrainment period is referred to as the phase angle of entrainment and reflects the true phase of entrainment. Although mice of wild-type C57Bl/6J background have a phase angle of almost zero, other strains exhibit a significant phase angle of entrainment, which might arise from abnormal phototransduction mechanism, core circadian clock function, or masking. During the subsequent days under DD, with no scheduled lighting or other timing cues, the temporal activity is said to “free run” entirely under the control of the circadian clock (figure 17.1). The temporal activity pattern still exhibits tight circadian control, so that the activity is mostly consolidated to the “subjective night” time. C57Bl/6J mice typically exhibit a DD free-running period length of about 23.5 hours, which truly reflects the daily rhythm in individual SCN neural activity and the rhythm of the molecular oscillator (Welsh et al., 1995; Yoo et al., 2004). Because the DD period length is slightly different from that of the geophysical day, the temporal activity is no longer expressed with reference to the local time. Instead, the temporal measures are referenced as the endog-

Figure 17.1 Circadian photoentrainment in mice. Wheelrunning activity record of a mouse shows daily activity entrainment to an imposed light-dark cycle, free running under constant darkness, and the activity phase-shifting effect of a single light pulse. Each horizontal line shows the temporal activity pattern over one geophysical day. The mouse was entrained to LD cycle for 10 days and subsequently released into constant darkness. On day 20, the animal received a 30-minute light pulse approximately 4 hours after activity onset. Phase shift (if any) in response to the light pulse is usually determined by calculating the time difference (on the day of light pulse) between two extrapolated regression lines fitting daily activity onsets before and after the light pulse.

enous circadian time (CT), where the free-running period length is normalized to 24 hours. In this convention, the daily activity onset is referenced as CT12, so that the subjective day spans from CT0 to CT12 and the subjective night extends from CT12 to CT24 or CT0. A pulse of light administered during the subjective night causes a shift in the phase of the clock, so that the wheel-running activity onset of the mouse shifts during the subsequent days. Such a lightinduced phase shift in the circadian activity rhythm is a widely used approach when measuring the interaction between light input and the circadian oscillator. This interaction is also assessed by measuring the free-running period length under constant light. After entrainment to the LD cycle, the mice are released into constant light (LL). Constant light has a period-lengthening effect in mice, such that the free-running LL period length is longer than 24 hours. In combination, these assays form a powerful set of tools for distinguishing between perturbations of the circadian clock and perturbations of the photoentrainment mechanism. For instance, mice with mutations in the core clock components exhibit a DD period length that is different from that of littermate controls, while photoentrainment mutations typically produce no change in DD period length but a significant

change in the phase angle of entrainment, light-induced phase shift, or LL period length.

Other adaptive photic responses

While the interaction between the day-night cycle and the circadian oscillator helps organisms adapt to daily fluctuation in ambient light, several other adaptive responses are also directly modulated by light. For example, some circadian outputs are directly regulated by light. As mentioned earlier, general activity in mice is suppressed by light. Similarly, melatonin synthesis and release by the pineal gland is both clock-regulated and strongly light-suppressed. These effects of light are independent of photoentrainment of the clock and are observed within minutes of light exposure. Finally, a much faster photoadaptive response is constriction of the pupil diameter in response to increased light intensity, which helps the outer retina photoreceptors adapt to environmental changes in irradiance. These adaptive responses, along with circadian photoentrainment, can persist in the absence of image-forming visual responses and are commonly referred to as nonvisual, non-image-forming or adaptive light responses (Van Gelder, 2001). These responses exhibit similar action spectra as photoentrainment and are valuable to the elucidation of the molecular basis of the latter.

Circadian photosensitivity

Photoentrainment in mice and humans is entirely mediated by retinal photoreceptors. When rodents are subjected to experimental bilateral enucleation, their circadian oscillator is rendered unresponsive to light and, as a consequence, free runs even under constant light conditions (Yamazaki et al., 1999). Despite the dependence on retinal photoreceptors, the sensitivity of circadian photoentrainment is quite distinct from that of patterned vision. The administration of discrete light pulses of varying intensities, wavelengths, and duration at different circadian time to rodents held under constant darkness has been extensively used to study features of circadian photosensitivity (Takahashi et al., 1984). Intriguingly, the phase-shifting effect of light exhibits strong circadian modulation, such that a bright light pulse administered during the subjective day produces no or only small phase shifts, while the same light pulse administered during the subjective evening causes significant phase delays, and during subjective late night causes phase advances (Pittendrigh, 1981). Although the molecular mechanism of such circadian gating of entrainment in mammals remains unclear, it highlights a novel mechanism for integrating both time and light information in appropriate resetting of the phase of the oscillator.

