The Circadian Visual System, 2005

BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

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Review

The circadian visual system, 2005

L.P. Morina,⁎, C.N. Allenb aDepartment of Psychiatry and Graduate Program in Neuroscience, Stony Brook University, Stony Brook, NY 11794, USA bDepartment of Physiology and Pharmacology and Centre for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

ARTICLE INFO ABSTRACT

Article history: The primary mammalian circadian clock resides in the suprachiasmatic nucleus (SCN), a Accepted 9 August 2005 recipient of dense retinohypothalamic innervation. In its most basic form, the circadian Available online 5 December 2005 rhythm system is part of the greater visual system. A secondary component of the circadian visual system is the retinorecipient intergeniculate leaflet (IGL) which has connections to Keywords: many parts of the brain, including efferents converging on targets of the SCN. The IGL also Circadian provides a major input to the SCN, with a third major SCN afferent projection arriving from Suprachiasmatic the median raphe nucleus. The last decade has seen a blossoming of research into the SCN anatomy and function of the visual, geniculohypothalamic and midbrain serotonergic Intergeniculate leaflet systems modulating circadian rhythmicity in a variety of species. There has also been a IGL substantial and simultaneous elaboration of knowledge about the intrinsic structure of the SCN. Many of the developments have been driven by molecular biological investigation of the circadian clock and the molecular tools are enabling novel understanding of regional function within the SCN. The present discussion is an extension of the material covered by the 1994 review, “The Circadian Visual System.” © 2005 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: +1 631 444 7534. E-mail address: [email protected] (L.P. Morin).

Abbreviations: 5-HT, serotonin CALB, calbindin CALR, calretinin CCK, cholecystokinin ENK, enkephalin GABA, gamma amino butyric acid GRP, gastrin releasing hormone IGL, intergeniculate leaflet NPY, neuropeptide Y NT, neurotensin PACAP, pituitary adenylate cyclase-activating polypeptide SCN, suprachiasmatic nucleus SP, substance P SS, somatostatin VIP, vasoactive intestinal polypeptide VP, vasopressin

Contents

1. Introduction and caveats ...... 3 2. Psychophysicsofthecircadianrhythmsystem...... 4 3. Organization of the suprachiasmatic nucleus ...... 5 3.1. Nomenclature ...... 5 3.2. Distribution of cell phenotypes ...... 5 3.3. Intra-SCN connections ...... 8 4. Retinal photoreceptors and projections ...... 8 4.1. Melanopsin ganglion cell characteristics ...... 8 4.2. Retinal ganglion cell projections and bifurcation ...... 9 4.3. Classical and ganglion cell photoreceptor contribution to regulation of circadian rhythms, masking, the pupillary light reflex and melatonin suppression ...... 11 4.3.1. Circadian rhythms ...... 11 4.3.2. Masking, pupillary light reflex and melatonin suppression...... 11 5. Neuromodulators of the retinohypothalamic tract ...... 12 5.1. Glutamate ...... 12 5.2. N-Acetylaspartyl glutamate ...... 12 5.3. Glutamate receptors ...... 13 5.4. Nitric oxide and RHT signal transduction ...... 13 5.5. PACAP...... 14 5.6. Substance P...... 15 6. SCN structure–function relationships ...... 17 6.1. Function of cellular neuromodulators in the SCN ...... 18 6.1.1. GABA...... 18 6.1.2. VIP and VIP/PACAP receptors...... 18 6.1.3. Calbindin and the central subnucleus of the hamster SCN ...... 19 6.1.4. Bombesin and gastrin releasing peptide...... 21 6.1.5. Neuromedin U and neuromedin S ...... 21 6.1.6. Vasopressin ...... 21 6.1.7. Diurnality–nocturnality ...... 22 6.2. Acetylcholine ...... 22 6.3. Neurophysiology of the SCN action potential rhythm...... 23 6.4. Inter-SCN differences in gene expression ...... 25 6.5. Regional expression of Fos ...... 25 6.5.1. Hamster ...... 25 6.5.2. Rat ...... 25 6.6. Regional clock gene expression ...... 26 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 3

6.6.1. Mouse...... 26 6.6.2. Rat ...... 27 6.6.3. Hamster ...... 27 6.7. Stimulus induction of gene expression ...... 28 6.7.1. Photic stimuli ...... 28 6.7.2. Nonphotic stimuli ...... 28 6.7.3. Inconsistencies between phase response and gene expression ...... 29 6.8. Polysialic acid and SCN function ...... 30 7. SCN connectivity ...... 31 7.1. Humoral factors...... 31 7.2. Efferent anatomy ...... 31 7.3. Afferent anatomy ...... 32 8. Intergeniculate leaflet ...... 34 8.1. Nomenclature ...... 34 8.2. Development ...... 34 8.3. Anatomy...... 34 8.3.1. Geniculohypothalamic tract...... 34 8.3.2. Connectivity and relationship to sleep, visuomotor and vestibular systems ...... 34 8.4. Function ...... 35 8.4.1. Neurophysiology ...... 35 8.4.2. Nonphotic regulation of rhythmicity ...... 36 8.4.3. Photic regulation of rhythmicity ...... 37 9. Serotonin and midbrain raphe contribution to circadian rhythm regulation ...... 38 9.1. Effects of serotonin-specific neurotoxins ...... 38 9.2. Serotonin receptor function ...... 38 9.3. Regulation and consequences of serotonin release ...... 39 9.4. Serotonergic potentiation of light-induced phase shifts ...... 40 10. Failure to re-entrain ...... 41 11. Masking...... 42 12. The future ...... 43 Acknowledgments ...... 43 References...... 43

1. Introduction and caveats The purpose of the current review is to continue the theme of the original, but to do so with minimal redundan- There have been a large number of new developments with cy. The focus of the current presentation will be on respect to knowledge about the anatomy and physiology of necessary modifications to the perspective presented 10 circadian rhythm regulation since publication of our 1994 years ago, and to the substantial additions to the general review, “The Circadian Visual System” (Morin, 1994). As is body of knowledge relating to the circadian visual system. nearly always the case with such projects, many of the The title of the 1994 review, “The Circadian Visual System,” developments were well underway, and in some instances remains of some import. We believe it is worth emphasizing completed, prior to the actual publication of that review. Most that, taken as a whole, the research topic under consider- notably, much of the presentation in the 1994 review ation is “circadian,” is “visual” and certainly is a “system.” concerning the serotonergic system was incomplete or That is an aspect of the research topic of importance that wrong and understanding of the issues changed substantially should not be underestimated. soon after publication of the review. Other issues emerged as We do not intend to cover all facets of circadian rhythm unanticipated major developments. One of these has been the research and we also expect to have inadvertently overlooked discovery of the photopigment, melanopsin, in retinal gangli- research worthy of inclusion here. To the extent that has on cells and the subsequent establishment of those ganglion happened, we apologize. On a technical note, when specific cells as a special class of photoreceptors. cell types are mentioned as containing a particular neuromo- The steady growth of molecular biological contributions dulator, the comment refers to the fact that it has been to the understanding of circadian clock mechanisms in demonstrated by immunohistochemical means. invertebrates has been translated into parallel evaluation of The work that is particularly ignored concerns the molec- clock mechanisms in mammals. This development has ular biology of the SCN or other tissues. There will be no effort created new avenues for architectural analysis of the to review the burgeoning literature concerning the molecular suprachiasmatic nucleus (SCN) function according to the mechanisms of the central circadian clock or of molecular regional distributions of cell phenotypes comprising the oscillations in peripheral structures. Rather, we will touch on circadian clock. this literature when it can be used to amplify understanding of 4 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 the system properties governing circadian rhythm regulation. or Joules/cm2) are equivalent. Most impressively, the photic One issue at the forefront of the list of mysteries presently intensity information in a series of light pulses appears to be confronting that understanding is the spatial distribution of summed across the series (Nelson and Takahashi, 1991), as SCN neurons exhibiting clock-like behavior. The general long as the individual pulses are of sufficient duration (i.e., question is, “Are all SCN cells pacemakers?” While some minutes). experimental work suggests that the answer is no, the topic is The integrative capacity of the circadian system appears to an area of intense research and is discussed in some detail be time-limited to the extent that, under test conditions below. explored to date, if a photic stimulus is sufficient to produce a A second question is, “Do all pacemaker cells in the SCN maximal phase shift, more photons at the same time or up to 2 oscillate with the same phase?” Evaluation of a variety of h later have no additional effect on phase shift magnitude molecular oscillations in SCN cells has given the impression (Nelson and Takahashi, 1999). In such cases, the circadian that the molecular behavior of a whole SCN reflects the system is deemed to be “saturated” with respect to its ability to behavior of the individual cells. While this may be true on show a further phase response to light. average, many individual SCN neurons do not express An unexpected result (van den Pol et al., 1998), demon- rhythmicity in phase with the average of the whole (Quintero strated the important point that a form of “temporal et al., 2000). This development in the analytical approach to integration” may occur even when the light stimulus is pacemaker activity among groups of cells emphasizes the subthreshold. For example, in the mouse or hamster, a brief importance of molecular activity within individual cells and (2–3 ms), bright (1013 photons/cm2/s) flash will not elicit a points to the need to evaluate spatial relationships and phase shift (Nelson and Takahashi, 1991; van den Pol et al., connectivity among oscillatory neurons within the SCN. 1998), but a series of flashes totaling 120 ms will yield a normal When microelectrode arrays are used for long-term phase response (van den Pol et al., 1998). Thus, some form of recordings, the action potential firing rhythm periods of summation has occurred to elicit the phase shifts. It is not yet individual of SCN neurons show a greater diversity in clear, however, that there is a systematic relationship between dispersed cultures compared to organotypic cultures. These the number of flashes and the magnitude of the phase shift. data suggest that interneuronal communication is required to Evidence from studies employing a series of 5 to 45 light fine tune the period of the clock (Nakamura et al., 2002; Herzog flashes, each only 10 μs long, suggests otherwise (Arvanito- et al., 1998; Liu et al., 1997a; Albus et al., 2002). The final giannis and Amir, 1999). Each such series effectively induces behavioral period is an average of these individual neuronal FOS-IR in the SCN and yields 75 min phase shifts when periods (Liu et al., 1997a). The demonstration of phase delivered to rats at CT13. Thus, the fact of a light-induced differences among SCN neurons is a variant on the larger phase shift may be separable from the process of theme indicating differences in phase between the two SCN in “integration.” a single animal (De la Iglesia et al., 2000). Thus, from the point The entire visual system has been intact in all studies to of view of this review, utility of the molecular analysis of date that involve “temporal integration.” Therefore, there is cellular clocks serves to highlight the need to understand the presently no information regarding the extent to which the system properties intrinsic to the SCN, as well as its regulation integration mechanism includes or excludes the retina, SCN, of efferent response to afferent information, whether that IGL, visual midbrain, the serotonergic or other systems. information be visual or some other sensory modality. Unilateral enucleation eliminates 50% of the retinal photoreceptors and their associated RHT projections, but had no effect on hamster re-entrainment rate (Stephan et al., 1982). 2. Psychophysics of the circadian rhythm system On the other hand, the same publication showed that both advance and delay shifts of rats were retarded following The Pickard et al. (1987) IGL lesion paper was one of the first to unilateral enucleation. D. Earnest (unpublished work cited by employ nonsaturating light stimuli to elicit circadian rhythm Pickard and Turek, 1983) failed to find an effect of unilateral phase responses. The method eliminated ceiling effects enucleation on hamster circadian period in LL. Similarly, caused by saturating light, providing a more accurate view of unilateral enucleation appears to have no effect on light- psychophysical sensitivity to light, rather than a simple induced FOS in rat (Beaulé et al., 2001a) or hamster SCN statement that animals respond (or not) to the stimulus. (Muscat and Morin, unpublished data), or in SCN of hamster Subsequently, the psychophysics of circadian rhythm phase with one eye occluded, except in response to high irradiance response to light presented at CT19 has been explored in or long duration light (Muscat and Morin, in press). In each of greater detail. The hamster (Nelson and Takahashi, 1991, 1999; these investigations, saturating stimuli were employed and Zhang et al., 1996), mouse (van den Pol et al., 1998) and gerbil the experimental methods not sufficiently sensitive to deter- (Dkhissi-Benyahya et al., 2000) circadian systems estimate the mine whether there is an effect of unilateral enucleation on importance of an acute photic stimulus by effectively “count- rate of re-entrainment or any other measure of circadian ing” photons. It may be more appropriate to refer to this rhythm regulation (but see Tang et al., 2002 for a broad set of activity as “temporal integration” which is a broader term. The results suggesting a large effect of unilateral enucleation). process is demonstrated by the fact that there is a clear Unilateral enucleation is also reported to shorten the relationship between the stimulus duration and irradiance circadian period of rats in LL, while a unilateral SCN lesion (photons/cm2) with respect to the magnitude of the phase has no similar effect (Donaldson and Stephan, 1982). Given shift. That is, dim, long duration stimuli have the same effect that the IGL regulates 50% of the hamster circadian period as brief but bright stimuli, if the irradiances (i.e., total photons response to LL (Pickard et al., 1987; Morin and Pace, 2002) (no BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 5 equivalent studies have been done in the rat), the effect of species; (B) is not consistent with the apparent anatomical enucleation could be attributable exclusively to loss of the differences between species; (C) has been employed with retinal input to the IGL. different meanings in different species, as well as within the Light induction of FOS-IR in the gerbil SCN (Dkhissi- same species; (D) unduly simplifies a structure which has a Benyahya et al., 2000) has been interpreted as obeying the still-emerging level of organizational complexity and (E) if temporal integration rule found in the hamster for phase accepted in an unquestioning manner, will have a direct shifts and melatonin suppression. However, over the same negative influence on experimental design, while augmenting irradiance range, the effect of light on FOS in ganglion cells of the likelihood of erroneous and incomplete interpretation of the gerbil retina appears to be all-or-none, rather than new and available data. additive. Similar results have been found for light-induced Anotherreasonforcautioninusingthecore/shell FOS in hamster IGL cells (Muscat and Morin, unpublished data) terminology is that they are based primarily on the localiza- and imply that neither the ganglion cell nor IGL neuron tion of the neuronal cell body. SCN neurons have a soma that properties are altered by light in a manner consistent with is approximately 10–15 μm in diameter and generally contain “photon counting” responses of the circadian rhythm system. 1–3 dendrites. These dendrites extend for a distance of about 250 μm. Given the small size of the SCN (300–400 μm wide and 400–500 μm tall), a dendrite has the ability to project across 3. Organization of the suprachiasmatic nucleus much of the SCN (van den Pol, 1980, 1991; Silver and Brand, 1979; Jiang et al., 1997). This implies, for example, that a cell 3.1. Nomenclature body in the dorsal part of the SCN could send a dendrite ventrally into the region where the densest retinal terminals An accepted anatomical nomenclature affords the opportuni- are located. Indeed, individual dendrites from the dorsal SCN ty to standardize the description of brain structures. This has have been shown to extend into the ventral SCN and further not yet happened with respect to the SCN intrinsic anatomy. into the optic chiasm (Silver and Brand, 1979). Rejection of Historically, there have been two approaches to SCN organi- “core and shell” nomenclature specifically does not mean zation, one based on the rat model and the other based on the that there is no specialized organization within the SCN. hamster. The rat SCN has been commonly divided into However, we believe the research goal should be to determine dorsomedial and ventrolateral divisions. Although these are anatomical reality with a view toward consistent and geographic designations, the conceptualization is based on constructive use of a nomenclature that is sufficiently fluid the fact that, in this species, the vasoactive intestinal to permit revisions reflecting the evolution of knowledge polypeptide (VIP) neurons plus most of the geniculohypotha- about the SCN. lamic tract (GHT) and retinal afferents occupy the latter, while the former is the location of most vasopressin (VP) neurons. 3.2. Distribution of cell phenotypes The hamster SCN has a caudocentral region containing neurons identified by several different peptides and this has Essential to the understanding of circadian rhythm system been referred to as the “central subnucleus” which partially function is knowledge about the intrinsic organization of the overlaps with the region containing VIP neurons. In this SCN, site of the predominant circadian clock in mammals. It species, there has been no anatomical attempt to designate a has been commonly stated that all SCN neurons appear to “ventrolateral” SCN division, although some investigators contain GABA (Moore and Speh, 1993; Morin and Blanchard, have applied rat-derived descriptors to the hamster. As 2001; Abrahamson and Moore, 2001), although an immuno- indicated in Section 3.2, identification of a dorsomedial area histochemical, electron microscopic study of the rat SCN based upon the location of VP-immunoreactive neurons is of suggests that only 40–70% contain GABA (Castel and Morris, dubious value, even in the rat. 2000). There is a dense GABAergic plexus throughout the The nomenclature has become further confused with nucleus, but a clear spatial organization of the SCN is references to a “core and shell” SCN organization. For evident if other cellular characteristics are evaluated. The example, in a 1996 review, one of us (LPM) used the terms as two most obvious are the distribution of cell phenotypes and an analogy to facilitate understanding of the relationship the distribution of cells projecting to non-SCN targets. The between the hamster central subnucleus and its surround former can be ascertained by immunohistochemical means, (Miller et al., 1996). However, another contributor to that while the latter relies largely upon retrograde tracer article employed the same terms in a different review, also injections. published in 1996, as labels of regions more or less equivalent A cogent argument has been made supporting the view to the previously used dorsomedial and ventrolateral divi- that the SCN consists of two major divisions, a dorsomedial sions in the rat SCN (Moore, 1996). Given the reasonable area related to the presence of VP neurons and a ventral area likelihood that a common SCN organizational plan will related to the presence of VIP cells, dense retinal afferents and emerge and be applicable to rodents, and that apparent neuropeptide Y-containing terminals of the GHT (Moore et al., structural differences between rat and hamster SCN will be 2002). However, the evidence seems to suggest the presence of reconciled, we believe it is premature to adopt the “core and more than two subdivisions within the SCN, with some more shell” terminology based upon their application to anatomical expressly related to cell phenotypes than others. For example, description in one species or another. More important, and as the rat SCN has VP cells distributed in a crescent extending discussed below, we are of the opinion that the “core and from a ventral location, medially, dorsally and laterally, shell” terminology (A) is not supported by the data in any almost encircling a region that contains the VIP neurons. 6 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

These two areas are distinct from, but appear to overlap with, a predominantly dorsolateral region containing calretinin (CALR) and enkephalin (ENK) neurons (Moore et al., 2002). Encompassed by the region of VIP cells is a smaller domain of neurotensin neurons. In the mouse, the regional situation resembles that of the rat to the extent that there appear to be multiple SCN areas characterized by one or more particular cell phenotypes. VP cells are scattered across the rostral SCN, are located predominantly in the typical dorsomedial aspect of the central SCN and are again scattered through the entire caudal SCN. Some cells are also present in the ventral and ventrolateral SCN. ENK cells are grouped as a collection in the dorsal SCN, while VIP neurons are generally localized to the ventral part of the nucleus. GRP cells occupy a more central region, slightly dorsal to the VIP neurons and ventral to the ENK cells (Abrahamson and Moore, 2001; Silver et al., 1999). A green fluorescent protein reporter which minimizes interference from GRP immunoreactivity in cell processes, indicates that these neurons occupy a large percentage of the central mouse SCN and that the GRP region extends dorsolaterally to the perimeter of the nucleus (Karatsoreos et al., 2004). Interpretation of regional distinctiveness based on cell phenotype may be limited by the experimental methods. For example, recent data suggest that discrete VP and VIP regions may be more apparent than real. Immunohistochemistry for VP peptide generally reveals dense staining in the dorsomedial hamster SCN where there is a mix of cells and processes in which cells are often difficult to identify (e.g., Miller et al., 1996; Kalsbeek et al., 1993). In marked contrast, in situ hybridization methods detecting VP mRNA enable clear visualization of individual neurons. This procedure reveals that, at certain circadian times, the gene is active over a much wider portion of the SCN (Silver et al., 1999; Hamada et al., 2001), including areas that do not contain neurons clearly identifiable by VP peptide content. In fact, at certain circadian times, virtually no VP mRNA at all is detected in the SCN (CT20), whereas at CT8, the VP gene appears to be active in the majority of anterior SCN neurons (Hamada et al., 2004a). In situ hybridization methods also demonstrate a much larger distribution of VP Fig. 1 – Schematic organization of the hamster SCN at a level mRNA in the rat SCN than expected from comparable results through the central subnucleus identified by SP, CALB, CALR based on immunohistochemistry (Dardente et al., 2002). The and GRP immunoreactive cells. (A) Approximate locations of method-related differences are also apparent when evaluat- cell types. VP, SS and CCK are found in a generally ing rat VIP. These results plus those for VP mRNA or peptide dorsomedial location, although this may vary according to indicate clearly different, but extensively overlapping, dis- time of day (indicated by shading). CCK cells are also evident tributions of VP and VIP cells in the rat SCN. The data have ventrolaterally (black dots); (B) terminal fields of the three implications for all allegations of discreet VP or VIP regions major afferent pathways. PACAP and glutamate from retinal and to any specific functions related to those cells or the areas projections occupy the entire SCN, to a varying degree (see in which they are purportedly found. Part C of this figure). 5-HT terminals from the median raphe Despite the foregoing caution, analysis of the hamster SCN nucleus fill essentially the entire nucleus, but only sparsely in indicates that it shares with other rodents the apparent the central and dorsolateral regions. NT, NPY, ENK and GABA separation of a VP region from a VIP region (Fig. 1) because arrive from the IGL, although the GABA terminals from the IGL there is no question that the most persistently evident have not been explicitly identified (see text) and (C) dorsomedial part of the VP region does not overlap with the identifiable zones of retinal projections. Black = region of VIP region (Hamada et al., 2004a; Card and Moore, 1984). The dense retinal innervation, predominantly from the hamster SCN also has a dorsolateral region generally devoid of contralateral retina. Cross-hatched = region of modest a presently known specific cell phenotype. The hamster SCN innervation predominantly from the ipsilateral retina. Shaded also contains a feature initially thought to be unique to this gray = region of lesser, approximately equal, innervation from species, namely a small, fairly central region identifiable by its each retina. This diagram is a guide, not a rule. The lines do distinct sets of neurons. The region was originally identified as not represent absolute boundaries. BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 7 the location of substance P (SP) cells (Morin et al., 1992) within the SCN, with a distribution not identical to another visualized with the use of colchicine. However, the same known phenotype (Dardente et al., 2002 but see Moore et al., region contains numerous cells synthesizing calbindin (CALB) 2002 for a different perspective). The group of rat GRP cells (Silver et al., 1996a) which is more easily visualized. The region appears to be smaller than the group of VIP cells and also contains neurons immunoreactive to gastrin releasing positioned within the distribution of those cells. A small peptide (GRP) (Miller et al., 1996) and calretinin (CalR; Silver et number of NT neurons has been described in the rat SCN in a al., 1996a; Blanchard and Morin, unpublished data). Further- distribution that overlaps with the lateral collection of VIP more, this caudocentral SCN subnucleus overlaps to some cells (Moore et al., 2002). Another candidate rat SCN homolog degree with the VIP region (Aioun et al., 1998). of the caudocentral hamster SCN subnucleus consists of the The complexity of the caudocentral region of the hamster region occupied by CalR cells. These are present in a location SCN is borne out by colocalization studies. About 8% (Silver et distinctly different from the other cell phenotypes in this al., 1996a) or 65% (LeSauter et al., 2002), 37 and 33% of CalB cells species, overlapping the dorsomedial VP cells to some extent, have colocalized SP, GRP and VIP, respectively. About 80–90% but largely occupying a separate region in the dorsal part of of SP cells and 43% of GRP cells also contain CalB and 60% of the nucleus (Moore et al., 2002). VIP neurons located within the region of the CalB neurons As has been noted previously (Morin, 1994), there are sub- actually containing CalB (LeSauter et al., 2002). GRP has also stantialspeciesdifferencesinSCNstructure.However,thereare been observed colocalized with VIP (Aioun et al., 1998). at least two organizational characteristics which many species Colocalization of CalB and cholecystokinin (CCK) does not seemtoshare.ThesearetherespectivegenerallocationsoftheVP occur (LeSauter et al., 2002). CalR has not been examined for and VIP cell groups. The former tends to occupy a dorsomedial the possibility of colocalization in the SCN, nor have most SCN position, while the latter group sits centrally in the ventral other combinations of antigens. part of the nucleus (Fig. 1). This spatial characteristic has been The region containing VP neurons is matched reasonably observedintheSCNofrat(Moore,1983),hamster(CardandMoore, well by the locations of CCK cells. Nevertheless, to the limited 1984), grass rat (Smale and Boverhof, 1999), golden mantled extent that the issue has been examined, there is no groundsquirrel(Smaleetal.,1991),Europeangroundsquirrel(Hut colocalization of VP and CCK in the hamster (Blanchard and et al., 2002), Octodon degus (Goel et al., 1999), blind mole rat Morin, unpublished data). In the most ventrolateral part of the (Negroni et al., 1997), monkey (Moore, 1993)andhuman(Moore, nucleus, CCK cells are evident at a location containing VP 1993).However,theminkandmuskshrewarestrikingexceptions fibers (LeSauter et al., 2002). to this rule as these species have no VP neurons present at the The caudocentral SCN subnucleus may be special with location normally identified as the SCN (Martinet et al., 1995; respect to circadian rhythm regulation. This issue is consid- Tokunaga etal.,1992). ered separately below. From the comparative anatomical Cassone et al. (1988) evaluated comparative SCN organi- perspective, it is useful to question the extent to which other zation by analyzing retinal projections to the SCN and the species have a clearly defined region of likely homology. The relative distribution of various peptidergic neurons. They answer is not clear. The only other species known to date with distinguish “visual” and “nonvisual” regions of the SCN, with SP cells at a similar location is the Djungarian hamster (Reuss the latter receiving a much reduced density of retinal and Bürger, 1994). The grass rat has CALB-IR neurons clustered projections compared to the former. The “visual” region in the SCN (Mahoney et al., 2000), apparently more centrally usually contains VIP cells, although it is noteworthy that located than those in the hamster in which the subnucleus is the specific location of this region varies. In the eutherian located in the caudal half of the SCN. In rats, CALB-IR neurons mammals examined (house mouse, guinea pig, cat, pig), the are more prevalent in the ventral SCN, but are not as tightly “visual” region resides in the ventral or ventrolateral SCN. clustered as in the hamster (Mahoney et al., 2000; Arvanito- Among marsupials, there are no VIP cells evident in the SCN giannis et al., 2000). In mice, CALB neurons are present within of the kowari or stripe-faced dunnart, while in the bandicoot, the general central SCN area, the surrounding region and VIP cells are codistributed medially and dorsomedially with generally throughout the entire nucleus (Abrahamson and the VP neurons. The retinal projection of the Virginia Moore, 2001). However, the mouse SCN does have a clearly opossum and gray short-tail opossum distributes ventrally defined central region notable because of the absence of a and medially in the caudal half of the SCN (Cassone et al., specific set of identifiable neurons (LeSauter et al., 2003). This 1988). In these two species, VIP and VP neurons are region corresponds most closely to the location of neurotensin codistributed through the retinorecipient region and medially and GRP neuron clusters (Abrahamson and Moore, 2001). A in more rostral SCN. Thus, in general, VIP neurons tend to be region with reasonably clear homology to the hamster central associated with the densely retinorecipient region, whereas subnucleus has been described in the golden mantled squirrel, the VP neurons are also located there in some species, but not but is recognized by a distinct cluster of ENK, rather than SP, in others, including most of the eutherian mammals. cells (Smale et al., 1991). Whether these differences in peptidergic cell distribution The rat SCN is more compressed along the dorsoventral reflect the functional organization of the SCN remains to be axis than it is in the mouse or hamster. A cell grouping demonstrated. It is also worth noting that the laterodorsal obviously homologous to those in the hamster caudocentral SCN of most mammals is a “specialized” region remarkable subnucleus has not been described in the rat. However, an because of the general absence of non-GABA cell phenotypes argument can be made that cells containing GRP occupy a (Abrahamson and Moore, 2001; Moore et al., 2002; Silver et al., similar position. The GRP cells, in particular, meet the criteria 1999; Card and Moore, 1984; Smale and Boverhof, 1999; Smale of being tightly distributed, more or less centrally located et al., 1991; Goel et al., 1999; Cassone et al., 1988). 8 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

3.3. Intra-SCN connections all, of the SCN. An injection of virus into the subparaventricular zone initially labels cells largely in the ventral two Studies of intra-SCN connections fall into two basic categories. thirds of the rat SCN, but by 54 h, labeled cells densely One involves filling individual cells and mapping processes populate the whole nucleus. The data suggest that cells in the within the nucleus. The second involves the use of tract dorsal SCN receive projections from cells in the ventral SCN tracers applied iontophoretically or by small volume injection. and vice versa (Leak et al., 1999). It has not been determined The first procedure is limited by the fact that relatively few whether all SCN cells become labeled with virus after injection cells can be examined, usually only one per SCN. A successful into a target of SCN efferents. Comparable studies do not exist application of the cell filling technique has demonstrated that for other species. dendritic processes of CALB neurons in the hamster SCN have a predominantly dorsomedial orientation (Jobst et al., 2004). This suggests that CALB neurons receive input from dorsome- 4. Retinal photoreceptors and projections dially located neurons, many (but not all) of which contain VP or CCK (LeSauter et al., 2002). In nonmammalian species, the existence of photoreceptors The tract tracer application method has the advantage of not specialized for classical visual function has long been labeling more cells, but the disadvantage of usually spilling recognized (Gaston and Menaker, 1968; Menaker, 1968; Mena- label into regions adjacent to the intended target. With that ker and Keatts, 1968; Menaker and Underwood, 1976; Enaker et caveat in mind, small iontophoretic injections of biotinylated al., 1970; Underwood and Menaker, 1970). These photorecep- dextran amine (BDA) into the SCN have provided a good initial tors regulate photoperiodism and entrainment of circadian appraisal of connectivity within the nucleus. Confocal micro- rhythms in nonmammals. For the same purposes, mammals scopic analysis of the injected brains shows that most, but not require photoreceptors in the eye (Foster et al., 2003; Yamazaki all, of the cell phenotypes are connected to CALB neurons (Fig. et al., 1999). A critical study by Foster et al. (1991) demonstrated 2), the focus of this particular study (LeSauter et al., 2002). that rd/rd mice, sustaining the near-total loss of rods and cones Particularly noteworthy is the absence of efferent or afferent along with their respective photopigments, retain substantial- connections between CALB and VP neurons. Interconnections ly normal phase responses to light. In addition, the rd/rd mice among other pairs of neuron phenotypes have not been perform similarly to controls even after 767 days, although examined. there are no electroretinogram responses after 210 days of age Use of the Bartha pseudorabies virus as a tract tracer has (Provencio et al., 1994). The implication of these studies was provided a novel perspective on intrinsic SCN connectivity. For that a novel retinal photoreceptor was sufficient for light- example, an injection into the dorsomedial hypothalamus induced phase shifts in mammals. In 1998, Provencio et al. retrogradely labels, by 48 h postinjection, many neurons in the (1998a) identified the molecule, melanopsin, and described its dorsal third of the ipsilateral rat SCN (Leak et al., 1999). About presence in Xenopus brain, eye and photosensitive skin 20–24 h later, labeled cells are present throughout much, if not melanophores. The human and mouse melanopsin genes were then cloned and demonstrated in cells of the mouse and monkey retinal ganglion cell layer (Provencio et al., 2000).

4.1. Melanopsin ganglion cell characteristics

The results of Provencio and colleagues were rapidly adapted to specifically identify photoreceptive retinal ganglion cells (Berson et al., 2002) with additional work demonstrating that melanopsin behaves as a photopigment capable of activating a G-protein (Newman et al., 2003; Melyan et al., 2005; Qiu et al., 2005; Panda et al., 2005; Isoldi et al., 2005). Although there is debate concerning the shape of the action spectrum, the best indications are that the melanopsin action spectrum peaks at about 480 nm. Several studies have been designed to evaluate light sensitivity of photoreceptive cells contributing to the retinohypothalamic tract (RHT). The results clearly demonstrated that a special class of ganglion cells projecting to the Fig. 2 – Connectivity of cell types within the hamster SCN. suprachiasmatic nucleus (SCN) are both photoreceptive in CCK = cholecystokinin; GRP = gastrin releasing peptide; VIP = the absence of known synaptic input (Berson et al., 2002) and vasoactive intestinal polypeptide; VP = vasopressin. Arrows contain melanopsin (Hattar et al., 2002; Gooley et al., 2001). In indicate the direction the cell types project. Numbers indicate the hamster retina, there are approximately 1600 melanopsin- the percentage of cells with afferent contacts. Dashed arrows containing cells (Morin et al., 2003), comprising an estimated indicate SCN-afferent projections containing serotonin (5-HT) 1.5% of all ganglion cells (Tiao and Blakemore, 1976; Hsiao et and neuropeptide Y (NPY) from the median raphe nucleus al., 1984; Rhoades et al., 1979). In the rat, the estimate is 2.5% (Mn) and intergeniculate leaflet (IGL), respectively. Data (about 2455 melanopsin cells) and, in the mouse, 1% (about 730 drawn from Table 1 in LeSauter et al. (2002). cells) (Hattar et al., 2002). These cells provide the retina with BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 9 what has been described as a “photoreceptive net” provided by outward rectification. These data are consistent with the overlapping, large arbors of beaded dendrites (Berson et al., current being mediated by channels of the trp family (transient 2002; Provencio et al., 2002). Apparently, all portions of the receptor potential) of channels (Warren et al., 2003). Properties melanopsin-containing cells are photoreceptive (Berson et al., of SCN-projecting ganglion cells (identified by retrograde 2002). Melanopsin of retinal origin has been described in the tracer) recorded from a cat eyecup preparation in which there SCN of rats (Beaulé et al., 2003b), although it has not been was no elimination of rod/cone contribution indicate that they observed in the SCN of mouse or hamster (Provencio, exhibit sustained responses of the “on” or “on–off” center unpublished data; Blanchard and Morin, unpublished data). variety; have a spectral sensitivity peak at about 500 nm and In the hamster, the melanopsin cells are spread homoge- response is optimal with motionless or slow-moving stimuli neously throughout the retina, as they also are in the cat (Pu, 2000). (Semo et al., 2005). The mouse may have a higher density of melanopsin in the superior retina (Hattar et al., 2002), a feature 4.2. Retinal ganglion cell projections and bifurcation that has been reported in the rat (Hattar et al., 2002; Hannibal et al., 2002) and the diurnal species, the Nile grass rat Retinal projections to the SCN were definitively described in (Blanchard, Novak and Morin, unpublished data). 1972 (Moore and Lenn, 1972; Hendrickson et al., 1972) and Nearly all the melanopsin-containing cells are present in focused scientific attention squarely on that nucleus as the the ganglion cell layer of the retina. However, there is a small probable site of the neural circadian clock. Evaluation of the percentage of the cells found as “displaced” to the inner RHT has continued as methods have improved and more nuclear layer (Berson et al., 2002). In either case, the dendritic species are evaluated. The extent to which the retina projects arbor is most extensive along the border between the inner to each SCN is highly variable across species. The diurnal nuclear and inner plexiform layers. The predominant location grass rat has bilateral retinal innervation of the SCN, although of the dendrites is in the OFF sublayer of the inner plexiform a preponderance arrives from the contralateral retina (Smale layer. The frequency histogram of soma diameters of mouse and Boverhof, 1999). This differs from what is seen in the melanopsin ganglion shows a mean of about 16.3 μm(Hattar et diurnal golden mantle ground squirrel in which RHT inner- al., 2002). In the cat, SCN-projecting ganglion cell diameter is vation is exclusively from the contralateral retina (Smale et similar to the rat (17.2 μm) with dendrites ramifying primarily al., 1991). A recent report shows modest ipsilateral SCN in the inner plexiform layer (Pu, 1999). The size distribution is innervation by the retina in the diurnal California ground not symmetrical, but skewed toward larger diameters for both squirrel (Major et al., 2003). In contrast, following analysis of species. darkfield images, the hamster RHT was described as bilater- Photoreceptive ganglion cells were first described following ality symmetrical (Johnson et al., 1988a). The issue of laterality intra-SCN injection of a retrograde tracer. This permitted and pervasiveness of the retinal projection in the hamster identification of ganglion cells contributing to the RHT and SCN has now been evaluated with confocal microscopy could be combined with recording from those cells in the (Muscat et al., 2003). The data provide several novel observa- absence of input from other retinal neurons (Berson et al., tions. First, nearly the entire SCN is innervated by the RHT, 2002). With or without the presence of drugs blocking synaptic consistent with earlier HRP results (Pickard and Silverman, input, the cells are slow to respond to light stimuli with 1981). Second, despite the extent of innervation, it is not saturating light pulses requiring hundreds of milliseconds, and homogenous within the SCN. And third, the axons from one near threshold stimuli requiring about 60 s, to response onset. retina project to most or all of the SCN bilaterally. The central Latency to maximal depolarization is reduced as stimulus and dorsal SCN receive dense innervation predominantly irradiance increases. Subsequently, the depolarization slowly from the contralateral retina, while the ventromedial SCN decays to a plateau that more or less endures for the duration receives moderate innervation largely from the ipsilateral of the stimulus, with the plateau proportional to the stimulus retina. Thus, innervation of the SCN by one retina is generally energy. The cells are wavelength-sensitive with a peak pervasive, but with clear regional specificity. Of particular response at about 484 nm, similar to the results from action interest is the presence of the densest retinal innervation both spectra for rodent circadian rhythm phase response (Takaha- to the region of the CALB-containing central subnucleus of the shi et al., 1984; Provencio and Foster, 1995). For a fixed SCN and to a region slightly more dorsal. The heavily wavelength stimulus, peak depolarization increases linearly innervated dorsal area corresponds to a zone in which until reaching a saturating irradiance. Spike frequency appears phosphorylated extracellular signal-regulated kinase (pERK) related to the extent of depolarization. Subsequent to stimulus is found (Lee et al., 2003), a characteristic that is apparently offset, repolarization is very slow, taking minutes after stimuli important to pERK activity. of high irradiance. Spike discharges are maintained until Other species also appear to have overlapping, but pre- polarity returns near baseline. The membrane depolarization ferred SCN regions of innervation by retinal projections. This is is produced by an inward current that is activated by the light apparent in the rat, but not the mouse (Shivers and Muscat, pulse (Warren et al., 2003). The current activates slowly 2004). One apparent difference between the RHT terminal reaching a peak several seconds after the application of a fields of these species and that of the hamster is the light pulse. The time course of the membrane depolarization substantially absent innervation of the dorsomedial SCN that and the inward current are similar. The signal transduction largely corresponds to the area in which most VP cells are pathways coupling melanopsin to the activation of the inward present. current are not known. The current was unaffected by The retina projects to the SCN via the RHT, a major direct substitution of extracellular Na+ and shows both inward and projection, and through a secondary, indirect pathway 10 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 throughtheIGLandtheGHT.Inaddition,thereare brain areas. The initial report of melanopsin cell targets numerous retinal projections to disparate brain regions showed X-gal staining for β-galactosidase activity in a many of which have potential, reciprocal connections to transgenic mouse strain with tau-lacZ targeted into the the SCN through the IGL (Morin and Blanchard, 1998). One melanopsin gene locus (Hattar et al., 2002). This allowed such is a direct retinal projection to the dorsal raphe visualization of β-galactosidase fused to a protein antero- nucleus, with an initial description in cats given in 1978 gradely transportable to axon terminals. The blue reaction (Foote et al., 1978). Interest was further heightened with the product emphatically revealed projections to the SCN, IGL demonstration, based on both anterograde and retrograde and olivary pretectal nucleus (OPT). Subsequently, retrograde tracing data, of a similar pathway in rat (Shen and Semba, tracing studies have confirmed melanopsin-containing gan- 1994). This pathway has also been observed in gerbil (Fite et glion cells projecting to these retinorecipient targets, as well al., 1999, 2003) and O. degus (Fite and Janusonis, 2001). The as to the ventral lateral preoptic area, and the subparaven- pathway has not been found in hamster, although it has tricular zone of the hypothalamus (Morin et al., 2003; Gooley been sought several times (Morin and Blanchard, unpub- and Saper, 2003; Sollars et al., 2003). Melanopsin cells in the lished data; Fite, unpublished data). hamster (Morin et al., 2003), but not the rat (Gooley and Bifurcation of ganglion cell axons and subsequent projec- Saper, 2003), have been reported to project to the superior tion of these cells to the SCN and IGL (Pickard, 1985) appears to colliculus. The retrograde studies confirm the projections be a common characteristic of the circadian visual system. from melanopsin cells to initially defined major visual The presence of ganglion cells that bifurcate to innervate both targets, but suggest that not all the ganglion cells projecting the SCN and IGL has been recently confirmed, with the to these areas contain melanopsin. Estimates are that 10– additional information that similar bifurcation is present for 20% of ganglion cells projecting to the SCN do not contain individual cells projecting to both the SCN and superior melanopsin (Morin et al., 2003; Gooley and Saper, 2003; colliculus or OPT (Morin et al., 2003). Injection of two Sollars et al., 2003). retrograde tracers, one into each SCN, also demonstrates The suggestion that melanopsin cells and their projec- that at least some ganglion cells bifurcate and send at least tions create a “nonimage forming” visual system has been one axon branch to each SCN. Further, at least some of these recently put under pressure with the observation that at cells contain melanopsin, as do some that project to the OPT. least some of these cells project to the monkey dorsal lateral In contrast, individual ganglion cells apparently do not geniculate nucleus (Peterson et al., 2003; Dacey et al., 2005). bifurcate and project bilaterally to the IGL, OPT or the SC. These cells appear to provide both irradiance and color Other retinorecipient regions have not been simultaneously information to the dorsal lateral geniculate nucleus, a site examined for both bifurcation and melanopsin cell afferents. involving in the processing of feature-related information, Nor has the question of individual ganglion cells projecting to i.e., images. Melanopsin cell projections to dorsal lateral more than two visual targets been examined. The prospect is geniculate nucleus have not been found in the rat (Gooley likely that individual ganglion cells have multiple axonal and Saper, 2003). As indicated above, the melanopsin cells processes (beyond two) that would allow a rather small are known to project to several other retinorecipient nuclei number of photoreceptors to commonly influence a broad and bifurcation may be a common feature of axons range of visual functions. projecting through the RHT to the SCN. Therefore, it is likely A novel contribution to the analysis of retinal projections that the melanopsin cells projecting to the dorsal lateral to the SCN comes from two studies by Abe and Rusak (1992). geniculate nucleus also bifurcate and innervate many, if not In the initial paper, hamsters were electrically stimulated in all, other subcortical visual regions. the IGL with the result that FOS protein synthesis was An unexpected development has arisen from a thorough induced in the centrodorsal SCN. The preliminary interpre- evaluation of an engineered version of the Bartha strain tation was that GHT activation was causal to FOS expression pseudorabies virus (PRV152), as a retrograde transsynaptic (Abe et al., 1992). However, a subsequent study (Treep et al., tracer (Pickard et al., 2002). Previously, viral tracing unequiv- 1995) showed that FOS expression could not be stimulated in ocally demonstrated a subset of retinal ganglion cells follo- bilaterally enucleated animals. This result suggested an wing injection into the contralateral eye. These cells alternate interpretation, namely, electrical stimulation may presumably gave rise to the RHT (Moore et al., 1995; have elicited antidromically activated retinal ganglion cells Provencio et al., 1998b; Hannibal et al., 2001a). However, a which produced orthodromic activation of the SCN via the new interpretation of these results is based on the fact that collaterals from bifurcating axons in the RHT (Morin et al., PRV152 is exclusively a retrograde tracer that makes its way 2003; Pickard, 1985). The resulting pattern of FOS protein to the SCN multisynaptically through the nucleus of expression would uniquely represent the distribution of Edinger–Westphal in the midbrain oculomotor complex retinal projections that bifurcate and project to both the (Pickard et al., 2002). Cells in the SCN, IGL and OPT are IGL and SCN. The extent to which such bifurcating projec- infected, as are several ganglion cell types in the contralat- tions originate from ganglion cells that do or do not contain eral retina. Only a subset of these contribute to the RHT. melanopsin is unknown. Viral transsynaptic tracing is a powerful method and has the Photoreceptive ganglion cells contributing to the RHT added advantage of revealing second order retinal neurons, contain melanopsin (Hattar et al., 2002; Gooley et al., 2001; bipolar and amacrine cells, that synapse with the primary Morin et al., 2003; Hannibal et al., 2002; Gooley and Saper, ganglion cells (Belenky et al., 2003). This observation has 2003). To date, however, there has not been a comprehensive significant implications for the manner in which light description of melanopsin ganglion cell projections to other regulates circadian rhythm phase. BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 11