The threshold sensitivity of circadian photoentrainment is significantly higher than that of patterned vision, which

protects the clock from photic noise in nature. For example, a brief light pulse of several seconds, as occurs during lightning, or a low-intensity light, such as moonlight, rarely causes a phase shift of the circadian clock. Circadian entrainment also exhibits characteristic features of spectral integration over time (Takahashi et al., 1984). Light pulses of defined wavelengths and intensities have been used to generate irradiance response curves and action spectra. The action spectrum of photoentrainment fits an opsin nomogram with a peak sensitivity around 480–500 nm (Takahashi et al., 1984; Yoshimura and Ebihara, 1996). Comparable action spectra are also observed for the pupillary light reflex (PLR) in mice (Lucas et al., 2001) and pineal melatonin suppression in human (Brainard et al., 2001; Thapan et al., 2001). Circadian photoentrainment, PLR, negative masking (light suppression of activity), and photic suppression of pineal melatonin synthesis remain intact in mice with outer retina degeneration (Keeler, 1927; Foster et al., 1991; Mrosovsky et al., 1999). Furthermore, in these mice, the sensitivity of circadian phase shift, as well as the phase angle of entrainment, does not show any significant difference from those of normal mice. This suggests that non-rod, noncone photopigments play a dominant role in circadian photoentrainment (Foster et al., 1991).

Neuroanatomy of the circadian photoentrainment pathway

The circadian system in mammals, including mice, is hierarchical in nature, with a master circadian oscillator resident in the 10,000–20,000 neurons of the paired SCN coordinating tissue autonomous oscillators in the rest of the body. The SCN neurons in turn are entrained to the ambient light-dark cycle via a direct synaptic projection to a small subset of RGCs that constitute what is known as the retinohypothalamic tract (RHT) (Abrahamson and Moore, 2001). This basic organization of the SCN and RHT is well conserved among mammals, with some minor variations. In most mammals, the axon termini of RHT neurons are restricted to the ventral portion of the SCN, whereas in mice they extend more dorsally (Cassone et al., 1988). Such neuroanatomical organization implies that only a small fraction of all SCN neurons receive direct retinal input, and therefore intercellular communication among SCN neurons is important to achieve complete resetting of the master oscillator.

The evolutionary conservation of direct projection of the RHT to the SCN and the observation that functionally active mRGCs make synaptic connections to the SCN during prenatal life in mice raise the possibility that tonic light input and/or interaction with the neurons of the RHT may influence the ontogeny and function of the master circadian oscillator of the SCN. In support of this,

anophthalmic mice or mice experimentally enucleated immediately after birth have an aberrant SCN morphology (Silver, 1977; Holtzman et al., 1989; Nagai et al., 1992). In these mice, the SCN either is reduced in size or is unilateral. Consistently, these anophthalmic mice exhibit no circadian photoresponse, but intriguingly, their DD period length is significantly lengthened compared with that of the normal mice. These results had earlier suggested a role of functional light input to the SCN in the development of normal circadian system. However, results from other genetic models with specific disruption of RGCs showed little effect of ocular light input on SCN oscillator development and endogenous function. Math5-null mutants (Math5−/−) have been useful in addressing this question. The Math5 gene encodes a basic helix-loop-helix (bHLH) class of transcription factor that plays a key role in RGC differentiation. Math5−/− mice lack fully differentiated RGCs and a functional optic tract. Circadian behavioral assessment from two different laboratories conclusively established that RGCs are necessary and sufficient for circadian photoentrainment (Wee et al., 2002; Brzezinski et al., 2005); however, the effect of this mutation on the endogenous circadian oscillator appears to be strain specific. On a C57Bl/6J.129S1/SvIMJ mixed background, the mutation leads to almost 90% reduction in the number of RGCs, including mRGCs, and the mice exhibit a freerunning period length that is significantly longer than that of wild-type littermates (Wee et al., 2002). However, the same mutation on a C57Bl/6J background does not affect the endogenous pace of the oscillator, which indicates that the SCN oscillator can develop normally in the absence of any signal input from the retina (Brzezinski et al., 2005). This view is further supported by the observation of normal circadian function but lack of entrainment in mice lacking both rod/cone and melanopsin function (Hattar et al., 2003; Panda et al., 2003). In summary, the RHT plays a major role in circadian photoentrainment of the SCN oscillator. However, light signaling from the retina is not necessary for normal development of the SCN or for its endogenous circadian rhythm.

Other circadian and light-regulated responses, such as pineal melatonin rhythm and negative masking, involve more complex neuronal connections. A multisynaptic connection from the SCN to the pineal gland that traverses the paraventricular nucleus (PVN), spinal cord, and superior cervical ganglion (SCG) mediates both SCN and light regulation of pineal melatonin synthesis and release (Larsen et al., 1998; Teclemariam-Mesbah et al., 1999). The neuroanatomic circuit regulating negative masking is less clear. The SCN is dispensable for negative masking, and several brain regions, including hypothalamic and visual centers, modulate negative masking (Edelstein and Mrosovsky, 2001; Redlin et al., 2003).

Melanopsin-expressing retinal ganglion cells play a key role in circadian photoentrainment

The existence of a novel inner retina photopigment with a dominant role in entraining the SCN oscillator via the RHT has long been suggested from the observations that mice with outer retinal degeneration have an intact circadian photosensitivity and that a small subset of RGCs make direct synaptic connections to the SCN. The search for this novel photopigment led to the discovery of melanopsin (Provencio et al., 1998, 2000, 2002). The majority of the SCN-project- ing RGCs express melanopsin and are intrinsically photosensitive. Mice deficient in melanopsin (Opn4−/−) entrain their circadian activity rhythm to an imposed light-dark cycle, yet the magnitude of circadian phase shift in response to discrete light pulses of varying intensities is significantly attenuated. Such a reduced circadian photosensitivity is also evident in their period length when placed in LL, which is significantly shorter than that of their littermate wild-type controls (Panda, Sata, et al., 2002; Ruby et al., 2002). Furthermore, the negative masking response is also attenuated in the Opn4−/− mice. When exposed to prolonged illumination during subjective night, wild-type (WT) and Opn4−/− mice stop activity immediately after light onset. While the activity of wild-type mice remains suppressed for the entire length of the light pulse, the Opn4−/− mice gradually become more active (Mrosovsky and Hattar, 2003). In addition, Opn4−/− mice exhibit reduced PLR at high light intensity, while at low to medium intensity light their responses are indistinguishable from those of their wild-type littermates (Lucas et al., 2003). The reduced photosensitivity phenotypes in Opn4−/− mice correlates with the loss of intrinsic photosensitivity of the SCN-projecting RGCs, whose number, overall morphology, and projections remain intact (Lucas et al., 2003), while the residual photosensitivity in the Opn4−/− mice is mediated by rod/cone photoreceptors. Indeed, mice deficient in both rod/cone and melanopsin function fail to entrain to the imposed light-dark cycles and exhibit no masking, and their pupils remain completely open even under high-intensity light. Irrespective of the light environment, the circadian activity rhythm in these mice always free runs with a period length similar to the DD period length of wild-type mice (Hattar et al., 2003; Panda et al., 2003). Such a phenotype further highlights the modular nature of the circadian system, in which the cellular development and function of the core oscillator are independent of any mechanisms responsible for mediating light input.

The behavioral and cellular phenotypes of rod/coneand melanopsin-deficient mice bear several implications for circadian photoentrainment and other adaptive photoresponses. The complete loss of ocular photosensitivity in rod/coneand melanopsin-deficient mice highlights the

necessity and sufficiency of these photopigment systems for all ocular photoresponses. These genetic analyses also unraveled an important role for rod/cone photoreceptors in circadian photoentrainment, which may account for the residual entrainment of Opn4−/− mice to the imposed LD cycle. Further genetic analysis may elucidate the relative contribution of rods or any of the specific cones to circadian photoentrainment. However, the neuroanatomical basis of signal integration from rod/cone and melanopsin photopigment remains unclear. It is highly likely that the mRGCs that remain intact in Opn4−/− mice also transmit the rod/ cone-initiated photic signal to target brain regions, including the SCN. The rod/cone response may also be transmitted to the SCN via non-mRGCs, some of which directly project to the SCN, and some which make indirect connections to the SCN via the IGL or the OPN (Hannibal and Fahrenkrug, 2004; Hattar et al., 2006). In either case, the molecular bases of signal integration from inner and outer retina photopigments remain undiscovered. Finally, the high threshold sensitivity of circadian phase shift in Opn4−/− mice (Panda, Sato, et al., 2002; Ruby et al., 2002) suggests that this property of circadian photoentrainment is independent of the threshold sensitivity of the photopigments and may be encoded in the signal transduction pathway or in the oscillator itself.