4.3. Classical and ganglion cell photoreceptor contribution cells contribute, approximately equally, to circadian rhythm to regulation of circadian rhythms, masking, the pupillary regulation by light. light reflex and melatonin suppression Subsequently, two sets of investigators, again using somewhat different methods, addressed the issue of photo- 4.3.1. Circadian rhythms receptor types and circadian rhythm regulation using animals Three classes of photoreceptive cell types reside in the defective in both melanopsin production and classical photo- mammalian retina. The current view is that while all effects receptor function. Again, the results are substantially the of light on the circadian visual system are accounted for by same: animals lacking melanopsin but bearing the rd/rd the three types, no single photoreceptor is necessary for genotype (Panda et al., 2003) or undergoing rod and cone entrainment. The rd/rd mouse strain suffers postnatal degeneration plus having defective phototransduction in degeneration of rods and cones. These individuals have cones (Hattar et al., 2003) free-run under normal light–dark essentially normal phase response to light (Foster et al., photoperiod conditions. Lack of entrainment was not altered 1991; Provencio et al., 1994; Freedman et al., 1999) and have by light intensity nor was circadian period under LL different an action spectrum for phase shifts that peaks at 480 nm from the circadian period under DD. In addition, light was (Yoshimura and Ebihara, 1996; Hattar et al., 2003) (but see unable to inhibit nocturnal melatonin synthesis (Panda et al., Provencio and Foster, 1995). Recently, entrainment stability 2003). It should be noted that the defects in rhythm response at threshold levels of illumination was assessed (Mrosovsky, to light are not the result of loss of ganglion cells that normally 2003) and, with this simple procedure, only 10% of rd/rd synthesize melanopsin or their projections (Hattar et al., 2003). mice were able to remain entrained to a 12:12 LD photoperiod when the daytime light intensity was 15 4.3.2. Masking, pupillary light reflex and melatonin suppression stops (b2 lx), compared to about 80% of wild-type mice. Other accessory visual functions are also modified by the This result supports the view (Yoshimura et al., 1994) that absence of melanopsin. Although light-induced negative there is a circadian system response to light that is masking is reduced, particularly at lower light intensities, in exclusively rod and/or cone-sensitive. Rods may regulate rd/rd mice, the melanopsin KO alone yields masking that is hamster entrainment under very dim lighting conditions about 30% greater than in the rd/rd mice (Panda et al., 2003). (Gorman et al., 2005). Neurophysiological methods also When the melanopsin KO and rd/rd traits are combined, the support the view that both rods and cones (primarily 510 mice do not exhibit negative masking to light (Hattar et al., nm green and secondarily 375 nm near-ultraviolet) provide 2003; Panda et al., 2003). Melanopsin does not seem to be photic information to the SCN (Aggelopoulos and Meissl, involved in the positive masking that occurs in response to 2000). dim light exposure (Mrosovsky and Hattar, 2003), but appar- A flurry of investigations followed the discovery of ently contributes to negative masking in the presence of light melanopsin photoreceptive retinal ganglion cells. Melanop- ≥10 lx (∼6.7 μW/cm2). sin gene knockout (KO) mice were made by two different Pupillary constriction in response to photic stimuli is much methods, yet the effects on light-induced rhythm responses reduced in rd/rd mice and the response has a longer latency. At were very similar (Panda et al., 2002; Ruby et al., 2002a). The high irradiances, pupillary response equals that in wild-type animals entrain and free-run properly in normal light–dark mice, but the response of rd/rd mice is markedly attenuated at photoperiod and constant dark conditions, respectively. intermediate and lower irradiances (Hattar et al., 2003; Panda However, phase response to light is attenuated in the KO et al., 2003; Lucas et al., 2001). Unlike masking and phase animals. With a 480 nm stimulus, phase shift magnitude response to light, the pupillary light reflex is relatively was only about half that of wild-type mice at each of three, unmodified in the absence of melanopsin. Only at irradiances nonsaturating irradiance levels (Panda et al., 2002). A N10 × 1012 photons/cm2/s is there a modest sensitivity deficit. saturating, white light pulse also yielded a diminished The full effect of light on the pupillary light reflex can be phase shift from the KO animals, although it was about accounted for by the additivity of rod/cone and melanopsin two thirds normal (Ruby et al., 2002a). A second defect in photoreceptor contributions (Panda et al., 2003; Lucas et al., response to light by the melanopsin KO animals was the 2003), an observation supported by the absence of any failure of constant light (LL) to elicit a properly lengthened pupillary light reflex in melanopsin KO animals lacking rod circadian period. In both strains, LL (white light) effectively and cone function (Hattar et al., 2003; Panda et al., 2003). A increased the period, but in melanopsin KO animals, the single opsin/vitamin A-based photopigment with a peak change was only about 55–65% of controls (Panda et al., 2002; sensitivity around 480 nm appears to drive the pupillary Ruby et al., 2002a). It is worth noting that similar effects on light reflex in rodless, coneless mice (Hattar et al., 2003; Lucas light-induced circadian period lengthening can be achieved et al., 2001). by IGL lesions (Morin and Pace, 2002), possibly because such A contribution of the pupil to circadian rhythm regulation lesions would destroy the functional effectiveness of mela- has not been studied and is seldom considered as a factor nopsin cell projections to the IGL (Hattar et al., 2002). Light regulating circadian rhythm response to light. However, it is administered at CT16 was able to induce c-fos mRNA in SCN useful to note (a) that both classical and melanopsin photo- cells of melanopsin KO animals, although there has been no receptors contribute to both responses (Hattar et al., 2003; test of the normality of this expression (Ruby et al., 2002a). Panda et al., 2003; (b) that, within limits, phase response An important outcome of the melanopsin KO studies is the magnitude is proportional to the total number of photons implication that both classical photoreceptors and intrinsi- assessed by the circadian visual system across the duration of cally photoreceptive, melanopsin-containing retinal ganglion light exposure (Nelson and Takahashi, 1991; (c) that the pupil 12 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 area can vary by a factor of 16 (Hattar et al., 2003; Lucas et al., tion of excitatory amino acid. The initial study using 2003) and (d) that the pupil acts to gate exposure of the glutamate generated a PRC more of the NPY-type (Meijer et circadian visual system to the very stimulus type to which it al., 1988), while aspartate had no effect (De Vries and Meijer, optimally detects and responds. 1991). Subsequently, NMDA was shown to elicit phase delays Mice which cannot synthesize melanopsin or which bear when administered at CT13.5 and advances at CT19, consis- the rd/rd mutation show, to the extent tested, normal light- tent with a light-type PRC (Mintz and Albers, 1997) and a full, induced suppression of pineal melatonin (Lucas and Foster, light-type PRC (although the subjective night is longer and 1999; Lucas et al., 1999). The two photoreceptor classes appear maximal delays equal maximal advances) to direct SCN redundant to the extent that loss of both eliminates melatonin application of NMDA, have been demonstrated (Mintz et al., suppression by light (Panda et al., 2003). 1999). The effects of NMDA on circadian rhythm phase were blocked by NMDA receptor antagonists. In vivo glutamate yields a PRC that differs from that for NMDA for reasons that 5. Neuromodulators of the retinohypothalamic are not clear. tract Injection of NMDA onto the SCN during the early subjective night elicits light-like phase delays which cannot be blocked 5.1. Glutamate by simultaneous tetrodotoxin (TTX) application (Gamble et al., 2003). NMDA treatment during the subjective day has no direct It is generally believed, based on abundant indirect evidence, effect on rhythm phase (Mintz et al., 1999), but it can inhibit that glutamate is the primary neurotransmitter of the phase advances induced by the GABAA agonist, muscimol retinohypothalamic tract. This topic (up to 1996) has been (Gamble et al., 2003; Mintz et al., 2002). In this respect, NMDA extensively reviewed (Ebling, 1996; Hannibal, 2002a) and will during the subjective day mimics the effect of light (Mintz et not be considered in depth here. However, despite the al., 2002). However, the action of NMDA during the day is numerous studies of function involving use of glutamate blocked by TTX treatment suggesting that sodium-dependent receptor agonists and antagonists, there is much less certainty action potentials are necessary for effects of light on rhythm regarding its actual presence in the RHT. Most convincing in phase during the subjective day, but not during the subjective this regard has been the electron microscopic demonstration night. that glutamate immunoreactivity occurs in cholera-HRP A corollary to the issue of glutamate in the RHT concerns identified terminals of retinal projections to the mouse and the biochemical pathway that is likely necessary for proces- rat SCN (De Vries et al., 1993; Castel et al., 1993). Electrical sing the neurotransmitter in the SCN. This pathway involves stimulation of the optic nerve induces SCN release of [3H]- the de novo synthesis of glutamate in neurons and glia, plus glutamate or [3H]-aspartate (but not [3H]-GABA) loaded onto the recycling of extracellular glutamate through glia back to rat slice preparations (Liou et al., 1986). However, direct neurons (Hertz, 2004). Extracellular glutamate concentration stimulation of the SCN yields even greater release of the two in the peri-SCN region is greatest during the hours of darkness amino acids, suggesting their presence in nonretinal term- (Rea et al., 1993a; Glass et al., 1993). Glial fibrillary acidic acid inals and SCN cells, in addition to RHT terminals. Pharmaco- concentration in SCN astrocytes is lowest after lights off (Glass logical studies suggest that aspartate may be the most potent and Chen, 1999). excitatory neurotransmitter in the rat RHT (Shibata et al., 1986). Two laboratories have evaluated the uptake and 5.2. N-Acetylaspartyl glutamate retrograde transport of [3H]-aspartate injected into the rat SCN. While the two investigations agreed with respect to A significant question that has not been resolved is whether nonretinal projections to the SCN, one identified retinal glutamate is the sole amino acid transmitter in the RHT ganglion cells contributing to the RHT (Devries et al., 1995) contributing to circadian rhythm regulation. N-Acetylaspartyl and the other did not (Moga and Moore, 1996). More recently, glutamate (NAAG) is also present in the RHT (Moffett et al., glutamate has been immunohistochemically identified in the 1990, 1991; Tieman et al., 1991), although at the synaptic cleft, RHT colocalized with PACAP which may identify most, if not NAAG is converted to its constituent amino acids and each is all, retinal projections to the SCN (Hannibal et al., 2000). available for postsynaptic activity (Baslow, 2000; Neale et al., Vesicular glutamate transporters, VGluT1 and VGluT2, 2000; Coyle, 1997). However, unlike NAAG, neither aspartate account for nearly all the glutamate transporters in the brain nor glutamate is released from retinal ganglion cell terminals and are generally abundant in retinorecipient nuclei in a calcium-dependent manner (the RHT has not been (Fujiyama et al., 2003; Land et al., 2004), for example, in the specifically tested; see Tieman and Tieman, 1996 for a dorsal lateral and ventral lateral geniculate nuclei. However, review). Whether this constitutes the source of photically they are not abundant in either the IGL (where their presence induced glutamate release or the transmitter is independent- is weak, at best) or the SCN. The two transporters are weakly ly released by RHT terminals remains to be discovered. The evident in the SCN, but not at the identical locations (Fujiyama most recently available information (Moffett, 2003) suggests et al., 2003). Unilateral enucleation has little, if any, effect on the presence of NAAG in a portion, but not all, of the rat RHT transporter visibility in the SCN, although bilateral enucle- (ventrolateral part of the SCN, i.e., not the whole retinoreci- ation may do so. pient region). Analysis of prospective NAAG function is One emergent difficulty resulting from the studies of complicated by the presence of numerous NAAG-IR neurons glutamate function has been the general inability to obtain a in the SCN with particularly dense expression in dorsomedial light-type phase response curve (PRC) to direct SCN applica- cells (Moffett, 2003). BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 13

5.3. Glutamate receptors nitric oxide generators to the slice (Ding et al., 1994). Sodium nitroprusside, S-nitroso-N-acetyl-penicillamine and hydrox- One presumption, given that the RHT is likely to use glutamate ylamine application yielded equivalent, glutamate-like phase as a transmitter and that glutamate agonists or antagonists advances and delays during the subjective night, but no phase alter circadian clock function, is that there are glutamate change during the subjective day. Further, the glutamate- receptors in the SCN. The most comprehensive study of induced phase shifts could be blocked by any of several types glutamate receptor localization in the SCN has been per- of competitive substrates for nitric oxide synthase. The formed with hamsters (Stamp et al., 1997) and demonstrates procedures were extended to the in vivo situation in which that the GluR1 receptor is most widely distributed in the SCN L-NAME was able to completely block hamster phase advances with greatest density in a region corresponding approximately induced by light. This blockade was reversed by increasing the to that which receives dense innervation from the contralat- availability of L-arginine substrate (Ding et al., 1994). L-NAME eral retina (Muscat et al., 2003). Both the GluR2/3 and GluR4 was also able to attenuate hamster phase delays (Watanabe et receptors are distributed primarily in the dorsomedial SCN, al., 1995). The data suggest that light acting through the RHT although there are also substantial GluR4 receptors in the releases glutamate in the SCN, activating the NMDA receptors central and ventral part of the nucleus. on SCN neurons. This increases intracellular calcium concen- One important consideration regarding glutamate recep- tration, followed by nitric oxide synthase stimulation and tors is the fact that they can also be abundant on astrocytes nitric oxide production (Ding et al., 1994). Nitric oxide may act (Porter and McCarthy, 1997), and may indirectly modulate as a short distance neuromodulator in the photic signal neuronal activity by altering glial response to neurotransmit- transduction pathway. The effects of melatonin and serotonin ters (Haak et al., 1997). Astrocytes may also be a source of on rat circadian rhythm phase may also require nitric oxide glutamate that can activate neuronal glutamate receptors (Liu synthesis activation in the SCN (Starkey, 1996). et al., 2004). Astrocytes may be part of a route yielding The foregoing basic principles have been linked to light- glutamate receptor-activated release of astrocytic endothelial induced phosphorylation of the cAMP response element nitric oxide synthase that can act in parallel with glutamate- binding protein (CREB) in SCN neurons (Ginty et al., 1993). activated neuronal nitric oxide synthase through which light The appearance of phosphorylated CREB (pCREB) is the can induce NO release in the SCN thereby altering circadian earliest known postsynaptic biochemical marker of photic rhythm phase response (Caillol et al., 2000). signal transduction in the SCN. In vivo light-induced pCREB immunoreactivity in rat SCN cells attained maximal expres- 5.4. Nitric oxide and RHT signal transduction sion within 10 min following a 10 min 150 lx light pulse at CT19 and this could be mimicked by glutamate application to a slice Early articulation of the prospect that nitric oxide might preparation (Ding et al., 1997). The effects of glutamate on contribute to circadian rhythm regulation was provided by pCREB immunoreactivity were blocked by an NMDA receptor Amir (1992) who tested whether the SCN mediated the blocker (APV) or a nitric oxide synthase inhibitor (L-NAME). As increased heart rate response to acute photic stimulation. yet, the steps between postsynaptic glutamate action and G Infusion of L-N -nitro-arginine-methyl ester (L-NAME), a induction of CREB phosphorylation or nitric oxide synthesis/ competitive inhibitor of nitric oxide synthase, over the SCN release have not been determined. It is important to note that greatly attenuated the heart rate increase. This attenuation light- or glutamate-induced pCREB and nitric oxide activity are could be overcome by simultaneous infusion of excess L- dependent on circadian clock phase indicating that the arginine, the natural substrate for the enzyme. Similar effects responsiveness of postsynaptic targets of the RHT is limited on the heart rate response were also obtained with a at certain times by feedback from the circadian clock. Whether glutamate NMDA-type receptor antagonist and it was sug- this means the postsynaptic cells are intrinsically “clock” cells gested (Amir, 1992) that nitric oxide mediates the effects of or they receive rhythmic inhibitory feedback from elsewhere light on the SCN. is not known. Subsequently, Ding et al. (1994) published an extensive Nitric oxide synthase activity in hamsters is rhythmically series of experiments probing the contribution of nitric oxide related to the presence of an alternating light–dark cycle. Light to photic signal transduction in the SCN. As a precursor to the administered at any time of day elevated enzyme activity and work, the investigators evaluated phase response of the SCN the day–night enzyme rhythm did not persist in DD (Ferreyra neuronal firing rhythm to acute perfusion with glutamate et al., 1998; Agostino et al., 2004; Chen et al., 1997). This is applied to a rat brain slice preparation. This yielded a very tidy consistent with the view that the consequences of nitric oxide glutamate-induced PRC (Ding et al., 1994) that closely mim- release, but not the release itself, are regulated by the icked the normal rat light PRC (Summer et al., 1984), with circadian clock. glutamate yielding concentration-dependent phase shifts There is no complete consistency in the literature − − across a range of 10 6 to 10 2 M glutamate. The phasic effects concerning nitric oxide function in circadian rhythm regula- of glutamate were mimicked by in vitro and in vivo application tion. Light-induced FOS protein, thought to be an event of NMDA (Ding et al., 1994; Colwell and Menaker, 1992; Colwell downstream of CREB phosphorylation (Kornhauser et al., et al., 1990, 1991; Takeuchi et al., 1991), a specific agonist for 1990), has been blocked in rat SCN, but not IGL, by peripheral one type of glutamate receptor. injection of L-NAME (Amir and Edelstein, 1997). In contrast, the Using in vitro activation of the NMDA glutamate receptor same procedure in hamsters has blocked light-induced sequelae as the indicator of a functional photic signaling activity rhythm shifts, but not light-induced FOS in the SCN pathway, the photic effects were mimicked by administering (Weber et al., 1995). This may indicate a species difference. 14 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

However, in the hamster, it is further recognition of the fact fashion, PACAP administered at CT6 onto a rat SCN slice that FOS protein expression is not a necessary component of preparation could induce 3.5 h phase advances (tests at CT14 phase response to light (Colwell et al., 1993a,b). or 19 yielded no phase change). VIP, which can act on The anatomical evidence in support of the SCN as a site for receptors similar to those for PACAP, also yielded phase nitric oxide modulation of circadian rhythmicity is not advances, but the concentration sensitivity and resulting particularly encouraging. The earliest descriptions of potential phase response were much lower (Hannibal et al., 1997). The nitric oxide synthesizing substrate based on NADPH-diapho- argument was offered that PACAP constitutes a neuromodu- rase staining in the circadian rhythm system revealed no cells lator in the RHT which, along with glutamate, normally acts to in the rat SCN (Vincent and Kimura, 1992) and sparse cells in modify circadian rhythm phase in response to retinal the Siberian hamster SCN (Decker and Reuss, 1994). Occasion- photoreception. Subsequently, large, concentration-depen- al neurons immunoreactive for the neuronal form of nitric dent, PACAP-induced (CT6) phase advances were observed in oxide synthase have also been observed in rat SCN, and some hamster SCN slices (Harrington et al., 1999). However, of these contain VIP (Reuss et al., 1995). More recently, a dense substantial phase delays were also observed if 1 nM PACAP plexus of processes immunoreactive for the neuronal form of was administered at CT14, but lesser or greater concentrations nitric oxide synthase has been identified surrounding a few had progressively diminished effects. In addition, small phase immunoreactive cells in ventral rat SCN (Chen et al., 1997), but delays or advances were obtained in vivo following infusion of this has not been seen by other investigators (Caillol et al., 1 nM PACAP onto the hamster SCN at CT14 and CT18, 2000; Wang and Morris, 1996). The most convincing anatom- respectively (Harrington et al., 1999). Thus, PACAP in vivo ical study indicates no cells in the SCN of rat or mouse mimicked the effect of light in terms of the direction of the identifiable by NADPH-diaphorase staining, but in both elicited phase shift (tests during the subjective day were not species, immunoreactivity for neuronal nitric oxide synthase performed). revealed distinct cell populations within the SCN. In the Replication of the foregoing PACAP effects has proven mouse, the labeled neurons are evenly distributed through the elusive. Additional in vivo hamster tests have usually, but SCN with about one tenth of the SCN neurons labeled. In not always, yielded phase delays following administration of contrast, all labeled rat neurons are grouped laterally in the various PACAP concentrations at CT14. Only phase delays ventral SCN among immunoreactive neuronal processes. were obtained at CT18, and small phase delays (no Neither labeled cells nor processes were evident in the advances) were observed with one PACAP concentration dorsomedial SCN (Wang and Morris, 1996). However, under given at CT6 (Piggins et al., 2001a). Recently, a third special slice conditions, no nitric oxide synthase immunore- investigation of hamster in vivo effects obtained small activity was observed in the rat SCN although the ventral phase delays following a moderately high concentration of lateral part of the nucleus is densely labeled with cyclic GMP PACAP administered at CT14 (Bergstrom et al., 2003). As is immunoreactivity. NADPH-diaphorase is not always a useful obvious, there are numerous inconsistencies in the available indicator of nitric oxide synthase (see Wang and Morris, 1996 data which require reconciliation before a function of PACAP for discussion). A subsequent report using similar methods, can be accepted. but different antisera, identified no cells containing neuronal The Hannibal lab has developed a monoclonal PACAP nitric oxide synthase in the hamster SCN and a few in the antibody (Hannibal et al., 1995) that has enabled production of ventral rat SCN. Nonneuronal cells (probably astrocytes) and a large amount of information regarding the anatomy of processes immunoreactive for endothelial nitric oxide structures containing this peptide (see Hannibal, 2002a for a synthase were observed in the SCN of both species (Caillol et review). PACAP is present in the RHT (Hannibal and Fahrenk- al., 2000). Despite the biochemical manipulations indicating rug, 2004; Hannibal et al., 1997, 2002; Bergstrom et al., 2003) that nitric oxide synthase activity is necessary for phase where it colocalizes with glutamate (Hannibal et al., 2000). response to light or glutamate, mutant mice lacking the gene PACAP-ir neurons in the rat SCN have also been reported coding for endothelial (Kriegsfeld et al., 2001) or neuronal (Piggins et al., 1996), although there may have been cross- (Kriegsfeld et al., 1999) nitric oxide synthase apparently reactivity with VIP. In addition, PACAP appears to be found in entrain normally to the light–dark cycle and light induces axons and terminals in most of the retinorecipient regions of normal FOS expression in the SCN. As suggested by Caillol et the rat brain (Hannibal and Fahrenkrug, 2004) including the al. (2000), it is possible that there are parallel neuronal and IGL, ventral lateral preoptic area and subparaventricular glial pathways by which photic information gains access to hypothalamus. Two exceptions may be the commissural the circadian clock. If this is the case, then the only effective pretectal area and the dorsal lateral geniculate nucleus, knockout might require deleting the genes coding for both although that information is difficult to discern. PACAP is endothelial and neuronal nitric oxide synthase. widespread in brain (Hannibal, 2002b) and it is not always clear whether the PACAP observed is actually present in 5.5. PACAP retinal projections or is from other sources. PACAP immunoreactivity, in addition to identifying projec- Pituitary adenylate cyclase activating polypeptide (PACAP) did tions to the SCN, identifies a small population of retinal not become a molecule of interest to the circadian rhythm ganglion cells in the rat most of which are found in the field until 1997 when Hannibal et al. (1997) described its superior retina (Hannibal et al., 2002). Most, if not all, of these presence in retinal ganglion cells plus a projection of these cells also contain melanopsin and glutamate. Hannibal et al. cells to retinorecipient regions of the SCN and IGL. The same (2002) has suggested that all ganglion cells projecting to the paper demonstrated that, in a concentration-dependent SCN contain both PACAP and melanopsin. However, the issue BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 15 has not been directly evaluated using retrograde tracers 5.6. Substance P injected into the SCN. The retrograde tracer procedure has shown that 10–20% of RHT neurons do not contain melanop- Confusion also abounds with respect to the presence of SP in sin (Morin et al., 2003; Gooley and Saper, 2003; Sollars et al., the RHT. In contrast to PACAP, there is more positive evidence 2003). The apparently close relationship between PACAP and regarding function of SP than there is for its anatomical melanopsin raises the possibility that PACAP released by presence in the RHT. SP has been suggested as a peptide native melanopsin cell electrical activity may generate prolonged to retinal ganglion cells projecting to the SCN (Takatsuji et al., postsynaptic activity in the SCN that endures beyond the rapid 1991; Mikkelsen and Larsen, 1993) and the distribution of SP release effects of glutamate. fibers and terminals in the rat SCN is similar to that for retinal The suggestion that all melanopsin-containing ganglion input (Piggins et al., 2001b). Other attempts to confirm a SP cells contain PACAP also implies a major role for that peptide projection have not succeeded (Otori et al., 1993; Hartwich et in normal photic regulation of circadian rhythms, assuming it al., 1994), and the presence of a SP terminal plexus in the SCN acts as a neuromodulator. In the context of light and varies substantially across species (Piggins et al., 2001b). A glutamate effects on circadian rhythm phase, in vivo applica- fairly comprehensive analysis of the rat RHT has demonstrat- tion of PACAP antibody or receptor antagonists to the SCN ed PACAP and anterograde tracer in the SCN terminal plexus attenuates the phase delaying or advancing effects of light of the RHT, but apparently there is no colocalization with SP (Bergstrom et al., 2003; Chen et al., 1999). Ordinarily, glutamate (Hannibal and Fahrenkrug, 2002). Similarly, SP does not applied to an SCN slice preparation mimics the effect of light colocalize with PACAP in the rat retina. According to one on the phase of the neuron firing rate rhythm with phase report, most retinal SP cells are present in the inner nuclear delays and advances induced during the early and late layer and outer part of the inner plexiform layer. The few SP subjective night, respectively (Ding et al., 1994; Chen et al., cells in the rat ganglion cell layer differ in size from the PACAP 1999). When glutamate and PACAP are coinfused onto the slice neurons and are considered to be “displaced amacrine cells” preparation, phase delays are enhanced and phase advances (Hannibal and Fahrenkrug, 2002). This contradicts an earlier are abolished. In contrast, the PAC1 receptor antagonist, report demonstrating SP-containing ganglion cells identified PACAP6-38, enhanced glutamate-induced phase advances by retrograde label as projecting to the SCN (Takatsuji et al., while blocking phase delays (also blocked by PACAP antibody 1991). However, the results of Hannibal and Fahrenkrug (2002) infusion). The implication is that glutamate cannot induce (Negroni et al., 1997) are consistent with the presence of SP in phase delays without cofunctioning PACAP, whereas gluta- the SCN surviving bilateral enucleation (Otori et al., 1993). A mate is better able to induce phase advances in the absence of similar conclusion has been reached by other investigators functional PACAP (Chen et al., 1999). following bilateral enucleation of either rat or hamster The role of PACAP in circadian rhythm regulation has been (Hartwich et al., 1994). recently addressed through the use of PACAP KO mice (Colwell The SP receptor, neurokinin 1 (NK1), is generally absent et al., 2004; Kawaguchi et al., 2003). The results are mixed, both from the ventral SCN of both rat and hamster (Mick et al., 1994, within and between studies. Absence of the gene coding for 1995), with low levels of NK1 receptor present in neurons PACAP does not modify rate to re-entrain to an advanced or which are found along the dorsolateral margin of the rat SCN. delayed LD12:12 photoperiod, phase response to dark pulses at Another report (Takatsuji et al., 1995) also indicates a general CT6, the circadian period in LL or the negative masking effect absence of the receptor from the ventral rat SCN, but of light on wheel running (Colwell et al., 2004). Nevertheless, demonstrates its presence on a few cells in the most ventral the same study showed that, in the PACAP KO mice, both part of the nucleus and in the underlying optic tract. Most evi- advance and delay phase shifts to normally saturating or dent receptor is on neurons and dendrites scattered through nonsaturating light stimuli administered at CT16 or 23 are the dorsal and lateral part of the nucleus. Dendrites of the greatly reduced, the circadian period is shorter in DD and the labeled neurons are described as extending ventrally and, in phase angle of entrainment is earlier. The second study enucleated animals, NK1 is found in dendrites contacting (Kawaguchi et al., 2003) failed to find a difference in circadian degenerating retinal projections via asymmetric axo-dendritic period during DD or an effect of the KO on phase delays to light synapses (Takatsuji et al., 1995). This suggests that SP-contain- at CT15, although the phase advances to light at CT21 were ing retinal fibers terminate on cellular elements in the SCN about half of the expectation. To make matters more with NK1 as a receptor. A detailed evaluation of NK1 receptor confusing, wild-type mice had greater light-induced FOS distribution in the rat, mouse, golden and Siberian hamsters protein in the SCN at CT15 than did PACAP KO mice, but not generally confirms the above observations as holding across at the time at which light yielded reduced phase advances species (Piggins et al., 2001b). The major point of difference is (CT21). At the present time, it is difficult to reconcile the the absence of receptor from the ventral SCN in all species observation (Colwell et al., 2004) of (a) a large effect of the gene except the rat. In all species, the SCN is surrounded by a clear KO on phase response to light pulses with the absence of an margin of dense NK1 receptor and there is a distinct cluster of effect on rate of re-entrainment; (b) the presence of reduced cells identifiable by NK1 immunoreactivity just inside extend- FOS at a time at which the same stimulus yields a normal ing to just outside the dorsolateral border of the SCN. rhythm phase shift; (c) the presence of a reduced phase shift at SP is present in fibers and terminals of the rat, mouse, time at which the same stimulus yields normal FOS expres- golden and Siberian hamster IGL (Morin et al., 1992; Piggins et sion and (d) a failure of the two studies (Colwell et al., 2004; al., 2001b), although the origin of this input is not known. The Kawaguchi et al., 2003) to find equally deficient phase delays NK1 receptor is densely distributed throughout the entire IGL in PACAP KO mice. of the same species (Piggins et al., 2001b). 16 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