Intrinsically photosensitive retinal ganglion cells

As mentioned earlier, the majority of the SCN-projecting RGCs express melanopsin and are intrinsically photosensitive (Berson et al., 2002; Hattar et al., 2002). The photoresponses of the ipRGCs is distinct from that of the outer retina photoreceptors. Their responses are characterized by long latencies approaching almost 1 minute, with a depolarizing current exhibiting fast action potentials sustained for the duration of the light pulse, relatively slow turn-off rates, and resistance to bleaching. The photoresponse in ipRGCs is resistant to synaptic blockade and persists in physically isolated cells, thus conclusively establishing the intrinsic nature of the photosensitivity. The action spectrum of the ipRGC photoresponse shows peak sensitivity around 480 nm, which is distinct from the peak sensitivity of rod/cone photoreceptors in rodents.

The sustained depolarizing current of the ipRGCs faithfully reflects the light intensity, which sets them apart from other RGCs. No other mammalian RGCs can encode ambient light level in this way. Although the ipRGCs were characterized in detail only recently, they may have been described as a luminance unit almost three decades earlier (Barlow and Levick, 1969). Their rare occurrence, accounting for about 1% of the total number of RGCs in adult rodent retina, might have been a major reason for the lack

of their detection and characterization in the intervening years.

ipRGC Morphology, Ontogeny, and Functional

Diversity The unique efferent projections of the ipRGCs, coupled with the power of retinal multielectrode array recording and the availability of genetic resources and specific antibodies identifying these cells, have catalyzed their cellular and functional characterization. Direct projection of a majority of the ipRGCs to the SCN allows retrograde labeling of these cells by injecting fluorescent dyes into the SCN (Berson et al., 2002; Hattar et al., 2002). Furthermore, specific staining of these cells and axons in a mouse strain carrying a functional lacZ gene in the melanopsin locus (Hattar et al., 2002), as well as the fortuitous tropism of adenoassociated virus-2 serotype (Gooley et al., 2003), has facilitated the comprehensive characterization of mRGC cell distribution and their brain targets. High-quality antimelanopsin antibodies (Provencio et al., 2002) have also been instrumental in immunohistological and ultrastructural characterization of these cells in the inner retina.

In the mouse CNS, melanopsin expression is extremely restricted, present only in 1%–3% of RGCs. The cell bodies of mRGCs are almost uniformly distributed throughout the retina, with occasional crowding along the periphery. Most of the cell bodies reside in the ganglion cell layer; however, approximately 5% of the cell bodies are found in the inner border of the inner nuclear layer. All melanopsin-expressing cells send their axons to the optic nerve, thus establishing that all melanopsin-expressing cells are RGCs (Hannibal et al., 2002; Hattar et al., 2002, 2006). Melanopsin protein is almost uniformly distributed throughout the plasma membrane of the dendrites, cell bodies, and axons of RGCs (Provencio et al., 2002). Such spatial distribution of melanopsin is distinct from vertebrate rod/cone and invertebrate image-forming opsins, which are densely packed in defined membrane ultrastructures. The dendrites of mRGCs arborize heavily and overlap in the synapse-rich inner plexiform layer (IPL), thus creating what appears to be a photoreceptive dendritic net covering the entire retina (figure 17.2A). The dendritic field of individual ipRGCs is relatively large and can approach 500 μM or 15°, which closely matches the calculated receptive field of SCN neurons. Ultrastructural studies have indicated that the melanopsin immunoreactive dendrites in the ON sublayer of the IPL make synaptic contact with both bipolar and amacrine terminals, while those in the OFF sublayer receive only amacrine terminals (Belenky et al., 2003).

As is evident from the reduced number of the mRGCs in Math5−/− mice, it is clear that the development of mRGCs is also controlled by the bHLH protein Math5. RGC differentiation begins early in the mouse retina, around