With respect to function, SP yielded increased 2-deoxyglu- antagonists were unable to block glutamate-induced phase cose uptake in the rat SCN during the subjective night, but not delays (Kim et al., 2001). The latter results are interpreted as during the day. It also produced a light-like PRC in the action indicating that SP works upstream of glutamate. potential rhythm (Shibata et al., 1992; Hamada et al., 1999; Kim Intracranial SP application to the hamster SCN in the early et al., 2001). Both effects were blocked by the SP receptor subjective night elicited small phase delays. The effects were antagonists. Cycloheximide also blocked SP-induced phase not dose-dependent in the pico/nanomolar range and were not shifts in the slice preparation (Shibata et al., 1994). About 43% seen at other circadian times (Piggins and Rusak, 1997). The use of SCN neurons were excited by application of SP to rat brain of SP receptor agonists or antagonists has yielded more slices (11% inhibited), but there was no night/day difference consistent and sometimes larger effects. Light-induced FOS (Shirakawa and Moore, 1994). Similar results have been found was significantly reduced by pretreatment with the nonspe- in the hamster SCN (Piggins et al., 1995a). In both species, SP cific SP antagonist, spantide. The reduction was concentra- modulated neuronal response to glutamate (Shirakawa and tion-specific and varied according to SCN region. Greater Moore, 1994; Piggins et al., 1995a). SP also induced glutamate reduction occurred in the dorsocaudal SCN than in the release in rat SCN slices (Hamada et al., 1999). In optic nerve ventrocaudal SCN and in the rostral SCN than in the caudal stimulated slices, an SP antagonist reduced excitatory post- SCN (Abe et al., 1996). The authors compare the effects of synaptic currents in rat SCN neurons. The effect on NMDA and spantide to those of NMDA and non-NMDA antagonists with non-NMDA receptor-mediated responses was similar (Kim et respect to the distribution of light-induced FOS and concluded al., 1999). However, SP-induced phase delays could be blocked that the glutamate receptor antagonists were involved in light by NMDA or non-NMDA receptor antagonists, whereas SP responsiveness of the ventrocaudal SCN, whereas SP BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 17 contributed to light responsiveness of dorsocaudal SCN et al. (1993) provided evidence, using extracellular recording neurons. Intraperitoneal injection of an NK1 receptor antago- methods, that separated dorsomedial and ventrolateral parts nist had no effect on hamster rhythms in DD, but in LL, phase of the rat SCN oscillated more or less equally in vitro, advances were obtained following treatment at CT8 (Challet et although amplitude of the rhythm from the ventrolateral al., 1998a, 2001). These were not associated with increased region was reduced. This early attempt to distinguish wheel running at the time of injection. NK1 antagonists had no regions of functional significance in which one SCN area effect on light-induced phase delays at any of three intensities differs from another with respect to oscillatory spike (Challet et al., 1998a, 2001). However, at CT19, they attenuated discharge provided a negative result, but presaged a topic light-induced phase advances by about 30–45%, if adminis- that has evolved along with the availability of molecular tered prior to the light pulse. The antagonists were unable to biological tools. More recently, more refined methods have block the effect of light on phase response if administered after allowed greater detail to be gleaned from individual SCN the light pulse (Challet et al., 2001). The effect of spantide on slices (Schaap et al., 2001, 2003). Subtle differences in phase rhythm regulation in hamsters has apparently not been and waveform are apparent both between the bilateral SCN investigated. and across regions within a single rat SCN. Whereas most attempts to evaluate rhythmicity of SCN cells have been done with reference to location or phenotype, 6. SCN structure–function relationships Saeb-Parsy and Dyball (2003) evaluated cells based on their targets of projection. Cells projecting to the arcuate and/or the The structure–function relationship that appears to be emerg- supraoptic nucleus had two peaks in firing rate, one in the ing is one of both time and space. That is to say, the phases of early night and the other in the late night, in contrast with intrinsic oscillatory events vary according to SCN region. cells projecting elsewhere. Attempts to refine the designation, “region,” according to cell As described below (Section 6.1.3), there is accumulating phenotype are progressing, but must be considered as fluid. As evidence that at least some cells may not oscillate. However, is discussed below, the actual number of “regions” in the SCN attempts to identify individual SCN pacemaker cells, while is highly debatable (2, 3 or even 4 for the rat; Figs. 3A–E), even successful in doing so, have not been able to provide within a single species, as is the correspondence of a nominal information regarding the percentage of SCN cells that are region to a particular cell type or organizational attribute. intrinsically oscillatory or their particular cell characteristics Perhaps the dominant question pertaining to function of (Herzog et al., 1997, 1998; Liu and Reppert, 2000, Liu et al., SCN neurons concerns the extent to which each is an 1997a; Welsh et al., 1995; Aujard et al., 2001). Three generalities oscillator. The prevailing view, until fairly recently, has been arise from these studies (Liu and Reppert, 2000). One is that the that the SCN is composed of many intrinsically oscillatory behavior of oscillatory cells evinces a wider range of intrinsic neurons which interactively synchronize to yield a mono- circadian periods if the cells are isolated from one another phasic rhythm (Herzog et al., 1998; Liu et al., 1997a). Gillette than if they are coupled. The second is that GABA is able to

Fig. 3 – Suggested organization of the SCN varies according to species, rostrocaudal location within the SCN, time of day, laboratory, criteria for region designation and the evolution of ideas. RAT: (A1 is rostral to A2) Structure using the “core” and “shell” nomenclature (Leak and Moore, 2001). Note fairly substantial differences in position that depend on the level within the SCN. (B) Geographical structure using “dorsomedial” (DM) and “ventrolateral” (VL) designations. Drawn from histology in Nagano et al. (2003). The area VL′ set off by the dashed line has been added to indicate a portion of the SCN that behaved differently from either the DM or VL, but was not acknowledged by the authors. (C1, C2) Divisional structure as suggested from the variation in density of FOS-IR cells (dots). Drawn from the histology in (Beaulé et al., 2001b). Note that the smaller number of FOS-IR cells within the middle of the 3 divisions of (C2) results from light exposure. The dashed line in panels C1 has been added to demarcate a lateral region that did not appear to vary with respect to FOS induction. (D) Three divisions (dorsomedial periventricular, dorsomedial central, ventrolateral) derived from the expression of Per1 (Yan and Okamura, 2002). Dashed line has been added to set off a region that varies minimally with respect to Per1 expression. Compare to panels C1 and C2. (E) Three divisions (dorsomedial, ventrolateral main, ventrolateral medial) derived from the expression of VIP-IR and Per1. The VLmed receives sparse or no retinal innervation (Kawamoto et al., 2003). MOUSE: (F) Expression of Cry1-IR (dots; F1—ZT23, F2—ZT14-light induced) and Per1 (dots; F3—ZT23, F4—ZT14-light induced). Drawn from histology in Field et al. (2000). The circular central area was drawn to encompass most of the Cry1-IR cells and held constant in panels F2–F4. Note that light-induced Per1 tends to be absent from the region of rhythmic Cry1 expression (F1—ZT23), but has substantial overlap with the area showing rhythmic Per1 expression (F3—ZT23). (G) Regions indicated by antiphase rhythmic expression of Per2. Drawn from histology in (King et al., 2003). The central area in panel G1 was drawn to encompass most of the Per2-IR cell nuclei (dots; CT0) and retained in panels G2 which shows the widespread expression of Per2 in nuclei (dots) at CT12. (H) Compartments indicated by the location of VP-IR cells and fibers or GRP cells as indicated by a green fluorescent protein signal. Drawn from Karatsoreos et al. (2004). (I) Solid lines diagram the mouse SCN “core” and “shell” according to one formulation (Yan and Silver, 2004). The dashed line indicates the border of the “core” as presented in another formulation (Abrahamson and Moore, 2001). HAMSTER: (J) SCN and CALB-identifiable central subnucleus (drawn from Hamada et al., 2001). (K) Cells expressing VP mRNA (dots) in the rostral SCN at (K1) CT20 and (K2) CT4 or in the caudal SCN at (K3) CT20 and (K4) CT4 (drawn from histology of Hamada et al., 2004b), outlines included, with the enclosed ventrolateral area designating the region containing CALB neurons in this study). 18 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 shift the phase of individual, noninteracting SCN neurons in a complication to the interpretation of the functional analyses phase-dependent manner. Third, the phases of oscillations of GABA in the SCN is the presence of a GABAergic afferent among a collection of independent cells are synchronized by projection from the IGL (Morin and Blanchard, 2001). repeated bath application of GABA at the same time every day. The basic observation is that peri-SCN administration of

There is no information concerning the cell phenotype the GABAA agonist, muscimol, substantially reduces the whether from hamsters (Herzog et al., 1998), rats (Welsh et magnitude of light-induced phase shifts, while similar appli- al., 1995) or mice (Liu and Reppert, 2000; Liu et al., 1997a). cation of the GABAA antagonist, bicuculline, substantially The ability to evaluate the simultaneous rhythmic behav- increases phase shift magnitude (Gillespie et al., 1996). These ior of multiple individual cells has provided a new perspec- results are inconsistent with prior data obtained using tive on the oscillatory organization of the SCN. In mouse SCN systemic drug injections (Ralph and Menaker, 1987, 1989). with a green fluorescent protein (GFP) reporter of Per1 gene Nevertheless, the effects of intracranial (i.e., SCN) drug activity (Kuhlman et al., 2000), most neurons oscillate in application appear to be repeatable and consistent, although synchronized fashion, but with numerous distinct phases the effect of bicuculline may depend on circadian time (Quintero et al., 2003). An estimated 11% (LD) and 26% (DD) of (Gamble et al., 2003; Mintz et al., 2002; Gillespie et al., 1997). the cells evaluated were judged as nonrhythmic. Unexpect- Similarly, bicuculline augments and muscimol suppresses edly, cells tend to be grouped, having one of three preferred light-induced Fos protein in the hamster SCN (Gillespie et al., phases that differ if the animals have been entrained or free- 1999). running. In SCN obtained from mice in LD, 78% of neurons The GABAB receptor may also be involved in modulation of had peak GFP expression at ZT5, ZT8 or ZT11 (most cells light-induced phase shifts. Peri-SCN injections of a GABAB peaked at ZT8). With a history of DD, most cells peak at CT12 receptor agonist, baclofen, block light-induced phase delays and 92% had peaks in the CT12, CT15 or CT18 phase groups. and advances, but the antagonist, CGP-35348, has no effect The half-maximum phase plot indicates CT12 for the DD- (Gillespie et al., 1997). Baclofen also blocks glutamate-induced housed animals and ZT8 for the LD housed animals. Several phase delays and this action is not affected by TTX (Mintz et valuable observations are evident in this study (Quintero et al., 2002). The GABAB agonist also effectively blocks light- al., 2003): (1) not all SCN neurons are rhythmic; (2) different induced Fos at CT13.5 and CT19 (Gillespie et al., 1999), rhythmic neurons have different phases; (3) there are three although an antagonist has no effect. The effect of GABAB preferred phases of rhythmic SCN neurons, a characteristic receptor activation is the result of an inhibition of glutamate previously observed using multiunit in vivo electrophysio- release from the RHT terminals (Jiang et al., 1995). logical methods (Meijer et al., 1997); (4) the preferred phase of The GABAA agonist, muscimol, is also able to induce phase rhythmic neurons varies according to photoperiod history. advances when administered to DD-housed hamsters during The results support the conclusion that, assuming clock the middle subjective day (Novak et al., 2004; Smith et al., 1989) genes actually determine the pacemaker properties of SCN and this effect is inhibited by a concomitant light pulse (Mintz neurons, other factors control the phase relationship be- et al., 2002). tween the locomotor rhythm and circadian clock phase. This is demonstrated by the fact that the preferred phase of gene 6.1.2. VIP and VIP/PACAP receptors expression is about 4 h earlier in LD housed mice compared VIP neurons are clustered in the ventral SCN of most to DD housed mice when assessed relative to activity onset mammalian species and use VIP as a neuromodulator to (Quintero et al., 2003). communicate with other neurons in the SCN through activity An exception to the above evidence concerning the timing at the PAC1 or VPAC2 receptor, both of which are found in the of oscillations in SCN neuronal activity has been observed in SCN (Cagampang et al., 1998a,b). A complicating factor in the the hamster. Whereas there is a neuronal activity rhythm analysis of receptor function in the context of the circadian peak recorded approximately once every 24 h if the SCN tissue system is the fact that both receptors bind VIP and PACAP with is cut in the coronal plane, there are two distinct peaks if the approximately equal affinity. PACAP-containing terminals are SCN is cut in the horizontal plane (Jagota et al., 2000). The found in projections from the retina, as well as from other, phases of the two peaks are modulated by photoperiod history unidentified brain regions (Hannibal and Fahrenkrug, 2004; (Mrugala et al., 2000). This phenomenon appears to apply only Hannibal et al., 1997). VIP peptide has been implicated in the to hamsters, and has not been seen in mouse or rat (Burgoon regulation of circadian rhythm phase and period, although et al., 2004). These results support the view that there are conflicting observations have been reported (Gillespie et al., functionally distinct regions within the SCN; different regions 1996; Piggins et al., 1995b; Albers et al., 1991). More recent work oscillate differently; and the “standard” perspective (here, the has focused on function of the PAC1 and VPAC2 receptors with coronal view) is not always the fully correct and adequate respect to circadian rhythm regulation and the results have perspective. been quite provocative. Calcium imaging methods suggest that the effects of 6.1. Function of cellular neuromodulators in the SCN PACAP on SCN neurons are elicited through the PAC1 receptor because of the absence of response to equimolar VIP 6.1.1. GABA application (Kopp et al., 1999). The PAC1 knockout mouse As indicated above, GABA is the predominant neurotransmit- tends to have a shorter circadian period in DD than wild-type ter in cells of the rat, mouse and hamster SCN. It is, therefore, and light-induced phase shifts are less after stimulation at not a surprise that manipulation of GABA neurotransmission certain circadian times compared to the wild type (Hannibal et in the SCN alters circadian rhythm function. One potential al., 2001b). Fos protein and Per1/2 gene induction were also BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 19 much less in the knockout animals following a light pulse potentials that are enhanced by VIP. These cells are during the early subjective night, although they were not distributed throughout the SCN with no discernible region- different during the late subjective night. related pattern (Itri and Colwell, 2003). VIP infused into the The effects of PAC1 receptor knockout are small compared hamster peri-SCN region or onto the rat SCN slice during the to the consequences of losing a functional VPAC2 receptor. early subjective night induces phase delays, while infusions The latter appears to be an essential component of the during the late subjective night yield phase advances, with a circadian rhythm system, at least in mice. Its absence in the PRC similar to that for light (Piggins et al., 1995b; Albers et Vipr2 KO mouse is associated with nearly complete loss of the al., 1991; Reed et al., 2002) (although there is controversy circadian locomotor rhythm assessed in constant dim red concerning whether or not PHI and GRP are necessary for light (Cutler et al., 2003). Similarly, rhythms in SCN gene obtaining phase shifts). In mice lacking genes coding for PHI expression (Per1, Per2, Cry1, VP were lost, although a vastly and VIP, a variety of rhythm-related measures were altered. attenuated, but statistically significant, rhythm in BMAL1 In particular, the circadian period was shorter by about an expression was evident (Harmar et al., 2002; Cutler et al., 2002). hour, phase response to light was greatly reduced or In the Vipr2 KO mice, there was also very little light-induced eliminated, there was more variability in activity onset FOS protein compared to what is normal in the wild type. In under LD and cohesive rhythms were generally not main- the SCN slice preparation, the ensemble circadian rhythm in tained under DD (Colwell et al., 2003). Mice lacking only the neuronal firing rate is lost in the Vipr2 KO mouse (Cutler et al., gene coding for VIP had similar characteristics. Most notably, 2003). An additional feature of this study was the demonstra- about two thirds of the animals had running rhythms in DD tion that a specific VPAC2 receptor antagonist had the same that expressed multiple noncircadian components. Impor- effect on the ensemble neuronal firing rhythm of wild-type tantly, the behavior of VIP KO mice was strikingly similar to animals as the gene defect does on the Vipr2 KO animal. In mice lacking the gene (Vipr2) coding for the VPAC2 receptor contrast to the gene knockout model, a transgenic mouse (Aton et al., 2005). The importance of VIP to normal overexpressing the VPAC2 receptor has a much longer rhythmicity in the VIP KO mice has been established by circadian period and appears to be more sensitive to light as reinstating SCN rhythmicity and synchrony among SCN indicated by faster re-entrainment to an advance of the light– neurons by periodic application of VIP agonist to a tissue dark photoperiod (Shen et al., 2000). culture preparation, but this has no effect on VPAC2 KO mice At the level of individual SCN cells, there is also an absence (Aton et al., 2005). of rhythmic gene expression in Vipr2 KO mice (Harmar et al., 2002). An explanation for the function of the VPAC2 receptor 6.1.3. Calbindin and the central subnucleus of the hamster within the context of circadian clock structure and intercel- SCN lular pacemaker regulation has not been determined, al- There has been emphasis on the central subnucleus of the though the suggestion has been made that rhythmic VIP SCN and the role its constituent CALB cells may play in serves to maintain coupling between oscillating neurons in circadian rhythm regulation. A subset of SCN neurons the SCN (Harmar et al., 2002). This explanation does not expresses the Ca2+ binding protein CALB (Arvanitogiannis et account for the fact that about 15% of the congenitally al., 2000; LeSauter and Silver, 1999). CALB contains six high- anophthalmic ZRDCT-An mouse strain are arrhythmic despite affinity Ca2+ binding domains and contributes to intracellular the presence of VIP neurons in the SCN (Laemle and Ca2+ signaling and buffering (Leathers et al., 1990; Roberts, Ottenweller, 2001). Thus far, there is a demonstrable require- 1994). As described above, the CALB neurons are clustered ment for the presence of the VPAC2 receptor in order for mice within the caudocentral SCN. This region contains about 250 to display circadian rhythmicity, but no demonstration that CALB neurons and is approximately 300 μm long, 180 μm high VIP itself is explicitly necessary or greatly involved in rhythm and 130 μm wide (Silver et al., 1996a). generation or regulation. Partial lesions of the SCN that completely destroyed the Nearly half the mouse SCN neurons immunoreactive for VP CALB region yielded animals with arrhythmic locomotor also have the VPAC2 receptor as do about 31% of the VIP and activity. In contrast, if the lesions spared a portion of one or CalR neurons (Kallo et al., 2004). In general, there is a high both CALB regions, behavioral rhythmicity was maintained degree of regional correspondence between the distribution of (LeSauter et al., 1996). The circadian locomotor rhythm is mouse neurophysin (marker for VP) and for PHI-containing restored after implantation of fetal SCN tissue into the third (marker for VIP) neurons with the distribution of cells ventricle of previously SCN-lesioned adult hamsters (Ralph et containing VPAC2 receptor (King et al., 2003), although the al., 1990). Restoration of rhythmicity requires the presence of degree of colocalization was not established in this paper. The CALB neurons within the transplanted grafts and the strength regional correspondence is not perfect as a central zone in the of the restored locomotor rhythm is correlated with the mouse SCN is devoid of VIP or VP neurons, but contains a few number of surviving CALB neurons (LeSauter and Silver, cells with VPAC2 receptors. 1999). Small lesions of the SCN that damage the CALB region The specific importance of VIP for circadian rhythms has bilaterally eliminate a number of behavioral and physiological been emphasized in mice (Colwell et al., 2003; Aton et al., rhythms. If the CALB region on either side was partially or 2005). VIP neurons project widely from the SCN to various completely spared, the rhythms remained intact (Kriegsfeld et targets (Kalsbeek et al., 1993), but also appear to have intra- al., 2004). These data suggest that the region of the SCN SCN connections (LeSauter et al., 2002; Daikoku et al., 1992; containing these CALB positive neurons is important in the van den Pol and Gorcs, 1986). Two thirds of cells in the generation of circadian rhythms. An important question to be mouse SCN have spontaneous inhibitory postsynaptic answered is whether it is the CALB neurons (or other neurons 20 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 in this region) or whether it is the network connections significantly more CALB expression in young animals that between the cells in this region that are critical for the gradually decrease as the animals approach adulthood (Ikeda generation of circadian rhythms. et al., 2003). The central subnucleus receives dense input from the CALB expression is inversely correlated with the duration retina via the retinohypothalamic tract and the CALB neurons of the light cycle (Menet et al., 2003). Hamsters maintained on are directly contacted by retinal projections (Muscat et al., short duration photoperiods had significantly more CALB 2003; Bryant et al., 2000). A light pulse during the subjective expression than those maintained on long photoperiods night increased FOS expression in the majority of CALB (Menet et al., 2003). Hamsters reared in DD or short photope- neurons of the hamster SCN. These data led to the develop- riod conditions had more CALB than controls (Tournier et al., ment of a model in which the CALB neurons serve as a “gate” 2003). Similarly, rats maintained in LL conditions had fewer for photic input to the SCN (Antle et al., 2003a). This model CALB-containing neurons then in rats maintained in DD must be reconciled with anatomical data demonstrating that (Arvanitogiannis et al., 2000). Neonatal monosodium gluta- the RHT projects to all regions of the hamster SCN and the fact mate (MSG) exposure increased the number of CALB-expres- that the densest retinal terminal field extends well beyond the sing SCN neurons and prevented the disruption of circadian CALB subnucleus (Muscat et al., 2003). In contrast to results in rhythms produced by LL in rats (Arvanitogiannis et al., 2000). the hamster, CALB and FOS colocalization are rare in the grass The parallel effects of MSG on CALB and LL changes on rat (Mahoney et al., 2000). Destruction of CALB neurons in the rhythms suggest that CALB may be important in the regula- rat SCN does not alter light-induced FOS expression, free- tion of response to LL. running rhythms or photic entrainment in that species CALB appears to play a key role in the photic responses in (Beaulé and Amir, 2002). hamsters. CALB is present in the cell nucleus and cytoplasm Neurons in the hamster central subnucleus do not show a of central subnucleus SCN neurons during the day, but is rhythmic expression of messenger RNAs for Per1 and Per2 absent from the cell nucleus during the night (Hamada et al., (Hamada et al., 2001, 2004a). However, light can induce the 2003). Reduction of CALB expression by antisense oligonu- expression of both these clock genes in the central subnu- cleotides blocked light-induced phase shifts during the cleus. This has led to the conclusion that the central night, but light could produce shifts of both behavioral and subnucleus is a nonrhythmic region of the SCN, while the Per rhythms during the day (Hamada et al., 2003). Interest- dorsomedial region corresponding to the general location of ingly, these effects occurred despite the fact that the CALB VP neurons is rhythmic because the cells in this area express mRNA was only reduced 38% and the CALB protein only Per1 and Per2 in a rhythmic manner. However, it may not be reduced 28% (Hamada et al., 2003). Also interesting was the possible to determine the rhythmicity of individual neurons rapidity of the effect, which occurred only 4 h after the with in situ hybridization techniques if only a portion of the antisense injections. Therefore, while the CALB neurons do neurons in a region are rhythmic. Further, electrophysiolog- not have rhythmic clock gene expression or show rhythms ical evidence suggests that at least some neurons in the in action potentials, CALB expression in the nucleus may be central subnucleus fire action potentials in circadian manner. controlled in a circadian manner. Thus, these cells appear to The mean spontaneous firing rate (SFR) of non-CALB neurons be rhythmic according to at least one criterion, but not by in the central subnucleus has a peak at ZT6 and a nadir at ZT19 others. The mechanisms controlling this cellular regulation (Jobst and Allen, 2002). Whether the rhythmicity of these non- are unknown. They may involve rhythmic feedback from the CALB neurons is driven by endogenous clocks located in dorsomedial “rhythmic” SCN or may result from the neurons of the central subnucleus or whether it is due a interaction between rhythmic and nonrhythmic neurons in network phenomena of interconnections between rhythmic the central subnucleus. and nonrhythmic neurons is an important area for future The central subnucleus is heterogeneous and CALB cells research. In contrast, CALB neurons had a mean SFR of less constitute a minority of the neurons (LeSauter et al., 2002; than 1 Hz and showed no circadian pattern of action potential Jobst and Allen, 2002). A better understanding of mechanisms firing (Jobst and Allen, 2002). It is noteworthy, but of unknown underlying intercellular communication between non-CALB significance, that some CALB neurons contact a neighboring and CALB neurons and how they respond to synaptic input CALB or non-CALB cell via gap junctions (Jobst et al., 2004). In will be important to understand how the SCN generates a the mouse, increased firing frequency is correlated with functional circadian output. Double-label methods have increased expression of Per1 (Kuhlman et al., 2003). This demonstrated that 94% of CALB neurons also contained suggests that CALB neurons are a functionally distinct GABA (Jobst et al., 2004). Of note, most of the cellular profiles subpopulation within the SCN that do not contain a rhythmic in the central subnucleus contain GABA, although many GABA circadian clock. The data raise the intriguing possibility that neurons in the central subnucleus did not contain CALB. In the interactions between rhythmic and nonrhythmic neurons addition to CALB and GABA colocalization in the central are required to generate circadian locomotor rhythms. There subnucleus, glutamate decarboxylase 67 (GAD67), one of two is also the untested possibility that the GRP, SP and CALR related isoforms of the synthetic enzyme for GABA, was neurons that occupy the central subnucleus along with the detected in CALB cell bodies. Immunoreactivity for the GABAA CALB cells are likewise nonrhythmic. receptor α2 and β2/3 subunits was prominent in the SCN (Jobst CALB expression is dynamic depending on the genotype, et al., 2004). Confocal microscopic analysis demonstrated that developmental age and the lighting conditions. Tau hamsters β2/3 and α2 did not colocalize with CALB. Labeling for the β2/3 have significantly more CALB expressing neurons than wild- subunits was diffusely distributed around CALB neurons, type hamsters (LeSauter et al., 1999). In mice, there is whereas labeling for the α2 subunit appeared as discrete BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 21 puncta. The data indicate that α2 and β2/3 subunits contribute response to a 15 min 30 lx light pulse at CT16, but unlike to the composition of GABAA receptors in the CALB region. A wild-type animals, the delays were not greater if the light was limitation of this study is that the GABAA receptor subunits 300 lx. Small (approximately 20%) decrements were also seen were not localized to either postsynaptic or presynaptic in Per2 (but not Per1) and Fos response to the 300 lx light (Aida regions. GAD65, an isoform of GAD exclusively located within et al., 2002). synaptic terminals (Erlander et al., 1991), is expressed in a In ovariectomized, estrogen-primed rats, pituitary prolac- punctate pattern throughout the SCN, including the central tin is released in a daily surge timed by the SCN (Mai and Pan, subnucleus (Jobst et al., 2004). 1993, 1995; Freeman et al., 2000). Properly timed bombesin infusion into the peri-SCN region blocks the prolactin surge 6.1.4. Bombesin and gastrin releasing peptide (Mai and Pan, 1995). The implication is that bombesin neurons Bombesin contains 14 amino acids, GRP has 27 and they share participate in the efferent control of prolactin release timing. a 7 amino acid sequence homology. There is a very small This raises the general issue of whether individual SCN cell literature concerning the effects of bombesin on SCN function. phenotypes are concerned with the control of single or Immunoreactive cells are abundant in the Djungarian ham- multiple efferent systems. ster (Reuss, 1991), although cross-reactivity with GRP is a potential problem (Panula et al., 1984). Bombesin increased the 6.1.5. Neuromedin U and neuromedin S firing rate of about 50–60% of golden hamster neurons studied, Neuromedin U (NMU) is a 24 amino acid peptide that shares a 7 while inhibiting the rate in half of the remainder (Piggins and amino acid sequence homology with the somewhat larger Rusak, 1993; Piggins et al., 1994). Neuromedin B, another in the neuromedin S (NMS) (Mori et al., 2005). NMU and its receptors, class of bombesin-like peptides, activated about 40% of SCN NMU-R1 and NMU-R2, are both present in the rat and mouse neurons and inhibited very few (Piggins et al., 1994). The SCN (Mori et al., 2005; Graham et al., 2003, 2005; Nakahara et responses of individual cells to application of several different al., 2004). NMU and NMU-R1 have high amplitude rhythmic bombesin-like peptides were similar in more than 80% of the gene expression that persists in DD, whereas rhythmic tests. Responses to the peptides were attenuated by pretreat- expression of the NMU-R2 gene is lower amplitude (Graham ment with antagonists to the GRP-preferring receptor which is et al., 2003; Nakahara et al., 2004). NMU is present in some, but present in the SCN (Ladenheim et al., 1992, 1993). More cells not all, VP neurons and some, but not all, NMU-containing were activated by GRP and neuromedin B at ZT12-16 than at neurons also have VP; it apparently does not colocalize in VIP ZT4-8 (Piggins et al., 1994). Bombesin has not been similarly neurons (Graham et al., 2003). The distribution of NMU in rat is tested. most similar to the distribution of VIP neurons, but is present GRP was originally reported to induce light-like phase throughout much of the SCN. NMU-R1 and R2 receptor shifts in hamster only if infused onto the SCN along with VIP distributions are similar to that for the peptide. Phase and PHI (Albers et al., 1991), but may be equivalently advances of about an hour followed ventricular injection of functional if applied alone (Piggins et al., 1995b). The action NMU at CT0 into rats; injections at CT6 yielded phase delays of potential rhythm in rat or hamster slice preparations about 0.6 h; and from CT8 to CT23, there was no phase exhibited large, light-like phase responses to GRP and response. FOS protein expression was widely induced by these effects could be blocked by bombesin 2 receptor intraventricular NMU (Graham et al., 2003). Mice lacking the antagonists. Bombesin 2 and bombesin 1 receptors were gene coding for NMU have rhythmic feeding behavior, but the identified in SCN, although there is no evidence that phase angle of entrainment may be altered (Hanada et al., bombesin 1 contributes to rhythm regulation (McArthur et 2004). al., 2000). GRP injected into the hamster third ventricle at CT NMS is distributed in the rat SCN in a pattern similar to VIP 13 induces sizable phase delays in association with induc- and there was rhythmic expression peaking at ZT11 (Mori et tion of Per1, Per2 and FOS (Antle et al., 2005). Interestingly, al., 2005). The rhythm was lost in DD and a light pulse acutely the gene expression occurs almost exclusively in the densely reduced expression. Intraventricular injection of NMS at CT23 retinorecipient region in the dorsal SCN, just above the induced 1 h phase delays and, at CT6, about 2 h phase central subnucleus identified by CALB cell presence (Muscat advances with a dead zone between CT9 and CT22. FOS et al., 2003). This is the same area in which pERK is expression in dorsal SCN cells was also induced by NMS. The expressed, dependent upon an intact RHT, during the PRC for NMS was similar to that for NPY (Albers and Ferris, subjective night (Lee et al., 2003). And, phosphorylation of 1984; Albers et al., 1984), whereas that for NMU appeared to be ERK is apparently necessary for phase response to GRP somewhat different. The general shape of the PRC for NMS because if phosphorylation is pharmacologically blocked, the was very much light-like, but was shifted such that phase phase shifts are also blocked (Antle et al., 2005). Although delays began at CT24 with a cross-over point at CT3 and the spontaneously rhythmic ERK phosphorylation does not phase advance region ended at approximately CT8-9 (Naka- occur in the absence of the RHT, the effect of enucleation hara et al., 2004). on phase response to GRP has not been examined. In the mouse, GRP infused into the peri-SCN region at 6.1.6. Vasopressin CT16 elicited small phase delays and induced Per and Fos The Brattleboro rat is deficient in VP, yet has circadian gene expression (Aida et al., 2002). GRP receptor was also rhythms (Ingram et al., 1998). It has been concluded that VP found throughout most of the SCN, although density was is not necessary for circadian rhythm expression. In fact, clearly greatest in the dorsocaudal part of the nucleus. Mice transplanted SCN from Brattleboro rats will restore normal lacking the GRP receptor had normal phase delays in circadian rhythmicity in arrhythmic hosts (Boer et al., 1999). 22 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