embryonic day 11.5, and is almost complete by the first week after birth (P7; reviewed in Mu and Klein, 2004). The mRGCs are also detected by immunostaining starting at E12.5, and at P0, each murine eye contains almost 200 ipRGCs/mm2 that are fully functional and transmit light information to the SCN (Tarttelin et al., 2003; Sekaran et al. 2005; Tu et al., 2005). The number of melanopsinpositive RGCs in murine retina declines dramatically after birth during a period when bipolar, amacrine, and outer retina photoreceptor cells are differentiated. By P14, when the mouse eyes are open and rods and cones are functioning, almost 75% of the melanopsin-expressing RGCs are lost, leaving approximately 700–1,000 melanopsin-positive cells in each adult mouse retina. This decline in ipRGC population, along with changes in the physiological diversity among them, has been more thoroughly examined by multielectrode array recording. This approach has enabled the simultaneous recording of the photoresponses of sufficiently large numbers of ipRGCs and has led to the accurate measurements of their sensitivity, onset speed, and offset kinetics. In young, P8 mice, three distinct types of ipRGCs are observed: slow onset (>12 s), sensitive (irradiance yielding half-maximal response or log IR50 < 12.85), fast off (type I); slow onset, insensitive, slow off (type II); and rapid onset, sensitive, and very slow off (type III). The majority (ca. 75%) of the ipRGCs in P8 mice are type I, which are almost undetectable in adult retina (Tu et al., 2005). The functional significance of the observed physiological diversity among ipRGCs is currently unclear. The subtypes may differ in projections to the target brain regions controlling various adaptive responses. Identification of molecular marker to tag or specifically perturb ipRGC subtypes may help elucidate their functional significance.

Target brain regions innervated by mRGCs

The projections of the mRGCs differ significantly from those of the rest of the RGCs (figure 17.2B). In the optic nerve the axons of the mRGCs are almost uniformly distributed up to the optic chiasm, beyond which the fibers run mostly contralaterally and are clustered along the dorsal surface of the optic tract (Gooley et al., 2003; Hannibal and Fahrenkrug, 2004; Hattar et al., 2006). The SCN, resident above the optic chiasm, is the most densely innervated brain target of the mRGCs. An almost equal number of crossed and uncrossed fibers innervate the SCN. The nerve terminals are distributed throughout the SCN, and some extend dorsally to reach the ventral subparaventricular zone (vSPZ). Sparse projections also reach the lateral preoptic nucleus (LPO), ventrolateral preoptic nucleus (VLPO), medial preoptic nucleus (MPO), and supraoptic nucleus (SON). Direct mRGC innervations of these hypothalamic targets may form a basis for the well-characterized photic modulation of sleep,

Figure 17.2 Morphology and projections of mRGCs in mouse. A, Retina flat mount stained with a polyclonal antimouse melanopsin antibody showing specific staining of dendrites, somata, and axons of a small subset of retinal ganglion cells. B, Schematic drawing showing direct axonal projections of mRGCs to several brain regions. (Results from Hattar et al., 2006, are redrawn here.) Brain regions receiving significant projections are represented in large, bold letters. AH, anterior hypothalamic nucleus; IGL, intergeniculate leaflet; LGd, lateral geniculate nucleus, dorsal division; LGv, lateral geniculate nucleus, ventral division; LH, lateral hypothalamus; LHb, lateral habenula; MA, median amygdaloid nucleus; OPN, olivary pretectal nucleus; PAG, periaqueductal gray; PO, preoptic; pSON, perisupraoptic nucleus; SC, superior colliculus; SCN, suprachiasmatic nucleus; SPZ, subparaventricular zone. See color plate 6.

body temperature, and neuroendocrine outputs. Beyond the hypothalamus, most of the mRGCs projections are contralateral, and their major brain target areas include the intergeniculate leaflet (IGL) and olivary pretectal nucleus (OPN), both of which are also reciprocally connected to the SCN. The direct projection to the OPN forms the basis for the mRGC regulation of PLR. A small but detectable number of melanopsin-negative RGCs also send direct projections to the SCN. Additionally, nonmelanopsin RGC projections

to the OPN and IGL may suggest potential non-mRGC regulation of circadian photoentrainment (Gooley et al., 2003; Hannibal and Fahrenkrug, 2004; Hattar et al., 2006).

While the majority of the mRGCs project to brain regions implicated in non-image-forming photoresponses, involvement of mRGCs in visual processes cannot be ruled out. Sparse projections to the lateral geniculate, which forms the primary relay center to the visual cortex, and projections to OPN and superior colliculus, which sends projections to the visual thalamus, may suggest a potential role of mRGCs in visual processing. Furthermore, in the RGC layer, the ipRGCs maintain gap junction connections with other retinal cell types (Sekaran et al., 2003), raising the possibility that they may directly influence other intraretinal functions, including rod/cone initiated signaling events. In support of this, cone ERG of mice has been shown to be modulated in Opn4−/− mice (Barnard et al., 2006).