Although VP is not a necessity for rhythm expression in cholinergic input to the SCN is the demonstration that, Brattleboro rats, transplanted SCN from normal animals can following an intra-SCN injection of retrograde tracer, labeled restore rhythmicity in paraventricular hypothalamic neurons cells containing ChAT are found in several brain nuclei and this rhythmicity is likely to be the result of VP stimulation including the lateral dorsal and pedunculopontine tegmen- (Tousson and Meissl, 2004). The issue is rendered more tum, areas concerned with sleep regulation (Bina et al., 1993). complicated by the demonstration that rhythmicity of VP Retrogradely labeled cholinergic cells were also found in the cells evaluated in vitro shows advances and delays according basal forebrain. Excitotoxic lesions of the nucleus basalis to circadian time of VIP stimulation. Interestingly, the same magnocellularis destroyed about 75% of cholinergic cells in study failed to observe a phase shifting effect of glutamate this location yielding a decrease in cholinergic processes in which would be expected to act either directly on the VP cells the SCN (Madeira et al., 2004). Bilateral nucleus basalis lesions or on the VIP cells projecting to the VP cells (Watanabe et al., also diminished the numbers of countable VP and VIP cells in 2000). the SCN by about 30% and reduced volume and mRNA levels of VP and VIP cells by about 34%. 6.1.7. Diurnality–nocturnality The immunotoxin, 192 IgG-saporin, destroys cells bearing In the grass rat, a diurnal rodent, the above effect of GABA the low affinity nerve growth factor receptor, p75NTR, which agonists administered during the subjective day may occur at are generally cholinergic (Leanza et al., 1995). Intraventricu- phases opposite to those effective in the nocturnal hamster lar infusion of the immunotoxin abolished p75NTR in the rat (Novak and Albers, 2004a). In particular, muscimol injected in SCN without affecting VIP neurons (Moga, 1998). However, the peri-SCN region at this time induces phase delays rather intrahypothalamic immunotoxin injection into the rat great- than phase advances and these effects are not blocked by TTX. ly reduced numbers of CALB neurons (Beaulé and Amir, Opposite to the effect in nocturnal species, phase shifts 2002). The circadian period length was related to the extent caused by peri-SCN muscimol are not inhibited by simulta- of cell loss. Typically, the period was much shorter in neous light exposure (Novak and Albers, 2004b). However, lesioned rats and about a third of them became arrhythmic. nocturnal and diurnal species appear to respond similarly Animals sustaining immunotoxin lesions of cholinergic cells with respect to the ability of GABAA and GABAB agonists to also had about a 50% reduction in light-induced FOS cell inhibit phase response to light (Novak and Albers, 2004b; counts. In another study (Erhardt et al., 2004), 192 IgG- Novak et al., 2004). Assuming that the action of injected GABA saporin was injected into the ventricle or directly into the agonists is occurring within the SCN, then the antiphase GABA SCN, but neither procedure yielded deficits in CALB or VIP activity in diurnal vs. nocturnal animals is the first indication neurons and changes in circadian period or general rhyth- that the SCN contributes to this species-specific behavioral micity were not observed. Nevertheless, the immunotoxin trait. generally reduced the ability of light to produce phase delays In a subparaventricular region contiguous to the dorsal or advances. The rhythm-related effect of 192 IgG-saporin SCN, the lower subparaventricular zone, the grass rat has treatment may be related to disruption of effective retinal higher expression of FOS during the night than during the projections to the SCN. Unilateral enucleation reduces day, an effect not observed in rats (Smale et al., 2001; optical density of p75NTR in the contralateral SCN by about Schwartz et al., 2004; Nunez et al., 1999). Moreover, the day– 50% (Bina et al., 1997). night pattern of expression is roughly opposite that which As reviewed (Morin, 1994; O'Hara et al., 1998), the cholin- occurs in the SCN of either the Norway lab rat or grass rat. ergic agonist, carbachol, has generally yielded light-like phase The suggestion has been made that the neuron firing rate responses. However, the topic is not without controversy. For rhythm can become inverted in the peri-SCN region for example, peri-SCN carbachol induced robust phase delays nocturnal (Inouye and Kawamura, 1979), but not for diurnal when injected at CT14 and advances at CT22, but also (Sato and Kawamura, 1984), species and may contribute to produced large phase advances in about half the animals the manifestation of nocturnal or diurnal activity traits receiving the drug at CT6 (Bina and Rusak, 1996). The phase (Smale et al., 2003). shifts during the subjective night are consistent with a light- like action, but the shifts during the day are not. The responses 6.2. Acetylcholine were blocked by pretreatment with atropine, but not by mecamylamine, suggesting that a muscarinic receptor med- Cholinergic innervation of the SCN has been of interest for iates the cholinergic effects. The functional presence of many years and there are numerous reports of cholinergic muscarinic receptors in the SCN region is supported by receptors on SCN cells and modification of circadian rhythm ample additional data, particularly from SCN slice preparation function by cholinergic agonists or antagonists (see Morin, studies (Liu and Gillette, 1996; Liu et al., 1997b; Artinian et al., 1994 for a review). Several anatomical reports have described 2001) and there is evidence supporting a rhythm function choline acetyltransferase (ChAT) immunoreactive processes specifically for the M1 receptor (Gillette et al., 2001). in the SCN, although the histology has not been robust and the Despite the foregoing, the nicotinic receptor may also demonstrable fibers have been located in somewhat different mediate the effects of acetylcholine on circadian rhythmicity. places, depending upon the particular study (van den Pol and The nicotinic antagonist, mecamylamine, blocked the phase Tsujimoto, 1985; Bina et al., 1993; Ichikawa and Hirata, 1986; shifting effects of light at CT19 and greatly attenuated light- Kiss and Halasz, 1996). Cholinergic fibers have also been induced FOS in the dorsomedial hamster SCN with little shown to make contacts directly on SCN neuron (Kiss and inhibition more ventrally (Zhang et al., 1993). In the rat slice Halasz, 1996). Perhaps the most convincing evidence of preparation, the PRC to nicotine was phase-independent with BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 23 advances occurring throughout the circadian day and the potential firing. The presence of a diffusible messenger is phase shifts could be blocked by mecamylamine (Trachsel et supported by the observation that spontaneous firing of SCN al., 1995). In vivo, nicotine administered subcutaneously at neurons “washes out” during the dialysis of the intracellular CT16 elicited significant phase delays and induced FOS in the milieu that occurs during whole-cell recording (Schaap et al., SCN and mecamylamine greatly attenuated FOS expression 1999). Currently, the signaling pathways connecting the (Ferguson et al., 1999). A preliminary description of rat molecular clock in the nucleus to changes in membrane response to nicotine indicated that phase shifts were unreli- potential are unknown. However, significant progress has able following systemic administration, but were light-like been made toward describing the ion channels responsible for following nicotine intraventricular infusion (O'Hara et al., the regulation of spontaneous firing. 1998). Many membrane properties of SCN neurons show a diurnal There are also reports that a strain of rats bred for or circadian variation. SCN neurons have input resistances in cholinergic supersensitivity has a shorter circadian period the range of 1–3GΩ when measured with whole cell recording than controls, an altered phase angle of entrainment and techniques. The input resistance of SCN neurons is higher reduced FOS induction in SCN neurons (Shiromani et al., 1988; during the day compared to the night in slices maintained in Ferguson and Kennaway, 1999). either LD or DD (De Jeu et al., 1998; Kuhlman and McMahon, 2004). The resting membrane potential of SCN neurons has 6.3. Neurophysiology of the SCN action potential rhythm been reported to be more depolarized during the day than during the night (De Jeu et al., 1998; Kuhlman and McMahon, Action potential firing by SCN neurons is regulated as a 2004; Pennartz et al., 2002). These differences in membrane circadian rhythm. Higher frequencies are observed during the potential are maintained in rats maintained in DD consistent subjective day than during the subjective night with the mean with their regulation by the circadian clock (Kuhlman and population firing frequency peaking near CT6 (Gillette, 1991). McMahon, 2004). However, a significant day–night difference During the subjective day, action potential firing frequencies in the membrane potential has not always been observed range from less than 1.0 Hz to 14 Hz, compared to the (Schaap et al., 1999; Kim and Dudek, 1993; Teshima et al., subjective night when action potentials fire with frequencies 2003). Difficulties in estimating the “resting” membrane of 0.5 to 5.0 Hz. The presence of fast firing neurons during the potential of spontaneously firing neurons and dialysis of day is responsible for the increased mean population firing intracellular contents may contribute to these different frequency observed in SCN slices (Gillette, 1991). This circadi- results. The resting membrane potential has been proposed an rhythm of action potential firing is a property of individual to be set, in part, by a Ba2+-sensitive, fast-activating, outwardly neurons because the firing difference is maintained when SCN rectifying K+n current (De Jeu et al., 2002). Interestingly, this neurons are grown in dispersed cell cultures (Welsh et al., current did not show a circadian rhythm in activity, although 1995; Herzog et al., 1997; Abe et al., 2000; Honma et al., 1998a; given the high input resistance of SCN neurons, a small Shirakawa et al., 2000). change in this current could significantly change the resting A current hypothesis proposes that the molecular circadian membrane potential. The membrane potential of some SCN clock sends signals to regulate the activity of ion channels neurons has a low frequency (2–7 Hz) oscillation that is responsible for setting the membrane potential and action mediated by L-type Ca2+ channels and is observed when action potential firing rate. This hypothesis predicts that disruption potentials are blocked by TTX (Jiang et al., 1997; De Jeu et al., of clock gene function would alter the action potential firing of 1998; Pennartz et al., 2002; Jackson et al., 2004). SCN neurons. Mutations of clock genes alter the period of Action potentials in SCN neurons are characterized by a action potential firing rhythms. Microelectrode array record- rapid upstroke of membrane depolarization (∼+25 mV) fol- ings of slice cultures from Clock homozygous mice demon- lowed by membrane potential repolarization. An afterhyper- strated neurons with a period near 27 h that was significantly polarization (AHP; ∼−80 mV), a membrane potential that longer than neurons of wild-type or Clock heterozygous mice hyperpolarizes below the resting membrane potential and (period near 24 h) (Nakamura et al., 2002). In a similar study, the which is dependent on extracellular Ca2+, follows the action period of Clock heterozygous mice was longer than that of the potential upstroke (Cloues and Sather, 2003; Thomson, 1984; wild type mice (24.5 vs. 23.3 h), while the Clock homozygous Thomson and West, 1990). The rapid upstroke of the action mice were arrhythmic (Herzog et al., 1998). In the tau mutant potential is driven by current flowing through voltage-gated hamster, which has a shortened circadian period, the circadian Na+ channels and is completely blocked by TTX (Thomson and rhythm of action potential firing is also shortened to approx- West, 1990; Huang, 1993; Wheal and Thomson, 1984). imately 20 h (Liu et al., 1997a). SCN neurons from mice with During the interval between individual action potentials mutations of the cryptochrome 1 or cryptochrome 2 genes are (interspike interval), the membrane potential shows a steady arrhythmic (Albus et al., 2002). Also consistent with this model depolarization that reaches spike threshold (Kononenko et al., are the observations of McMahon and colleagues who used a 2004; Pennartz et al., 1997). This interspike interval can be transgenic mouse expressing a green fluorescent protein modulated by synaptic input, particularly inhibitory postsyn- driven by the Per1 promoter to demonstrate that the frequency aptic potentials generated by GABAergic input (Pennartz et al., of action potential firing was linearly correlated with Per1 1998; Kononenko and Dudek, 2004). These “depolarizing transcription (Quintero et al., 2003). ramps” are sensitive to the Na+ channel blocker, TTX, and to SCN neurons express a wide repertoire of ion channels and riluzole, a compound used to treat amyotrophic lateral regulation of the activity of these ion channels by signals from sclerosis, that blocks a persistent Na+ current. Voltage-gated the molecular clock would lead to regulation of action Na+ channels rapidly activate and inactivate during a 24 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

membrane depolarization. However, many neurons have a SCN neurons. Block of iberiotoxin-sensitive BKCa did not alter + small Na current that shows very slow or no inactivation (for firing frequency. An apamin- and iberiotoxin-insensitive KCa review see Crill, 1996). This “slowly-inactivating” or “persis- current was diurnally modulated and contributes to the spike ” + tent Na current (INa,S or INa,P) is TTX-sensitive and may frequency (Cloues and Sather, 2003). The AHP is dependent on represent current flowing through a noninactivating class of both small conductance (SKCa) and large conductance (BKCa) voltage-gated Na+ channels or a modal variation of the Ca2+-activated-K+ channels. normally transient Na+ current. In SCN neurons, a slowly Abundant experimental evidence demonstrates that Ca2+ inactivating Na+ current (in I =50–250 ms at − 45 mV) is blocked plays an important role in the activity of SCN neurons. First, 2+ by TTX, and riluzole similar to the INa,P. The INa,S may represent removal of extracellular Ca abolishes the circadian pattern of a third modal state of voltage-gated Na+ channels (Jackson et action potential firing (Shibata et al., 1984). Second, some SCN al., 2004). neurons have a regular pattern of action potential firing

It has been hypothesized that the INa,S is primarily characterized by small deviations in the interspike interval responsible for driving the membrane depolarization during (Thomson, 1984). Reduction of the extracellular Ca2+ concen- the interspike interval (Kononenko and Dudek, 2004; De Jeu tration disrupts this regular firing pattern, whereas an increase and Pennartz, 2002). Jackson et al. (2004) recorded from acutely potentiates the action potential spike amplitude and the rate of isolated dorsomedial SCN neurons and used action potential membrane repolarization (Thomson and West, 1990). Removal waveforms as voltage signals under voltage clamp to deter- of Ca2+ or block of Ca2+ channels with high concentrations of mine the currents flowing during the interspike interval. The extracellular Mg2+ reduces the spike amplitude, rate of ionic currents flowing during the action potential waveform membrane potential repolarization and the amplitude of the are the same as those that flow during the action potential AHP (Thomson and West, 1990). Third, there is a circadian firing. The majority of the current during the interspike rhythm of intracellular Ca2+ levels with higher levels during interval was carried by TTX-sensitive Na+ current. A nimodi- the subjective day than during the subjective night (Ikeda et al., pine-sensitive Ca2+ current was also present during the during 2003; Colwell, 2000). The Ca2+ rhythm is not blocked by TTX the interspike interval. However, the amplitude and charge although action potentials are completely eliminated. Ryano- carried by the Ca2+ current are only 26% of that carried by the dine, which depletes intracellular Ca2+ stores, significantly Na+ current (Jackson et al., 2004). Further, the Ca2+ current blocked both the intracellular Ca2+ rhythm and disrupted the turns on later in the interspike interval than the Na+ current action potential firing (Ikeda et al., 2003). and is not significant until the membrane potential GABA is the most prominent neurotransmitter in the SCN approaches threshold. The authors conclude that the slowly (Moore and Speh, 1993; Morin and Blanchard, 2001) and there inactivating Na+ current is primarily responsible for setting the are abundant inhibitory postsynaptic potentials that can be action potential frequency while contribution of voltage-gated recorded in SCN neurons (Jiang et al., 1997; Kim and Dudek, 2+ Ca currents to the action potential firing frequency is small 1992). Activation of GABAA receptors is traditionally thought to (Jackson et al., 2004). produce cellular inhibition due to activation of a chloride Other candidate ion currents have been proposed to conductance and membrane hyperpolarization or the shunt- contribute to setting the action potential frequency (Kim and ing of excitatory currents. However, in the SCN, descriptions of