The melanopsin photopigment

The mouse melanopsin gene spans 7.8 kb on chromosome 14 and encodes a protein of 521 amino acids (aa). The predicted seven transmembrane region (amino acids 72–350) shows sequence homology with opsins and contains a lysine residue in the seventh transmembrane region that may serve as the binding site for a retinal-based chromophore (Provencio et al., 2000). Targeted mutation of this site abolishes photoactivation of melanopsin (Newman et al., 2003; Kumbalasiri et al., 2007). Phylogenetic comparisons of this region show higher sequence similarity to rhodopsins from invertebrates than to those from vertebrates (Provencio et al., 2000). Specifically, the cytoplasmic regions of the opsin segment show sequence features suggestive of potential activation of the Gαq class of G proteins instead of transducin (Nayak et al., 2007). However, unlike mouse rhodopsin (full length 348 aa) or Drosophila rhodopsin-1 (373 aa), the mouse melanopsin contains an unusually long C-terminal sequence (351–521 aa) with no sequence similarity to any other known protein. This C-terminal region contains multiple putative phosphorylation sites and may serve as a target region for several regulatory proteins.

Melanopsin Phototransduction Mechanism The scarcity of melanopsin-expressing cells in the mouse has prevented a systematic characterization of its signaling properties. The ipRGCs exhibit light-evoked transient increases in intracellular calcium levels (Sekaran et al., 2003) and a depolarizing membrane current that bears characteristic features of nonselective cation channels, like those of the Trp class (Warren et al., 2003). Successful heterologous expression of functional melanopsin in cultured cell lines and in Xenopus oocytes has shed light on the potential phototransduction mechanism in ipRGCs. In both cultured cells and oocytes,

photoactivated mouse melanopsin signals through Gαq/Gα11, and phosphoinositide signaling pathways to trigger the release of intracellular calcium store (Panda et al., 2005; Qiu et al., 2005). When coexpressed with the TrpC class of ion channels, it can also trigger light-induced opening of Trp channels, leading to membrane depolarization. The photocurrent in these melanopsin-expressing cells exhibits a peak sensitivity at about 480 nm, which is similar to that of ipRGC and circadian phase shift. The heterologous expression experiments have also suggested that melanopsin, like Drosophila Rh1 opsin, may possess an intrinsic photoisomerase activity to photoconvert all-trans retinal photoproduct to its 11-cis isomer (Melyan et al., 2005; Panda et al., 2005).

Despite the successful demonstration of potential phototransduction mechanism in a heterologous system, the steps from photoactivation to membrane channel opening in the ipRGCs remain unclear. Both Gαq and Gα11 G proteins are almost ubiquitously expressed and can functionally compensate for each other’s loss. To further complicate the analysis, mammalian melanopsin has also been shown to signal through the Gαi/o class of G protein (Newman et al., 2003; Melyan et al., 2005), and melanopsin may likely activate promiscuous G proteins such as Gα14 and Gα16. Genetic analysis of the subsequent candidate steps, such as PLC and Trp channels, also suffers from a similar problem of functional redundancy among family members. Generation of ipRGC specific loss-of-function mutants may be a promising option to conclusively establish the melanopsin phototransduction pathway.

The biochemical characterization of melanopsin photopigment has been achieved with some success. Melanopsin purified from COS cells and reconstituted with 11-cis retinal can activate a G protein (Newman et al., 2003). However, the peak absorption spectrum of the purified melanopsin is significantly different from the peak action spectra of photoresponses of ipRGCs, and also different from the photoresponses of cells heterologously expressing mouse melanopsin. This disparity highlights the potential differences between the expression system and the chemical environment.

The nature of the native chromophore used by melanopsin in ipRGCs is currently unknown. Heterologously expressed melanopsin can use all-trans retinal and several cis-isomers of retinaldehyde, which implies that melanopsin photopigment can photoisomerize its chromophore and that the photosensitivity of ipRGCs may be independent of the RPE retinoid cycle. Consistently, mice carrying loss-of- function mutations in key components of the retinoid cycle still exhibit some, if reduced, circadian photosensitivity and PLR (Fu et al., 2005; Doyle et al., 2006; Tu et al., 2006). Although these mice can entrain to an imposed LD cycle, the discrete light pulse–induced phase shift during free running in DD is retained only at high light intensities. The