Dudek, 1993; Pennartz et al., 1997). The H-current (IH), a the actions of GABA on the SCN neuronal activity have been nonselective cation channel activated by membrane depolar- complicated and controversial. The majority of recordings had ization has been hypothesized as contributing a depolarizing shown GABA to be an inhibitory neurotransmitter during the current, but a direct test of this hypothesis did not yield any day (Bos and Mirmiran, 1993; Gribkoff et al., 1999; Liou and evidence for an IH contribution (Akasu et al., 1993; De Jeu and Albers, 1991; Liou et al., 1990). However, other investigators

Pennartz, 1997). The A-current (IA), a fast-activating, rapidly described the activation of GABAA receptors to be primarily inactivating K+ current present in all SCN neurons, may excitatory, producing an increase in firing activity or mem- contribute to setting the duration of the interspike interval brane depolarization during the day reducing action potential (Huang, 1993; Bouskila and Dudek, 1995). The low-threshold T- firing and producing a membrane hyperpolarization during type Ca2+ current has also been proposed as a contributor to the the night (Wagner et al., 1997, 2001). This diurnal rhythmicity setting of action potential frequency (Akasu et al., 1993). No was proposed to be caused by a daily change in the direct evidence for the IH or IT has been presented. intracellular chloride concentration (Wagner et al., 1997, The AHP that follows membrane potential repolarization 2001). A high daytime intracellular chloride concentration also contributes to setting action potential firing frequency. was proposed to account for the observations. An excitatory The AHP duration correlates with the spontaneous firing rate, GABA response would be observed when the equilibrium − the slower the AHP decay, the slower the firing rate (Cloues and potential of Cl (ECl)islessnegativethantheresting Sather, 2003). If the AHP is blocked, then the rate of firing of SCN membrane potential. Contrary to those results, a different neurons increases. In SCN neurons, the AHP is dependent on group of investigators reported that GABAA receptor activation Ca2+ entering the cell via predominately L- and R-type voltage- produces only inhibitory effects during the day and excitatory gated Ca2+ channels that activate both large-conductance effects in a subpopulation of SCN neurons during the night (De 2+ + (BKCa) and small-conductance (SKCa)Ca -activated-K chan- Jeu and Pennartz, 2002). The reversal potential during the nels. Blockade of apamin-sensitive SKCa channels (only SK2 night was more depolarized than during the day consistent subunits in SCN) has been reported to produce an increase or with an increased intracellular chloride concentration. The no effect on spontaneous action potential frequency. The specific reasons for these dramatically different observations difference probably reflects a diversity of SKCa expression in remain unknown. Technical issues such as style of whole cell BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 25 recording, duration of GABA application and the GABA 6.5. Regional expression of Fos concentration may all contribute to the different observations. 6.5.1. Hamster 6.4. Inter-SCN differences in gene expression An early observation (Rea, 1992) was that light did not homogeneously induce FOS expression throughout the ham- One of the more intriguing developments during the last 10 ster SCN. Rather, the protein was heavily induced in the years has been the observation that the two SCN can ventral half of the nucleus both at CT13 and CT18, but there oscillate out of phase (De la Iglesia et al., 2000). In male was also large induction of FOS in the dorsal half at CT18. This hamsters with split rhythms caused by LL exposure, the study lacked no-light controls, but the result has been putative circadian clock gene, Per1, is maximally expressed corroborated (Chambille et al., 1993; Guido et al., 1999). FOS in one SCN while another clock gene, BMAL1, is simulta- induction by light is similar in the dorsal and middle thirds of neously expressed maximally in the other SCN (normally, the hamster SCN at both ZT15 and ZT22 (no response and Per1 and BMAL1 are expressed about 180° out of phase and large response, respectively) and in the ventral third (very maximal expression cannot be seen simultaneously; Honma large responses at both times) (Guido et al., 1999). Also, the FOS et al., 1998b; Shearman et al., 2000). High Per1 expression response to light is greater in the ventral third of the SCN than normally occurs during the middle subjective day, a in the other two thirds at both ZT15 and ZT22. Much of the characteristic that is also true in SCN from hamsters with ventral third of the SCN, in which dense, light-induced FOS split rhythms. The genes coding for VP and FOS proteins are occurs, corresponds to the central subnucleus that can be also expressed rhythmically in normal animals at the same identified by the presence of CALB neurons and dense retinal phase as Per1. In the animals with split rhythms, activity of innervation. One point of the Guido et al. (1999) results is that these genes remains temporally synchronized with that for FOS induction by light occurs throughout the SCN, not just in Per1. Despite the continuous presence of light, photically the central subnucleus. induced Per or FOS expression does not appear to occur The effects of GABA agonists on facilitation or inhibition of during the subjective night (De la Iglesia et al., 2000). This light-induced FOS are proportionally equivalent in rostral, observation is curious because it implies that a photic action middle and caudal thirds of the SCN (Gillespie et al., 1999). normally occurring in DD-housed animals in is being When FOS counts were made through an entire SCN section at blocked, at least in the case of animals with split rhythms. the midcaudal level, there was no response at all to a 1 min Apparently, LL-housed animals with unsplit rhythms have light exposure at ZT15, whereas the same stimulus at ZT18, 21 not been tested to determine if there is light-induced gene or 24 yielded a near maximal result. A longer exposure to light expression during the subjective night. It would also seem at ZT15 greatly increased the FOS response, but never to the possible, although perhaps unlikely, that the rhythmic gene level seen at the other times. A complementary analysis expression observed in SCN of animals with split rhythms is (Chambille et al., 1993) has additionally demonstrated that induced by a temporally gated action of light rather than while light at CT14 induces a small increase in FOS in the being an expression of intrinsic cellular rhythmicity. rostral 3/4 of the SCN, there is substantial FOS induction in this The observation that the two SCN may oscillate in area when light is administered at CT19. The above-cited antiphase has raised the possibility that each SCN may papers demonstrate a region by time interaction with respect to have specific control over the timing of individual physio- gene induction by light stimulation, with region being defined logical events. This has been examined by evaluating the by rostrocaudal or dorsoventral level. Further, the Guido et al. control of the gonadotropin, luteinizing hormone (LH), in (1999) work indicates that the sensitivity of SCN neurons to animals with split running rhythms. Ordinarily, estrogen- photic input varies according to time during the subjective treated, ovariectomized female hamsters have a single daily night. Implicit, but not studied, is the likelihood of region by LH peak timed by a circadian clock (Alleva et al., 1971; time interaction with respect to sensitivity to light. The Bittman and Goldman, 1979), but in animals with split observations of region by time interaction with respect to rhythms, there are two such LH peaks in antiphase (Swann light-induced FOS expression have not had an impact on more and Turek, 1985). The circadian clock action occurs through recent developments focused on photic regulation of putative regulation of pituitary LH release by septal-preoptic neurons clock genes. synthesizing gonadotropin releasing hormone (GNRH) (De la Iglesia et al., 1995). Activation of GNRH neurons, as indicated 6.5.2. Rat by FOS expression, is maximal at the time of LH release Fos gene expression spontaneously oscillates in the SCN with (Urbanski et al., 1991; Doan and Urbanski, 1994). When most of the amplitude accounted for by the expression in the estrogen-treated, ovariectomized hamsters have split dorsomedial region (Schwartz et al., 1994; Koibuchi et al., rhythms in LL, there is daily FOS expression in GNRH cells 1992). However, a low amplitude oscillation is present in the predominantly (about 5:1) localized to the side of the ventrolateral region (Sumova et al., 1998). Regional FOS forebrain in which SCN FOS expression is also dominant oscillation persists in vitro (Prosser et al., 1994; Geusz et al., (about 5.5:1) (De la Iglesia et al., 2003). The implications of 1997). Multiple Fos-family genes are light-induced in the this form of lateralized circadian organization for regulatory ventrolateral region of the rat SCN. These include c-fos, fra-2 physiology are far-reaching and there are preliminary data and fosB. It has been suggested that each gene is a candidate supporting the view that each SCN controls the ipsilateral mediator of light effects on SCN neurons and regulation of expression of rhythmicity in peripheral organs (Yamazaki et circadian rhythm phase that may enable reasonably normal al., 2000). rhythm function in the absence of normal gene activity 26 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

(Schwartz et al., 2000; Honrado et al., 1996). A double label In the organotypic slice preparation, luminescence rhythm study has shown that only a small percentage of VP cells peaks of Per1 expression in cells of the dorsal mouse SCN have contain spontaneously expressed FOS and that a similarly a broad distribution of phases, but most are maximal in small percentage of cells expressing FOS also contain VP advance of those in middle and ventral thirds of the nucleus (Sumova et al., 2000). The daily profile of spontaneously (Quintero et al., 2003). The cells rhythmically expressing Per1 in expressed FOS is altered according to the length of the the dorsal third are unable to collectively sustain a cohesive entraining photoperiod, a phenomenon with implications for rhythm, unlike those more ventrally located. It is noteworthy, photoperiodism (see Sumova et al., 2004 for a review). however, that each dorsal cell continues a seemingly normal Analysis of light-induced FOS expression in various SCN oscillation uncoupled from the others in the same dorsal regions has been rendered more complex by the observation region. This suggests that the more ventral oscillatory cells are that light can inhibit it (Beaulé et al., 2001b). In the rat, light imparting a signal to those in the dorsal SCN, thereby induces large increases in FOS expression in neurons controlling phase of rhythmic cells in that region. In addition, throughout the ventral SCN. As indicated above, the dorsome- the investigators note that results from the horizontal slice dial region does not show light-induced gene expression, but preparation indicate the dorsal neurons are not necessary to does have constitutive FOS that varies according to circadian maintain either rhythmicity of the ventral cells or their time. A particularly novel result in this study is the demon- oscillatory synchrony (Yamaguchi et al., 2003). The presence stration of an actual drop in the number of cells expressing of preferred phases by regionally specific collections of FOS in an apparently specialized sector of the dorsomedial oscillatory neurons (Quintero et al., 2003; Yamaguchi et al., SCN (Fig. 3C1, 2) following light administered at CT2 or CT14, 2003) is consistent with the view described for hamsters or in entrained animals, at ZT1 or ZT2 (Beaulé et al., 2001b). (Hamada et al., 2004a)(discussedbelow),althoughthe interpretation may be substantially different. On the one 6.6. Regional clock gene expression hand, discussion of mouse SCN tends to emphasize the presence of many oscillatory neurons collectively synchro- 6.6.1. Mouse nized, but with different phases of peak Per1 gene expression. The advent of digoxigenin procedures for in situ hybridiza- On the other, the hamster work describes the “slow spread of tion has permitted analysis of light-induced clock genes at gene expression” from one area to another. The appearance of the cellular level. As indicated by these procedures, light “spread” may be the consequence of what has been described applied at CT16 induced Per1 mRNA in the ventral, but not in mice (Yamaguchi et al., 2003), namely, the presence of the dorsomedial, mouse SCN (Shigeyoshi et al., 1997). In oscillatory neurons with grouped or a gradient of phases. The contrast, the dorsomedial region exhibited a circadian mouse data have an implication for the direction of causation. oscillation of Per1 expression with a peak near CT4 and The dorsal oscillatory neurons cannot maintain synchrony similar rhythmicity in Per1 expression was present through- when separated from those more ventrally located. At least in out the mouse SCN. A two component model of SCN the mouse, this strongly suggests that the direction of operation was proposed in which the SCN is organized into causality with respect to phase angle and synchrony among dorsomedial “type A” cells and ventral “type B” cells oscillators is from the more ventral rhythmic cells to the dorsal (Shigeyoshi et al., 1997). The latter are strongly influenced rhythmic cells. by light, while the former are light-independent. Similar Other studies of the mouse SCN note a central region (Fig. results have been obtained in the rat (Yan et al., 1999). 3F3) with greatly reduced rhythmic Per1 and Per2 expression In the Per1-GFP mouse, the extent of regionality appears to (Karatsoreos et al., 2004; LeSauter et al., 2003; King et al., 2003; depend upon the lighting history. In LD housed animals, Reddy et al., 2002). This area corresponds closely to the approximately equal numbers of cells rhythmic for Per1 were distribution of GRP neurons (note: the GRP cells were identified found in the medial and lateral halves of the SCN (Quintero et in a CALB-GFP reporter mouse in which, for some unknown al., 2003). In contrast, in DD housed animals, about 66% of the reason, about 90% of the GFP cells in the SCN also contained medial neurons were rhythmic compared to 34% in the lateral GRP, and for an equally unknown reason, no cells in the SCN SCN. There were also differences in the dorsoventral distribu- known to contain CALB were identified by GFP (Karatsoreos et tions of rhythmic cells, with twice as many found in the al., 2004). However, the most central part of the region ventral SCN of LD housed animals compared to similar containing putative GRP cells has distinct expression of both percentages in the two halves of DD housed mice. As Per1 and Per2 at ZT2 or CT2, times when rhythmic expression is described above, the Per1-GFP mice have rhythmic neurons minimal elsewhere in the SCN (King et al., 2003). Previously, a with peak activity at one of three preferred phases. In DD similar sized, fairly discrete collection of neurons in the housed animals, the vast majority of cells peaking at CT18 are centrodorsal (not dorsomedial) mouse SCN was described as present in the medial SCN, whereas the medial–lateral expressing Per1 at ZT0 or CT0 (Hastings et al., 1999). difference becomes progressively less at CT15 and CT12, With respect to light-induced clock gene expression in the respectively. A similar relationship between peak phase and mouse, a light pulse at CT13-14 has been reported to cause laterality is evident in LD housed mice, but is not as Per1 expression throughout the central and dorsolateral SCN pronounced (Quintero et al., 2003). In organotypic slice in a region complementary to the medial area in which cultures of SCN from mice carrying the Per1promoter-driven rhythmic Per1 is expressed (Karatsoreos et al., 2004). There is a luciferase reporter gene, most cells identified by the reporter high degree of correspondence between light-induced Per1 show oscillatory gene expression and these cells are widely and the GRP cell distribution. The same appears true for FOS distributed throughout the SCN (Yamaguchi et al., 2003). expression. The region of light-induced Per1 and GRP cells is BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 27 completely filled with retinal projections which tend to be the ventrolateral division as the sole SCN region with retinal much sparser in the medial part of the SCN in which rhythmic innervation and VIP neurons, while the dorsomedial division Per1 or Per2 expression predominate. However, another report has no retinal innervation and contains VP neurons. However, indicates that light-induced Per1 is found in the ventral third convenience must give way to a more complex reality as of the SCN corresponding largely to the area containing PHI suggested by the presence of two functionally distinct parts of neurons (King et al., 2003). This is consistent with observations the dorsomedial rat SCN (Yan and Okamura, 2002). Similarly, that VIP neurons containing Per1 jump from 31% to 59% when the ventrolateral region containing VIP neurons has following a light pulse at ZT21 (Kuhlman et al., 2003). In been examined closely, it also can be subdivided into contrast, Per1 is expressed in about 35% of VP neurons at this differently functioning units. VIP cells identify an elliptical time, but is not elevated in response to light. VIP and VP cells area occupying the ventral half of the rat SCN. However, cells expressing Per1 comprise about 51% of the total neurons in the medial quarter of this region do not express light- spontaneously expressing that gene at ZT10, indicating that induced Per1 mRNA nor, as demonstrated in adjacent there is no specific correspondence between cell phenotype sections, does this area receive much, if any, retinal innerva- and rhythmic clock gene activity. The information also tion (Kawamoto et al., 2003). Thus, the nominal ventrolateral indicates that rhythmic gene expression is not solely the rat SCN containing VIP cells is divisible into a lateral part in province of dorsomedial neurons, as the VIP cells are which Per1 is induced by light and a medial part in which the consistently present in the ventral part of the SCN. cells do not have this response (Fig. 3E). Light-induced Per2 protein in the SCN is described (King The foregoing results are of significance because they et al., 2003) as being expressed with breadth and magnitude demonstrate, at least for the rat, that (a) cells throughout the equivalent to that which occurs at its maximal rhythmic entire SCN oscillate, although the magnitude is greater in some expression. One specific characteristic of the rhythmic regions than in others; (b) there are regional distinctions expression is its relative absence from the GRP cell region between the rhythmic expression of Per1 and Per2; (c) there are (Karatsoreos et al., 2004; LeSauter et al., 2003; King et al., at least four functionally distinct regions within the SCN; (d) 2003)(Figs. 3G1, 2; H). Cells identifiable as containing the the SCN appears to have a compartment in which cells rapidly VPAC2 receptor had both rhythmic and light-induced express Per genes in response to light, but this does not occur in expression of Per2 (King et al., 2003). There appear to be the other three compartments and (e) there is a large, but some differences in the results of two comparable mouse clearly imperfect, anatomical correspondence between known studies (Karatsoreos et al., 2004; King et al., 2003), but it is cell phenotypes and the three regions. In addition, it is difficult to determine whether or not they are real or simply noteworthy that the ventrolateral division, unlike the others, the result of substantially different methods. does not extend the entire length of the SCN (Yan and Okamura, 2002). 6.6.2. Rat One obvious burden imposed on investigators by the A comprehensive analysis of Per1 and Per2 gene expression in increased number of SCN subdivisions is the need to minimize the entire rat SCN has produced a modification of the confusion by using a common nomenclature characterized by dorsomedial/ventrolateral SCN divisional model to include fully understandable definitions. It is difficult to compare three functionally distinct regions (Yan and Okamura, 2002). studies when, in one study, a region may be referred to as the The single dorsomedial region has been divided into periven- “VP” area, a second study refers to the same area as the tricular and central parts (Fig. 3D). The former overlaps a fairly “dorsomedial region,” while a third study emphasizes a thin, dorsomedial portion of the SCN identifiable by VP difference between the “medial dorsomedial” and the “lateral neurons. The central portion overlaps the ventrolateral part dorsomedial.” The difficulty is compounded by the fact that, of the VP cell group as well as the dorsal part of the VIP cell because the information is so new, the laboratories making distribution. The third, ventrolateral, portion of the SCN most of the anatomical observations have yet to achieve a generally corresponds to the bottom 80% of the region standard nomenclature across publications. containing VIP cells. The three regions are reported to be distinct with respect to their expression of Per1. For example, 6.6.3. Hamster the peak expression in the periventricular division is at CT4, A functional analysis of SCN divisions has also been made in but at CT8 in the central division. Overall, the profiles of Per1 the hamster, although without the same level of detail. A two- expression are different in the three regions. A second compartment description has been demonstrated, with one distinguishing feature is the Per1 response to light which, for unresponsive to light and the other highly responsive to light the periventricular and central divisions, is nonexistent at (Hamada et al., 2001). The shape of the hamster SCN lends CT16, while extremely robust for the ventrolateral division. In itself to a descriptive terminology more closely related to contrast to Per1, rhythmic expression of Per2 is similar in the peptide content of cells than to the geographic descriptors of periventricular and the central divisions, thereby indicating a dorsomedial vs. ventrolateral. Thus, there is more emphasis different SCN divisional structure. on the central subnucleus and the fact that cells in this region An analysis of Per2 protein has shown rhythmic expression behave differently than the more dorsomedial cells. As in the in dorsomedial and ventrolateral rat SCN, but no induction in rat, there is a pronounced endogenous rhythm in Per1 and either location after a light pulse at CT13 (Beaule et al., 2003a). Per2 gene activity among neurons closely associated with the It has been conceptually convenient to consider the rat SCN distribution of VP cells in the hamster SCN. As a generality, the as having two parts, the dorsomedial and ventrolateral distribution of these cells wraps around the medial half of the “divisions.” Moreover, it has also been convenient to view collection of CALB neurons in the central subnucleus. Cells in 28 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 the CALB-containing region express the Per1 and Per2 genes there is a “periventricular” part of the hamster SCN, as there is robustly in response to light. It is also the case that neurons in for the rat (Yan and Okamura, 2002), remains to be determined. the CALB cell region do not have endogenous oscillations in Per1, Per2 or Fos gene expression unlike their more medial 6.7. Stimulus induction of gene expression counterparts (Hamada et al., 2001). It is not known to what extent the Per1 or Per2 gene expression occurs rhythmically in 6.7.1. Photic stimuli VP neurons or is light-induced in CALB neurons. Rea (1989) showed photic induction of FOS protein in SCN Further analysis of the hamster SCN has demonstrated neurons in 1989. During the past decade, there have been that while the most robust Per gene response to light occurs in many other investigations directed at the expression of a cells in and near the CALB cell region, a similar phenomenon variety of genes thought to be components of the circadian occurs among cells in the dorsomedial SCN, the region of VP clock. Substantial information exists which shows rapid neurons (Hamada et al., 2004a). The time course of light- induction of Per1 and Per2, but not Cry1, Cry2 or Per3, gene induced Per1 gene expression in cells of this region is expression following a light pulse (Shigeyoshi et al., 1997; Best significantly longer than in the CALB region, suggesting an et al., 1999; Field et al., 2000; Zylka et al., 1998; Shearman et al., altogether different pathway for induction. Nevertheless, the 1997). Brief (15 min) light pulses at ZT14 yielded Per1 and Per2 result is consistent with the smaller number of retinal gene expression increases that were 26 and 30%, respectively, projections to the dorsomedial region than to the central greater than controls when measurements were made 1 CALB cell region in the hamster (Muscat et al., 2003). It is also h later. However, the changes in mRNA levels occurred the case that the VP gene itself not only has an intrinsic without a noticeable increase in protein which is not evident oscillatory expression in SCN neurons, it is also light-inducible until many hours later (Field et al., 2000). These results in a slow onset manner similar to that for the Per1 gene in cells emphasize the investigators' point that there is not a reliable of the VP region (Hamada et al., 2004a). relationship between the phase shift magnitude and the level Rhythmic expression of VP mRNA in the hamster SCN of light-induced Per gene expression. appears to have at least two characteristics: there is always In a novel experiment, Paul et al. (2003) tested whether expression in the dorsomedial region (minimal at CT20), and is glutamate receptors mediate light-induced suppression of greatly increased in that region at CT8, while also appearing nocturnal pineal melatonin as well as light-induced expres- throughout a much larger part of the entire SCN (Hamada et sion of Per1 mRNA. As expected, both nocturnal environmen- al., 2004a). Per gene expression has similar rhythmic expres- tal light and the glutamate receptor agonist, NMDA, applied to sion characteristics, but Per1 and Per2 mRNAs are virtually the SCN suppressed pineal melatonin and elevated Per1 nonexistent at CT20. The investigators suggest that rhythmic mRNA. Joint application of AMPA or NMDA receptor antago- gene expression is initiated among a small number of nists with the NMDA blocked the effect of light on Per1 mRNA dorsomedial cells and spreads slowly through more caudal expression, but in the same animals, the antagonists were and ventrolateral parts of the SCN. unable to prevent the light-induced reduction in melatonin. There is clear intra-SCN organization to the temporal and This study demonstrates the existence of two different path- spatial rhythmic expression of the Per and VP genes. However, ways in the SCN. Both are light-responsive, with one presum- in an oscillatory system where there is no beginning and no ably related to the regulation of circadian rhythm phase end, it is not clear that an origin of the gene expression rhythm response and the other contributing to photic inhibition of can be defined. More important is the issue of whether cells in nocturnal pineal melatonin. The study also raises an issue that one part of the SCN transmit neural or humoral rhythmic should be of concern to all rhythm investigators. That is, information to other cells that is requisite for the expression whether the changes observed in the SCN are directly related or phasing of rhythmicity in those cells. Related to this issue is to the phenomenon under investigation or simply correlates. the question of cellular “gradients” in gene expression within This issue has been extended to evaluation of photic induction the SCN (Hamada et al., 2004a; Yan and Okamura, 2002). A of the clock genes, Per1 and Per2 (Paul et al., 2004). The results “gradient” implies gradual spatial change, a view contrary to indicate that both light and NMDA induced the expected gene observations of rhythmic gene expression occurring within expression in the SCN and also suppressed pineal melatonin. specific sectors of the SCN (see Fig. 3 in Yan and Okamura, Blockade of sodium-dependent action potentials with TTX 2002). The observations of temporal and spatial rhythmic or- blocked both light-induced gene expression and melatonin ganization within the SCN are likely to be of great importance suppression consequent to either light or NMDA. In contrast, with respect to understanding the movement of information induction of Per1 and Per2 by NMDA was not blocked by TTX within the SCN itself as well as to the understanding of (Paul et al., 2004). The results of the above studies are function attributable to the various sectors of the SCN. consistent with the view that there are two distinct mechan- To our knowledge, there has not been an investigation of isms regulating light-induced gene expression and light- light-induced clock gene expression comparable to the com- suppression of pineal melatonin. prehensive study of FOS by Guido et al. (1999). However, possibly unlike the situation in the rat (Yan and Okamura, 6.7.2. Nonphotic stimuli 2002), a CT19 light pulse first induces Per1 expression in an area Whereas light during the subjective night induces Per gene of the hamster SCN encompassing the central subnucleus, but activity in the SCN and causes rhythm phase shifts, it has this is followed, with time, by induction of gene expression in little, if any, effect on these two measures during the cells throughout most of the dorsomedial region as indicated subjective day. In contrast, phasically active nonphotic by the location of VP neurons (Hamada et al., 2004a). Whether stimuli administered during the subjective day elicit rhythm BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 29 shifts when applied during the subjective day which is al., 2003a for a discussion that considers a variety of issues associated with decreased Per gene activity in the SCN. related to the above inconsistencies). Several classes of nonphotic stimuli have this effect, including benzodiazepine treatment (Yokota et al., 2000), 6.7.3. Inconsistencies between phase response and gene novel wheel activity (Maywood and Mrosovsky, 2001), expression injection-related arousal (Maywood et al., 1999), peri-SCN Light-induced Fos gene activity is closely correlated with the infusion of NPY (Maywood et al., 2002; Fukuhara et al., 2001) ability of light to entrain circadian rhythms, but there is a and serotonin receptor agonists (Horikawa et al., 2000). general consensus that FOS is not an essential part of either Antisense oligonucleotide to the Per1 gene administered the clock or the input pathway mediating entrainment directly into the peri-SCN region during the subjective day (Colwell et al., 1993a,b,c). Moreover, FOS protein induction in suppresses Per1 mRNA expression by about 60% (Hamada et response to light is usually, but not always, predictive of al., 2004b). This treatment at CT6 induces a phase advance phase response. Quipazine injection at CT8 is significantly that averages about 60 min, a result consistent with the view reduced FOS expression in hamster SCN, but there was no that enhanced Per1 expression, in this case caused by a effect on phase shifts at this time and in mice, this drug had nonphotic stimulus, induces phase shifts. no effect on phase response, but increased FOS expression A proposed unifying hypothesis suggests that elevated during the subjective day (Antle et al., 2003b). In mice Per gene expression and Per protein synthesis is causal to entrained to a skeleton photoperiod of 1 h light pulses phase shifts induced either by light or by nonphotic stimuli presented to create a 1:14:1:8 LDLD photoperiod, some (Maywood and Mrosovsky, 2001). Moreover, the hypothesis animals ran during the long, and others during the short, makes an unconventional specific prediction that light interval of dark. The light manipulations revealed that only should be able to modify circadian clock function even the light pulse occurring near activity onset (ZT12) appeared during the “dead zone” of the PRC. The rationale is that, to influence locomotor rhythm phase. Irrespective of the during the normal dead zone, light cannot alter phase effect on entrainment, among animals running during the because Per expression is already high and further expres- long dark interval, light pulses at the beginning or the end of sion cannot be elicited by light during the subjective that interval equally induced Fos mRNA expression. More- daytime. However, light can be shown to alter SCN activity over, if animals ran during the short dark interval, Fos mRNA during the subjective day because light exposure at that time expression was induced by both pulses, but was much greater prevents the drop in Per gene activity normally induced by after the pulse at ZT21 (Schwartz et al., 1996). Thus, in this nonphotic stimuli (Maywood and Mrosovsky, 2001) or direct paradigm, there is a clear disparity between the entraining NPY infusion into the peri-SCN region (Maywood et al., 2002). capability of light and the magnitude of Fos gene expression The converse of this procedure is also demonstrable with in response to light. Molecular absence of Fos does not nonphotic stimuli presented during the subjective night able prevent entrainment of mice (Honrado et al., 1996). In vivo to attenuate light-induced phase shifts (Mistlberger and blockade of Fos availability is associated with an inability of Antle, 1998; Ralph and Mrosovsky, 1992). light to induce phase shifts in rats (Wollnik et al., 1995). According to the Maywood and Mrosovsky (2001) unifying However, JunB gene activity was simultaneously blocked in hypothesis, nonphotic stimulus should achieve its attenuating this study and the negative effects on light-regulated phase effects on light during the subjective night by reducing the control may have been the consequence of the suppression of extent of light-induced Per gene activity. However, direct tests the activity of both genes, rather than only Fos (see Schwartz of this proposition have yielded mixed results. In the first et al., 2000 for a discussion). instance, benzodiazepine treatment produced an approxi- Edelstein et al. (2003a) suggest that their data from mately 50% reduction in light-induced (5 lx, 15 min, CT20) nonphotic stimulus conditions are consistent with the view phase advances, but had no effect on phase delays (Yokota et that several different mechanisms contribute to the regulation al., 2000). The effect on phase advances was associated with an of circadian phase depending on the nature of the stimulus approximately 40–50% drop in Per1 or Per2 mRNA (the involved. Most importantly, they conclude that phase shifts corresponding test for phase delays was not performed). In can occur without necessarily modulating Per gene expression. contrast, access to a novel running wheel reduced light- This is contrary to the general view that phase shifts are induced phase delays without altering light-induced phase dependent upon Per gene activity, although not at all advances, but the novel wheel activity had no effect on light- inconsistent with the available literature. It is also the case induced Per1 mRNA (Christian and Harrington, 2002). In a that Per1 and Per2 expression is not the same in the SCN of similar investigation, access to a novel wheel for 1 h after a animals showing rhythm splitting in LL as it is in animals light pulse resulted in virtual abolition of phase advances in showing a form of induced splitting in DD referred to as response to less intense illumination and substantial attenu- “behavioral decoupling” (De la Iglesia et al., 2000; Edelstein et ation of response to more intense illumination. However, as in al., 2003b). In the latter case, “splitting” consisting of a daily the above study, there was no reduction in either Per1 mRNA or bout of induced locomotion is followed by a resting interval FOS protein associated with the reduced phase response to during which Per gene expression is increased throughout light (Edelstein et al., 2003a). As noted by the authors, Per1 most of the SCN. However, when the elevated gene expression appears to be insufficient for phase shifts in locomotor occurred during the rest phase following bouts of spontaneous rhythms and that, “The relationship between light and locomotion, Per gene expression was also elevated, but only in nonphotic stimuli is more complex than has been previously the dorsomedial part of the nucleus (Edelstein et al., 2003b). suggested (Maywood and Mrosovsky, 2001)” (see Edelstein et Also, despite having several characteristics similar to 30 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 spontaneous splitting in LL conditions, the DD-housed behav- animals so treated (Yannielli et al., 2002). On the other hand, if iorally decoupled animals had no indications of bilateral an additional 2 h of wheel access was permitted (with or asymmetry in Per gene expression in the two SCN, unlike the without additional running), a the firing rate rhythm advanced situation in animals showing LL-induced splitting (cf., De la about 2.4 h. Brains taken 5 h, rather than 3 h, after the initial Iglesia et al., 2000). access to wheels had reduced Per2 expression, but Per1 had There are several forms of dissociation between the returned to normal. One conclusion is that clock resetting in regulation of gene expression and the phasic effects of response to activity-related stimulation may require a 3–5 light on circadian rhythms. Perhaps the simplest is the h poststimulus latency (Yannielli et al., 2002; Antle and temporary loss of synchrony between gene expression Mistlberger, 2000), whereas other nonphotic stimuli or light rhythms during clock resynchronization to a shifted photo- may not require the same amount of time (Mead et al., 1992; period. The re-entrainment rate of certain gene rhythms in Sumova and Illnerova, 1996). mouse is faster than that of others and depends upon the direction of the phase shift. (Reddy et al., 2002). The rhythm 6.8. Polysialic acid and SCN function of mPer1/2 expression in the mouse phase advances rapidly in response to light, while re-entrainment lags for mCry1. In There are a variety of possible neurobiological roles that may contrast, the rhythms of mCry1 and mPer1 or mPer2 re- be played by the molecule, polysialic acid (PSA) and its entrain at the same rate during a phase delay. These gene carrier, the neural cell adhesion molecule (NCAM), which expression results match corresponding behavioral data form PSA-NCAM (particularly during development and in the showing slower advances and faster delays (Reddy et al., context of neural plasticity; see Rutishauser and Land- 2002). A variation on the theme of temporary desynchroni- messer, 1996). PSA-NCAM is distributed along plasma zation among gene expression rhythms is the demonstration membranes of appositions between neurons, at synapses that they (Per1, Per2, Cry1) shift rapidly in the ventrolateral and between glia (Shen et al., 1997, 1999). It is of interest that rat SCN, but more slowly in the dorsomedial region (Nagano the SCN of Siberian (Glass et al., 1994) and golden hamsters et al., 2003). (Fedorkova et al., 2002), mice (Shen et al., 1997) and rats A second form of dissociation has been demonstrated in (Prosser et al., 2003) contains concentrations of PSA that mice lacking the PAC1 receptor (Hannibal et al., 2001b). A light fluctuates rhythmically. In LD, PSA concentration drops pulse that induces a 100 min phase delay in wild-type mice precipitously and by more than 90% during the dark hours. elicits a larger phase delay in the mutant mice. However, the Although the shape of the curve describing PSA content in mutant mice have a vastly attenuated Fos or Per gene the SCN is substantially different during DD, clear rhythmic- induction response to light. A substantially different experi- ity persists (Glass et al., 2003a). Moreover, light greatly boosts ment has yielded the observation that imposition of a 6 transient PSA expression in the SCN (but not in hippocam- h photoperiod advance rapidly induces a large phase advance pus). NCAM rhythmicity is similar in the SCN, although its in the Per1-luc bioluminescence rhythm relative to unshifted rhythm in DD is much less robust than that for PSA (Glass et controls. This phase differential persists for at least 6 days in al., 2003a). DD. In contrast, such advances did not occur in in vivo SCN Mice lacking the NCAM-180 isoform that carries PSA have electrophysiological or behavioral rhythms (Vansteensel et al., a shorter than normal circadian period, a longer activity 2003a). phase and become arrhythmic within 3 weeks of DD (Shen et A third form of dissociation addresses the question of al., 1997). The mutant mice also show light-induced phase whether particular genes, or change in their expression, are delays smaller than control mice (Glass et al., 2000a). The always necessary for rhythm expression. One example is the role of PSA itself has been investigated using an enzyme, gene, Clock. Mice homozygous for a mutant form of Clock endoneuraminidase (endoN), to remove PSA. Animals trea- rapidly which when absent yields mice that rapidly become ted with endoN have shorter circadian periods in DD, arrhythmic in DD (Vitaterna et al., 1994). More recently, although they do not appear to become arrhythmic (Shen however, a similar test procedure has been applied to Clock et al., 1997). Similar to the mutant animals, the endoN- mutant mice, but with an LL background to the rhythm treated mice have diminished phase delays in response to expression. In this circumstance, many of the mice exhibit light; they also have reduced light-induced FOS expression clear circadian rhythms despite the absence of the Clock (Glass et al., 2000a). gene (Spoelstra et al., 2002). Other investigators have In contrast to the diminished effects of light in mice suggested that running may be an inadequate variable to sustaining endoN removal of PSA, hamsters so treated had assess in order to determine whether or not the Clock augmented phase response to nonphotic stimuli (61% mouse in DD is rhythmic, and present data supporting the increase to 3 h arousal; 120% increase to 8-OH-DPAT view that such mice are indeed fundamentally rhythmic and injection) applied at ZT6. In the PSA-depleted rat SCN entrainable, but with a much longer period than normal slice preparation, direct application of 8-OH-DPAT elicited a (Kennaway et al., 2003). 41% greater phase advance (Fedorkova et al., 2002). Also in In quite a different situation with the mixed in vivo/in vitro the slice preparation (Prosser et al., 2003), PSA levels preparation, novel wheel access for 3 hduring the subjective remain rhythmic. Glutamate treatment of the slice induced day rather quickly reduced Per1 expression in the SCN as a 100% increase of PSA in the SCN, but this was blocked by might be expected during the midsubjective daytime. Howev- endoN pretreatment. Treatment with endoN also blocked er, under the conditions of the experiment, there was no the phase shifting effects of glutamate or electrical associated shift in the phase of the SCN firing rate rhythm in stimulation of the slice. The strong implication of this BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 31 research is that retinal input to the SCN requires PSA to be 7.2. Efferent anatomy functional. A further analysis of the molecular requirements form Watts et al. (1987) identified six general regions to which the normal mouse rhythmicity was performed using three strains rat SCN projects. Subsequent studies using the anterograde deficient, to varying degrees, in PSA and NCAM. Animals tracer, Phaseolus vulgaris leucoagglutinin, yielded generally lacking both PSA and all forms of NCAM become arrhythmic in similar pathways in the hamster brain for which there is (1) DD and tended to run so much when lights are on that an anterior projection to the ventral lateral septum, bed entrained rhythms are often difficult to discern (Shen et al., nucleus of the stria terminalis and anterior paraventricular 2001). A similar strain, but not lacking NCAM isoform 120 has thalamus; (2) a periventricular hypothalamic projection to normal entrainment under LD, but is arrhythmic in DD, the medial preoptic region, subparaventricular zone, the suggesting that NCAM-180 and/or NCAM-140 are required to paraventricular, ventromedial and dorsomedial nuclei and avoid arrhythmicity in PSA deficient mice. The conclusion the premammillary area and (3) a posterior projection to the from these studies is that some combination of PSA and NCAM posterior paraventricular thalamus, precommissural nucleus is necessary for normal rhythmicity and entrainment. The and olivary pretectal nucleus (Kalsbeek et al., 1993; Morin et mechanisms through which this action is achieved are al., 1994). In the hamster, it is likely that the cells projecting presently unknown. to the olivary pretectal nucleus are actually located in the peri-SCN, as is indicated by retrograde tracer injections into the pretectum (Morin and Blanchard, 1998). Such cells are 7. SCN connectivity also seen following retrograde tracer injections into the IGL, ventrolateral geniculate, posterior limitans nucleus, com- 7.1. Humoral factors missural pretectal nucleus and medial pretectal nucleus (Morin and Blanchard, 1998; Morin et al., 1992; Vidal and One of the more surprising results during the last decade of Morin, 2005). In particular, cells projecting to the IGL from research on SCN function has been the demonstration that the SCN region do not reside within the SCN proper, but rhythmic events can be controlled, in all likelihood, by rather around its dorsal perimeter with a substantial nonneural linkage to the circadian clock. The availability of probability that their dendrites extend ventrally into the perinatal SCN transplant methods enabled this conclusion. nucleus. Watts identified an IGL-efferent projection in the The procedure developed with hamsters by Ralph et al. (1990) SCN of rats and the cells of origin can be seen within the used embryonic SCN to restore locomotor rhythms in lesioned, SCN in this species (Watts and Swanson, 1987). The hamster arrhythmic hosts, but SCN efferent connections were restored also differs from the rat in that it has a distinct caudal to an uncertain degree (Canbeyli et al., 1991; Lehman et al., projection that ascends to innervate posterior paraventricu- 1987). Subsequently, transplantation of viable, membrane lar thalamus (a separate projection innervates the anterior encapsulated SCN restored locomotor rhythmicity without paraventricular thalamus in both rats and hamsters) (Morin any apparent neural connections to adjacent hypothalamus et al., 1994). Given that retrograde tracer studies identify (Silver et al., 1996b). In contrast, rat to hamster or mouse to cells around, but not in, the SCN for some of the putative hamster SCN transplants restore rhythmicity in association SCN targets (IGL, VLG, pretectum), it is distinctly possible with substantial growth of donor SCN projections into host that others (such as the posterior amygdala or posterior hypothalamus, preoptic area and septum (Sollars and Pickard, paraventricular thalamus) might also receive afferents from 1993; Sollars et al., 1995; Lehman et al., 1998). It appears to be peri-SCN cells. the case that behavioral rhythms, but not endocrine rhythms, Lesion methods have been used in conjunction with are restored by transplantation of embryonic SCN (Meyer- immunohistochemistry to identify SCN projections having Bernstein et al., 1999; LeSauter and Silver, 1998). specific peptide content (Kalsbeek et al., 1993). In the Despite declarations that the SCN contains the timing hamster, VP cells project to the medial preoptic, parvicellular apparatus governing transplanted circadian periodicity, there paraventricular and dorsomedial hypothalamic nuclei and are other possible explanations of the phenomenon and there the anterior paraventricular thalamus. Cells containing VIP may be species differences in the extent to which period is the project to the anterior paraventricular thalamus, parvicellu- exclusive property of the SCN (Sollars et al., 1995). It is also the lar paraventricular hypothalamus, subparaventricular area case that the specific location of the transplant within the host and the dorsomedial hypothalamus. SCN efferent projections brain is relatively unimportant as long as the site is in or near containing GRP were identified in the subparaventricular the third ventricle (such as adjacent lateral ventricle) (LeSau- area, dorsomedial hypothalamus and supraoptic nucleus. ter et al., 1996; Lehman et al., 1987; Aguilar-Roblero et al., 1994). The topography of projection neurons in mouse SCN has In addition, transplants consisting of dissociated SCN cells in been inferred (Abrahamson and Moore, 2001)fromthe the anterior hypothalamic parenchyma or medial hypothala- presence of VP or VIP immunoreactive fibers in many of mus also restore rhythmicity (Silver et al., 1990). Precision of the areas identified as receiving SCN projections in other activity onset reinstated is related to the transplant distance species. caudal or dorsal (but not rostral) to the SCN. Based on these Analysis of SCN projection neurons by retrograde tracer data, the suggestion has been made that the target of a methods provides at least a preliminary indication of a neurohumor conveying period information from the SCN is structure–function relationship. Successful retrograde proce- located near or slightly rostral to the SCN (LeSauter et al., dures indicate both targets of SCN efferent projections and the 1997). location of neurons providing those efferents. Retrograde 32 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 tracing methods have provided much of the recent informa- SCN neurons have also been reported to project to, and make tion regarding the putative “core” and “shell” organization of likely contact with, both hypocretin (orexin) and melanin the SCN (Moore et al., 2002). In this regard, there are two concentrating hormone neurons in the rat lateral hypothala- references that need to be compared. The 1987 study of Watts mus (Abrahamson et al., 2001). and Swanson (1987) reached conclusions different from those Transneuronal viral tracers have been utilized to deter- of Leak and Moore (2001), although the two investigations mine polysynaptic routes from the SCN. Following virus were equally broad and used basically similar methods. As a injection into the rat locus coeruleus, cells are retrogradely result, it is not possible to determine which interpretation is labeled in the dorsomedial hypothalamic nucleus, a known correct. On the one hand, Watts and Swanson (1987) empha- target of SCN neurons. The transneuronal tracer also labels sized the result that, “...cells in dorsal as well as ventral parts SCN cells, but this does not occur following lesions of the of the SCh project to each of the terminal fields examined.” (p. dorsomedial hypothalamus. Furthermore, the circadian 230, abstract). Three cases showing labeled cells following rhythm in firing rate of locus coeruleus cells is abolished retrograde tracer injection into the paraventricular hypothal- after by the same lesions, suggesting that the SCN control of amus were illustrated. In Case E46, the labeled cells were rhythmic neuronal activity in the locus coeruleus is via a predominant in the dorsomedial SCN with few in the ventral two synapse circuit with a first order synapse in the part of the nucleus. In Case E47, there were many more cells dorsomedial hypothalamic nucleus (Aston-Jones et al., labeled and the great preponderance was found in the ventral 2001). Injection of virus into the hamster medial vestibular half of the SCN. In Case PVH33, fewer labeled cells were nucleus also labels a few neurons in the SCN. This is a present, and the greatest density of such cells was in the polysynaptic pathway that probably includes the locus ventrolateral quadrant of the SCN. On the other hand, a coeruleus which projects to the medial vestibular nucleus predominance of retrogradely labeled neurons in the dor- (Horowitz et al., 2005). somedial SCN compared to the ventral part following para- Viral tracers injected into fat pads (brown or white ventricular nucleus tracer injection has been reported (Leak adipose tissue) identified neurons in Siberian hamster and and Moore, 2001; Vrang et al., 1995a). The observation of rat SCN (Bamshad et al., 1998, 1999). The actual number of injection-related variability by Watts and Swanson is consis- retrogradely labeled SCN neurons is small, and an estimated tent with an equivalent observation by Leak and Moore who 2% of these also contain VP (Shi and Bartness, 2001). noted opposite patterns of dorsomedial:ventrolateral SCN cell Injections placed in the adrenal medulla (Ueyama et al., labeling that depended upon the precise location of tracer 1999), pancreas (Buijs et al., 2001) and other structures (see injection in the subparaventricular zone (medial injec- Ueyama et al., 1999; Bartness et al., 2001 for reviews) label tion = 5.6:1; lateral injection 1:5.6). In the hamster, tracer cells in the SCN. The general theme of these studies is that injection into the paraventricular hypothalamus yields a the SCN regulates sympathetic outflow, a feature evident in “pattern of retrogradely labeled cells in the SCN […] uniform the viral tract tracing of pineal innervation (Larsen, 1999; across cases […] scattered throughout the subdivisions of the Larsen et al., 1998; Teclemariam-Mesbah et al., 1999). How- SCN, and no tendency for backfilled cells to be located in the ever, it appears that there is also SCN control of parasym- dorsomedial SCN was observed” (Munch et al., 2002; p. 53), i.e., pathetic output (Ueyama et al., 1999; Kalsbeek et al., 2000). there is an absence of topographic organization in the species The combination of the two types of peripheral innervation in which it might be most expected. The Watts and Swanson is consistent with the presence of a circuitous polysynaptic study remains the most substantial examination of the extent route from the SCN through the midbrain to the eye to which individual SCN cells containing a particular neuro- identified by virus injection into that organ (Pickard et al., peptide project to various targets. In general, tracer injection 2002; Smeraski et al., 2004). into a target nucleus retrogradely labels SCN neurons contain- There is a large amount of convergence by SCN-efferent ing VP and VIP, although the great majority do not contain projections onto brain regions receiving IGL input (Fig. 4). In either peptide. addition, many of the same areas are also innervated by In the mouse, SCN cells projecting to the subparaventri- retinal and olfactory system projections (Johnson et al., 1988a; cular area have been shown to express FOS protein in Morin and Blanchard, 1999; Morin et al., 1994; Cooper et al., response to night time light exposure (De la Iglesia and 1994; Ling et al., 1998). Schwartz, 2002). In the hamster, SCN neurons projecting to the paraventricular hypothalamus have also been shown to 7.3. Afferent anatomy express FOS in response to light. The percentage of the cells expressing FOS is much greater at ZT19 than at ZT14 (Munch The SCN-afferent projections in the hamster were initially et al., 2002). It was also determined that some of the same cells described in a fairly comprehensive fashion by Pickard (1982) contained VP, VIP or GRP. Of these, cells with the combined in the early 1980s. There have been two additional such traits of projecting to the paraventricular nucleus, expressing investigations, and in another species, the rat (Moga and FOS in response to light and containing VIP were most Moore, 1997). In this study, a combination of retro- and common and the combination in which the cells contained anterograde tracing studies demonstrated additional projec- VP was rare. Direct SCN projections to gonadotrophin releas- tions to the SCN arising from infralimbic cortex, lateral ing hormone cells of the basal forebrain (De la Iglesia et al., septum, substantia innominata, ventral subiculum, subforni- 1995; Van der Beek et al., 1997) and to the rat paraventricular cal organ, ventromedial hypothalamus, arcuate nucleus, hypothalamic nucleus and corticotropin releasing hormone posterior hypothalamus, pretectal nuclei, median raphe and cells, in particular, have been reported (Vrang et al., 1995a,b). IGL, but not in several of the brainstem regions identified by BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 33

Fig. 4 – Convergence of SCN (solid lines) and IGL (dashed lines) projections onto diencephalic and basal forebrain regions (black areas) in the hamster. Gray areas receive innervation only from the IGL. Only the premammillary area (white) receives input solely from the SCN. Thickness of the lines indicates approximate density of the projection. Note that the hamster SCN does not project to the IGL, although peri-SCN cells do so. See text regarding further convergence of visual and olfactory input to many of these areas. Drawn after Morin et al. (1994) and data from Morin and Blanchard (1999).