ipRGC responses in Rpe65−/− mice as measured by microelectrode arrays remain normal. Yet both the number of melanopsin-expressing RGCs and dendritic staining for melanopsin are reduced. The diminished behavioral photosensitivity these mice display, as well as the weak melanopsin immunostaining seen in their retina, can be rescued by abolishing rod function (Rpe65−/−;rdta) (Doyle et al., 2006), which demonstrates that disruption of retinoid cycle in the RPE can indirectly influence the function of the ipRGCs. Intriguingly, double mutant mice (Rpe65−/−;Opn4−/−) with no functional RPE65 and melanopsin are largely diurnal under bright light/dark condition, while triple mutants (Rpe65−/−; rdta;Opn4−/−) show no photoresponses. The temporal niche switching in Rpe65−/−;Opn4−/− mice shows how interaction between the photic input pathway from the retina and the circadian oscillator can contribute to establishing diurnal or nocturnal behavior in animals.

Molecular basis of circadian photoentrainment

Our knowledge of the core molecular mechanism of circadian oscillation, the neurotransmitters of the RHT and their mode of action, has provided a framework for understanding the molecular bases of circadian photoentrainment (figure 17.3). In mammals, the core mechanism of circadian oscillator is composed of interlocked transcription-translation negative feedback loops. In a simplified model, the positive transcription factors Bmal1, Clock, and NPAS2 heterodimerize and drive transcription of the Period and Cryptochrome genes (Per1, Per2, Per3, Cry1, Cry2), whose protein products repress their own transcription. The transcriptional inhibitors are eventually degraded, relieving repression and allowing the start of another round of transcription; this ultimately produces oscillating levels of Per and Cry gene products

(Reppert and Weaver, 2002). The phase of Per1 or Per2 gene product roughly correlates with the phase of the behavioral rhythm. A discrete light pulse administered to mice in constant darkness causes transient induction of Per1 and Per2 transcripts and a concomitant change in the phase of their oscillations. This molecular event may underlie the change in phase of the behavioral rhythm (Yan and Silver, 2004).

Nonetheless, the components and signaling events that begin from light detection in the retina and progress to the acute induction of Per genes in the SCN are yet to be fully elucidated. It is thought that light-induced RGC membrane depolarization releases the neurotransmitters glutamate and PACAP, both of which are stored in the SCN-projecting RGCs, from their axon terminals in the SCN (Hannibal et al., 2000). Glutamate acts through ionotropic glutamate receptors in the SCN neurons (Ding et al., 1994). In addition, SCN neurons also express at least two receptors that can bind to PACAP: Pac1 receptor (Pac1r) and VPAC2 receptor (Vpac2r). The signaling events downstream of glutamate or PACAP receptors ultimately converge and include increases in cytosolic calcium, phosphorylation of CREB at serine 133, histone modification, and phospho-CREB- mediated transient induction of Per1 or Per2 gene products leading to a change in the phase of the molecular oscillator (reviewed in Meijer and Schwartz, 2003). In support of this model, mice that lack PACAP or Pac1 receptor exhibit an attenuated phase shift in response to light (Hannibal et al., 2001; Colwell et al., 2004). Vpac2r is also a receptor for vasoactive intestinal peptide (VIP) which is significantly expressed in the SCN. Signaling via Vpac2r is presumed to synchronize or entrain the individual cellular oscillators with each other. As a consequence, Vpac2r-deficient mice exhibit a more severe circadian dysfunction and are generally arrhythmic (Harmar et al., 2002; Maywood et al., 2006).

Figure 17.3 Model depicting major steps during photoentrainment of the SCN oscillator. Light received by the rod/cone and mRGCs ultimately causes depolarization of mRGCs and release of glutamate and PACAP from their axon termini in the SCN. These neurotransmitters activate their cognate receptors in the SCN neurons and ultimately trigger CREB phosphorylation, binding of pCREB to cis-acting CRE site and transcriptional induction of Per1

and Per2 genes. An increased level of Per1/Per2 proteins resets the phase of the oscillator. Under constant conditions, a negative feedback loop maintains oscillation of Per gene products. Transcriptional activators Clock and BMAL1 drive Per and Cry gene transcription by binding to cis-acting E-box. Per and Cry gene products are translated, phosphorylated, and translocated into the nucleus, where they inhibit Clock/BMAL1 function.

The linear model of circadian photoentrainment in its current format is too simplistic, because it does not take into account some signal integration steps. First, it is unknown whether rod and cone input is exclusively transmitted via mRGCs. If an alternate pathway, such as direct projection of non-mRGCs to the SCN or indirect projections via the IGL and OPN, contributes significantly to photoentrainment, the neurotransmitters and receptors transmitting light information through these pathways are not clearly known. Furthermore, it is known that several nonphotic cues also modulate the phase of the SCN oscillator. The molecular bases of integration of the photic and nonphotic entraining cues are just beginning to be understood.

Other mouse models for circadian photoentrainment

Mouse genetic studies have suggested that several additional genes may participate in circadian photoentrainment, although the causal genes and the mechanism are unknown. For instance, the mechanism of participation of cryptochromes in mammalian circadian photoentrainment is still unclear. Although cryptochromes from plants and flies have been demonstrated to be functional photopigments (Cashmore, 2003), the mammalian cryptochromes are best described as transcriptional repressors (Griffin et al., 1999; Kume et al., 1999), and evidence for direct light-dependent function, as has been demonstrated in plants and flies, is still lacking. Cryptochrome-deficient mice (Cry1−/−;Cry2−/−) lack a functional circadian oscillator, which prevents the evaluation of their role in circadian photoentrainment. However, other surrogate roles in negative masking and pupillary light reflex suggest some role in these processes. As mentioned earlier, rd mice entrain to LD cycle and free run with a wild-type period length under DD (Foster et al., 1991), and Cry1−/−;Cry2−/− mice entrain (negative masking) to the LD cycle but are arrhythmic under DD (van der Horst et al. 1999). On the other hand, triple mutant rd; Cry1−/−;Cry2−/− mice exhibit no negative masking, and their locomotor activity is arrhythmic under both LD and DD conditions, thus suggesting a role of cryptochromes in photic control of activity (Selby et al., 2000). Similarly, the triple mutants exhibit significantly lower photosensitivity in pupil constriction than either rd or Cry1−/−;Cry2−/− mice (Van Gelder et al., 2003). Despite these observations, extensive expression of Cry1 and Cry2 in the retina, SCN, and other brain regions, and lack of tissue-specific loss-of-function mutants makes it difficult to assess whether their function in the photoreceptive cells, in downstream cells, or in brain regions regulating masking or pupillary light reflex underlies the observed deficits.

Quantitative trait analyses in mice have implicated several loci regulating the phase angle of entrainment or masking

response (Shimomura et al., 2001; Panda et al., 2003). The genetic intervals for these loci are relatively large, spanning several megabases. None of these chromosomal regions harbors any known photopigment or light-signaling component. Some of these loci modulate the phase angle of entrainment (Shimomura et al., 2001). A different locus modifying melanopsin function does not affect general entrainment when the LD cycle is shifted by several hours, but it significantly attenuates masking, resulting in increased activity during the light period (Panda et al., 2003). Finally, a wild species of mouse, Peromyscus californicus (de Groot and Rusak, 2002), exhibits normal masking but impaired photoentrainment. Cloning and characterization of the relevant genes in these mouse lines is expected to offer new tools to understand the interplay between masking and circadian photoentrainment.

Summary

The identification of mRGCs and the demonstration that melanopsin is a functional photopigment have precipitated systematic studies of circadian photoentrainment. The rarity of mRGCs still poses a major challenge for the cellular and molecular understanding of photoentrainment. Although possible mechanisms of melanopsin phototransduction have been demonstrated in several heterologous expression systems, the exact mechanism, its preferred chromophore, and the effecter channels in ipRGCs are still unknown. Methods to isolate, culture, and extract RNA or protein materials from ipRGCs will have a profound impact on the systematic analysis of the photosensitivity of ipRGCs by verifying expression of candidate signaling molecules and channels in these cells.

Understanding the molecular properties of the melanopsin photopigment will still heavily depend on heterologous expression. Despite sharing sequence similarity with a group of fast-acting GPCRs, the slow photosensitivity of the ipRGCs and of cells that heterologously express melanopsin raises questions about the biophysical basis of slow activation of melanopsin photocurrent. It is unknown whether the slow kinetics reflects a property of the photopigment or of the signaling cascade.

Genetic analyses have shown that rod and cone photoreceptors also play a significant role in photoentrainment. Identifying the specific (if any) rod or cone photopigment that can largely compensate for the loss of melanopsin will also be a major finding. Furthermore, it is yet to be tested whether the photopotentiation effect on melanopsin function shown in cultured cells (Melyan et al., 2005) is also evident in the whole animal. Overall progress in understanding the mammalian photoentrainment mechanism has the potential to improve therapy for sleep and mood disorders.

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