Bina et al. (1993), as containing cells projecting to the SCN. The cortex, hippocampus, nuclei of the amygdala, thalamus, observations of Moga and Moore (1997) in the rat are hypothalamus, as well as nuclei in the midbrain and substantially similar, but broader than, those of the older, medulla from which there are very few reported direct SCN more limited hamster study (Pickard, 1982). As part of a larger projections. While this study is important, it should be noted analysis, Krout et al. (2002) evaluated monosynaptic inputs to that any uptake by terminals in the peri-SCN region is likely the rat SCN identified by CTB labeling. These investigators to identify regions not connected to the SCN. A similar observed about 40 regions sending direct projections to the thorough examination of polysynaptic projections to the SCN, largely in agreement with the results of Moga and Moore SCN has not been systematically completed in other species. (1997). However, Krout et al. (2002) also observed a more However, some information is available for the hamster and widespread distribution of labeled cells in areas such as the shows both similarities and significant dissimilarities with locus coeruleus and divisions of the parabrachial complex, the rat data. In particular, there appears to be little or among others, but did not see such cells in the substantia nothing in the way of a polysynaptic projection from the innominata and only a few or none in the olivary pretectal hamster tectum and pretectum to the SCN, yet more nucleus. In all studies involving tracer injection into the SCN, oculomotor midbrain structures contain labeled cells than the most likely cause of label disparity across injections is the in the rat SCN (Horowitz et al., 2004). A polysynaptic extent of label spread outside the nucleus, uptake by projection from the medial vestibular nucleus to the SCN processes in close proximity to the nucleus or uptake by cell has been reported in the hamster. processes extending significant distances to enter the nucleus Focused studies have documented SCN-afferent projec- (Morin and Blanchard, 1998; Morin et al., 1992, 1994; Gerfen tions from the pretectal complex, with cells identified in the and Sawchenko, 1984). olivary, medial and posterior pretectal nuclei (rat) (Mikkelsen The above-mentioned report (Krout et al., 2002) described and Vrang, 1994) and from the median raphe nucleus (Meyer- about 40 brain regions with cells monosynaptically project- Bernstein and Morin, 1996; Hay-Schmidt et al., 2003). Cholin- ing to the SCN. Injection of an exclusively retrograde ergic cells in the substantia innominata, preoptic nucleus, (Pickard et al., 2002), transneuronal virus label into the SCN diagonal band of Broca, medial septum, pedunculopontine, expanded the number of brain nuclei with cells projecting to laterodorsal tegmental and parabigeminal nuclei have also the rat SCN to about 100–140 regions (variation may relate to been reported to project to the SCN in the rat (Bina et al., 1993). label spread outside the SCN) (Krout et al., 2002). The regions As noted above, there is disagreement concerning which, if with likely polysynaptic projections to the SCN include any, these nuclei send projections to the SCN (Moga and numerous additional olfactory structures, divisions of the Moore, 1997; Krout et al., 2002). 34 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

(Botchkina and Morin, 1993) and precedes the initial retinal 8. Intergeniculate leaflet innervation of the SCN by about 2 days (Speh and Moore, 1993). The arrival of retinal projections in the lateral geniculate 8.1. Nomenclature region occurs on E13, but there is a delay in terminal arborization until about E15.5 (Jhaveri et al., 1991). The timing The IGL has been, and continues to be, confused with the of retinal innervation of the IGL is not known. ventral lateral geniculate nucleus. Focus on the term, “leaflet,” as a descriptor of the IGL has drawn attention away from the 8.3. Anatomy actual boundaries of the nucleus and, until fairly recently, has not had a clearly identifiable homolog in nonrodent species. It 8.3.1. Geniculohypothalamic tract is now established that the medial division of the ventral The NPY neurons were the first IGL cell phenotype shown to lateral geniculate nucleus is the cat homolog of the IGL project to the SCN (Card and Moore, 1982; Moore et al., 1984). (Nakamura and Itoh, 2004; Van der Gucht et al., 2003; Pu and The initial description of IGL projections to the contralateral Pickard, 1996). In the monkey, the homologous structure is nucleus in the rat indicated that many of the cells contained known as the pregeniculate nucleus (Van der Gucht et al., enkephalin, but none of this phenotype projected to the SCN 2003; Moore, 1989). However, there is uncertainty regarding (Card and Moore, 1989). There are substantial species differ- the neuromodulator content of the GHT across primate ences with respect to IGL organization and in the hamster, species (Chevassus and Cooper, 1998). only a very small percentage of NPY or enkephalin neurons In the hamster, the definition of the IGL has gradually project to the contralateral IGL (Morin and Blanchard, 1995). In evolved to include all regions of the lateral geniculate complex both species, GABA neurons are present in the IGL and project in which NPY neurons and cells projecting to the SCN may be to the SCN (Morin and Blanchard, 2001; Moore and Card, 1994). found. As indicated from the developmental data described In the rat, there is a fourth, unidentified, cell type (Moore and below, NPY cells found in medial areas near the acoustic Card, 1994). In the hamster, a fourth class consists of radiation and ventral to the leaflet portion of the IGL have not neurotensin neurons (Morin and Blanchard, 2001). It is useful completed full migration (Botchkina and Morin, 1995). The to recognize, as is further indicated below, that while ample region of the laggard migratory NPY cells also contains numbers of the IGL neurons also project to the pretectum, only neurons projecting to the SCN which are not found elsewhere a small percentage contain any of the various neuromodula- in the lateral geniculate complex outside of the IGL (Morin and tors thus far evaluated, implying existence of at least one Blanchard, 1995; Morin et al., 1992). There is also a other cell type in hamster. corresponding group of enkephalin-containing cells, especial- NPY cells can be used to identify the IGL as they occur ly in the medioventral part of the IGL (Morin and Blanchard, nowhere else in the lateral geniculate complex (Morin and 1995). This description of the hamster IGL is generally Blanchard, 2001; Moore and Card, 1994). In the hamster, consistent with that for the rat (Moore and Card, 1994), neurotensin, which is almost completely colocalized with although there are slight species differences with respect to NPY, can likewise identify the nucleus, albeit with somewhat its cross-sectional appearance at various rostrocaudal levels. greater difficulty because it is present in only about half the NPY neurons. There is colocalization ranging from 7% (percent 8.2. Development of GABA cells containing enkephalin) to 98% (percent of neurotensin cells containing NPY) of all identified peptides IGL development has been described for the hamster (Botch- with each other in hamster IGL neurons (Morin and Blanchard, kina and Morin, 1995). The IGL is probably best considered as 2001). part of the so-called ventral thalamus with a developmental origin different from the dorsal lateral geniculate nucleus 8.3.2. Connectivity and relationship to sleep, visuomotor and (Altman and Bayer, 1979a,b). The hamster IGL develops out of vestibular systems the reticular neuroepithelium with the first NPY cells evident Harrington (1997) has reviewed the substantial literature on embryonic day 9–10. These are evident against a back- concerning the ventral part of the lateral geniculate com- ground of radial glial cells extending dorsolaterally. NPY plex. This review was titled, with a good deal of foresight, neurons apparently migrate along a specialized set of radial “The ventral lateral geniculate nucleus and intergeniculate glial processes reaching their final destination in what will leaflet: interrelated structures in the visual and circadian become the IGL on E14. The radial glial cell bodies dislocate systems.” Most of the literature cited in that review was from the ventricular germinal zone on E15, and transform into unaware that an “intergeniculate leaflet” existed, let alone the astrocytes that become evident in the IGL across postnatal the possibility that it might have functions different from days 5–10. Some NPY neurons do not achieve complete those of the ventral lateral geniculate. At the time of the migration and remain relatively displaced in what is now review, it was established that the IGL contributes to the considered ventromedial IGL. Fiber outgrowth from NPY- photic regulation of circadian period and entrainment, and containing IGL neurons is evident by P0, and by P3, they that IGL lesions could block the ability of certain nonphotic penetrate the SCN. The adult-like NPY terminal plexus in the stimuli to induce phase responses normally consistent with SCN is achieved by approximately P10. an NPY-type PRC (Janik and Mrosovsky, 1994; Wickland and The P3 arrival in the SCN of the NPY component of the GHT Turek, 1994; see Morin, 1994; Harrington, 1997 for reviews). approximately coincides with the end of SCN synaptogenesis The prediction in Harrington's review, evident in the title, (Speh and Moore, 1990), arrival of serotonergic innervation that the IGL has functions beyond those of circadian rhythm BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 35 regulation has not yet been directly established, but the IGL has at least two classes of neurons identified by their groundwork is now in place for doing so. projections to the SCN, one being so connected and the other The most important anatomical feature of the IGL appears not. Those IGL cells that contribute GHT input to the SCN to be the enormity of its connections with the rest of the brain appear not to receive any input from the pretectum or superior (cf., Fig. 4, which only includes a portion of the ascending colliculus despite abundant IGL-afferent projections from projections). Not only are they widespread, but they are those areas (Morin and Blanchard, 1998; Horowitz et al., generally bilateral and often reciprocal. The magnitude of 2004). This further suggests that photic input to the pretectum IGL connectivity was initially suggested by an investigation of and tectum does not influence circadian rhythmicity by hypothalamic-IGL connectivity in the gerbil (Mikkelsen, 1990). passing through the IGL and GHT. This inference is supported This study, and others in hamster (Morin and Blanchard, 1999) by lesion studies indicating that loss of the IGL, but not visual and rat (Horvath, 1998; Moore et al., 2000) revealed a major midbrain areas afferent to the IGL, modifies circadian rhythm efferent target extending from the lateral optic chiasm response to light (Morin and Pace, 2002). medially through the lateroanterior hypothalamus to the There have been recent reports describing brainstem midline, including the SCN, and dorsally to include the origins of IGL-afferent connections (Horowitz et al., 2004; subparaventricular zone and adjacent perifornical anterior Vrang et al., 2003). Two of these, a serotonergic input from hypothalamus. In addition, IGL projections extend to several the dorsal raphe and a noradrenergic projection from the divisions of the bed nucleus of the stria terminalis and variety locus coeruleus, have been previously documented (Meyer- of basal forebrain areas extending laterally as far as the Bernstein and Morin, 1996; Kromer and Moore, 1980). Other piriform cortex (Morin and Blanchard, 1999). Projections to regions sending projections to the IGL include several nuclei of the midline thalamus are also fairly extensive (Morin nuclei of the oculomotor complex, the caudal cuneiform, and Blanchard, 1999; Moore et al., 2000). Many of the same pedunculopontine and laterodorsal tegmental nuclei (Hor- regions also send projections back to the IGL (Morin and owitz et al., 2004; Vrang et al., 2003). In addition, projections Blanchard, 1999; Vrang et al., 2003). from the pararubral nucleus (which also is retinorecipient) Comparison of regions receiving retinal projections and vestibular nuclei to the IGL have been described reveals convergence with IGL efferents. Four distinct routes (Horowitz et al., 2004). Several of these regions, including providing retinal innervation of rostral diencephalon have the medial vestibular nucleus, locus coeruleus, divisions of been described (Johnson et al., 1988a; Morin and Blanchard, the dorsal tegmental and pontine tegmental nuclei and the 1999): (a) to the SCN and anterior hypothalamus; (b) to the dorsal raphe, are also recipients of projections from the ventral lateral hypothalamic area; (c) rostrally to the ventral hypocretin/orexin neurons in the lateral hypothalamic area preoptic area and (d) to the bed nucleus of the stria (Horowitz et al., 2005; Mintz et al., 2001). These regions are terminalis (Cooper et al., 1994) (earlier identified as project- also thought to be involved in the modulation of sleep and ing to the anterodorsal thalamic nucleus; Johnson et al., arousal processes (Saper et al., 2001). Most also receive 1988a). With very few exceptions, IGL projections innervate projections directly from the IGL (however, the locus coer- the retinorecipient regions, although the extent of IGL uleus and vestibular nuclei do not) (Morin and Blanchard, projections is substantially greater than those from the 2005). Thus, it seems likely that the IGL contributes to the retina (Morin and Blanchard, 1999). In a similar vein, areas regulation of sleep and arousal in addition to its role in receiving IGL input are also very likely to receive projections circadian rhythm regulation and its hypothesized role as a from the SCN (Morin et al., 1994) and from the olfactory mediator of visuomotor function. system (Cooper et al., 1994). IGL connections with the midbrain and brainstem are just 8.4. Function as extensive as those for the forebrain. The concept of a “subcortical visual shell” has been invoked to facilitate 8.4.1. Neurophysiology discussion of connectivity of the IGL (Morin and Blanchard, A novel observation has been made concerning the firing rate 1998). The subcortical visual shell consists of 12 contiguous of IGL neurons. In a series of paper, Lewandowski and retinorecipient nuclei of the thalamus and midbrain. All but colleagues have described a slow oscillation in the rat IGL those projecting to cortex (lateral posterior and dorsal lateral that is not evident in adjacent thalamic structures. Maximal geniculate nuclei) receive both ipsi- and contralateral projec- firing rate is approximately 20 Hz occurring as a burst that lasts tions from the IGL. In addition, most of the regions also supply about 70 s with the overall oscillation of firing having a period reciprocal input to the IGL bilaterally (Morin and Blanchard, of about 106 s (Lewandowski et al., 2000). Unlike the firing 1998), although ipsilateral connections are more robust. The pattern observed in the dorsal lateral geniculate, there is no functional importance of IGL connections with nuclei of the correlation between IGL cell firing and the pattern of firing in subcortical visual shell has not yet been tested. However, the the visual cortex. A lesion of one IGL does not disrupt presence of bilaterally symmetrical connectivity strongly rhythmicity in the other (Lewandowski et al., 2002). The origin suggests involvement of the IGL in the regulation of visuo- of the oscillatory activity is unknown, but is not exclusively motor function (for a review, see Morin and Blanchard, 1998; intrinsic to the IGL as it fails to persist in a slice The function of Horowitz et al., 2004). this burst oscillation is not known, nor is it even known to be The further suggestion has been made that the IGL may related to circadian rhythm regulation. However, it is present contribute to two discrete classes of events, one concerning only in the light with bursting suppressed by dark (Bina et al., visuomotor, and the other circadian rhythm, regulation 1993). The bursting pattern can be obliterated and rendered a (Horowitz et al., 2004, 2005; Morin and Blanchard, 2005). The tonic discharge pattern by bicuculline or picrotoxin applied 36 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 intraventricularly (Blasiak and Lewandowski, 2004a). In addi- SCN mediates the phase shift response to two distinct tion, the IGL burst discharge frequency is increased about 100% stimuli, there are different anatomical substrates subserving by lesions of the dorsal raphe nucleus (Blasiak and Lewan- those stimuli. Whether the ability of DD to elicit phase shift dowski, 2003), although serotonin agonists injected intraper- responses corresponding to the NPY-type PRC depends upon itoneally unexpectedly induce an increase in discharge locomotion has not been determined. frequency as well. The implications of the slow oscillatory NPY, the first neuromodulator identified in the IGL (Card et bursting activity are not clear and may be related more to al., 1983), has also received the greatest amount of attention suspected visuomotor functions of the IGL. with respect to IGL and GHT function. Stimulation of the IGL Intriguingly, the ultradian rhythm in IGL neuron burst has been thought to elicit phase shifts because of NPY release discharge is very similar to what has been described as a burst from GHT terminals in the SCN (see Morin, 1994). This concept pattern in SCN neurons (Miller and Fuller, 1992). About 20% of has been reaffirmed by antagonism of NPY activity in vivo by the cells recorded in and near the SCN burst at a rate of about infusion of NPY antiserum onto the SCN which blocked phase 10 Hz with an average period of about 143 s. It may be that the shifts in response to novel wheel running (Biello et al., 1994). IGL burst rhythm is derived from the SCN region because the As indicated above, cells contributing to the hamster GHT IGL rhythm is lost in a slice preparation (Blasiak and also contain GABA, neurotensin and enkephalin. Neurotensin Lewandowski, 2004b). appears not to have been evaluated with respect to its ability to modify circadian rhythmicity in vivo. However, a CT6 8.4.2. Nonphotic regulation of rhythmicity application of neurotensin to the rat SCN in a slice preparation One major question concerning the influence of nonphotic generally reduced neuron firing (Coogan et al., 2001), while stimuli on circadian rhythm regulation is whether it is inducing large phase advances of the firing rhythm (Meyer- mediated by activity (i.e., muscle/joint movement) or a Spasche et al., 2002). correlate of activity. Dark pulses, triazolam and novel wheel A small literature exists concerning the function of locomotion each elicit large phase advances when adminis- enkephalin in the hamster GHT. The delta opioid receptor tered at CT6 (see Morin, 1994; Mrosovsky, 1996) and each of visualized by immunohistochemistry is densely distributed these is usually associated with increased wheel running. At on cells in the SCN, particularly in the ventral and medial parts least two experiments have now demonstrated that different of the nucleus (Byku et al., 2000). A few cells in the IGL also are parts of the brain mediate the phase shifts elicited by identifiable from the delta opioid receptor labeling. The different stimuli. In the clearest case (Marchant and Morin, observed distribution of receptor label in the SCN is consistent 1999), the benzodiazepine, triazolam, failed to elicit phase with the distribution of enkephalin terminals (Morin et al., shifts at any circadian time in hamsters sustaining neurotoxic 1992). Delta opioid agonists administered at CT8 or CT10 (but lesions of the deep superior colliculus. Lesions of the not at other times) will induce moderate phase advances that pretectum were at least partially effective in eliminating are not related to the amount of activity also induced by the such responses and, because of the lesion method, it has not drugs (Byku and Gannon, 2000a,b). Agonists of kappa or mu been possible to fully distinguish between collicular and opioid receptors apparently do not elicit phase shifts, although pretectal effects. However, lesions confined to the superficial other reports indicate that fentanyl, a mu opioid agonist, can layers of the superior colliculus had no effect on phase generate an NPY-type PRC (Meijer et al., 2000; Vansteensel et response to triazolam. al., 2003b). The delta (but not mu or kappa) opioid agonists are In contrast to the ability of midbrain lesions to block phase also able to prevent light-induced phase shifts when admin- shift responses to triazolam, the same lesions had no effect on istered in advance of a light pulse at CT19 (Tierno et al., 2002). phase shifts induced by prolonged running in a novel wheel. This result is consistent with a presynaptic inhibitory action Thus, the neural substrates for triazolam and novel wheel- on RHT terminals, as suggested by the receptor localization in induced phase shifts are different, although each appears to the SCN (Byku et al., 2000). However, electrophysiological require an intact GHT as a final common path conveying evidence suggests that the primary site of action of the information to the SCN (Janik and Mrosovsky, 1994; Wickland effective delta opioid agonists might be outside the SCN and Turek, 1994; Johnson et al., 1988b; Marchant et al., 1997). (Cutler et al., 1999). These results support previous observations that chlordiaz- The ability of delta opioid agonists to block light-induced epoxide (a benzodiazepine) and fentanyl (an opiate mu opioid phase shifts is similar to the effect of NPY. In vivo infusion of receptor agonist), neither of which enhances locomotion in NPY onto the SCN greatly attenuates light-induced phase hamsters, nevertheless induce equivalent phase shifts (Biello advances and delays (Lall and Biello, 2002, 2003a,b; Weber and and Mrosovsky, 1993; Maywood et al., 1997; Meijer et al., 2000) Rea, 1997). In contrast, infusion of NPY antiserum onto the to those induced by triazolam. SCN augments phase advances to light during the late The second experiment differentiating response to tria- subjective night (Biello, 1995). Wheel running, which is zolam from response to novel wheel involved serotonin- thought to release NPY into the SCN through the GHT (Biello specific neurotoxic lesions of the median or dorsal raphe et al., 1994), appears to attenuate light-induced phase nuclei. Loss of serotonin neurons from the median (but not advances, but not delays (Mistlberger and Antle, 1998; Ralph the dorsal) raphe eliminated phase shifts in response to and Mrosovsky, 1992). Inhibition of phase response to light is triazolam treatment (Meyer-Bernstein and Morin, 1998). observable in the slice preparation if the light pulse is Novel wheel access elicited equivalent phase shifts regard- administered in vivo followed by removal of the brain and less of dorsal raphe, median raphe or control lesions. Thus, acute treatment of the SCN with NPY (Yannielli and Harring- it is clear that while a final common path from the IGL to ton, 2000). Similarly, NMDA stimulation of the SCN slice yields BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 37 large phase shifts that are blocked by coapplication of NPY intraperitoneal injection of saline (Mead et al., 1992; Hastings (Yannielli and Harrington, 2001; Yannielli et al., 2004) and the et al., 1992) may reflect an action of this stimulus complex NPY-blocked phase response to NMDA can be restored by through an alternate route to the circadian clock not requiring simultaneous treatment with an NPY Y5 (but not Y1) receptor locomotion for phase shifts (cf., benzodiazepine; Marchant antagonist. NPY induces phase advances when applied by and Morin, 1999; Biello and Mrosovsky, 1993), or the response itself to the SCN slice during the subjective day, but no to saline may in fact include a locomotor component not observable phase shift during the subjective night (Harrington measured in those studies. and Schak, 2000; Biello et al., 1997b). In vivo studies using One variation on the theme of nonphotic stimuli capable of intracranial injections showed that a Y5 receptor antagonist inducing phase advances has involved use of “dark pulses” enhances light-induced phase shifts. If the locomotor rhythm administered at CT6. The dark pulse is associated with phase response to light is blocked by intracranial NPY, the increased locomotion and consequent phase shifts in ham- shifts can be restored to normal by coinjection of NPY and the sters housed in LL (Boulos and Morin, 1985; Boulos and Rusak, Y5 receptor antagonist (Lall and Biello, 2003a; Yannielli et al., 1982). Phase advance responses can be greatly increased (up to 2004). Additional in vivo studies also demonstrated probable 7 h) if the dark pulses coincide with the “gentle handling” involvement of the Y5 receptor in the NPY suppression of paradigm for sleep deprivation (Mistlberger et al., 2002). light-induced phase shifts (Lall and Biello, 2003a) and lend Although the reference to dark pulses as a nonphotic stimulus credibility to the view that light and NPY (consequent to implies that it is of the same class as benzodiazepines, novel locomotor activity) normally act together to regulate phase wheel, gentle handling and saline injection, the mechanism of shift magnitude (Lall and Biello, 2002). action of dark pulses is not necessarily the same. In particular, The Mistlberger lab has added a new twist to the topic of IGL lesions block the ability of triazolam (Johnson et al., 1988b), nonphotic control of circadian rhythm phase. Initially, there novel wheel (Janik and Mrosovsky, 1994; Wickland and Turek, were two significant observations. One was that 24 h forced 1994) and saline injection (Maywood et al., 1997) (gentle treadmill activity blocked light-induced phase delays, where- handling has not been tested) to elicit phase advances, but as simple treadmill activity simultaneous with the light pulse they apparently do not block such responses to dark pulses did not alter phase. The second observation yielded the (Harrington and Rusak, 1986). converse result, namely, a shorter interval on the treadmill The effect of restraint has also become more ambiguous (6 h) blocked phase response to light (Mistlberger et al., 1997). than expected when considered in the context of dark pulses. Studies were then performed, using the Aschoff type II Restraint during the midsubjective day has no effect on procedure, to elaborate on the ability of “sleep deprivation” to rhythm phase (Van Reeth et al., 1991; Rosenwasser and induce rhythm phase shifts. In these experiments, sleep Dwyer, 2002). However, during the late subjective day, brief deprivation was accomplished using gentle handling (not or 6 h restraint will induce phase delays. At both circadian confounded by simultaneous locomotion) beginning at CT6. A times, restraint blocks the ability of a dark pulse to induce a 3 h procedure elicited a significant phase advance shift and a 1 phase advance (Rosenwasser and Dwyer, 2002; Van Reeth and h procedure yielded an even greater (about 150 min) shift Turek, 1989; Reebs et al., 1989). Thus, on the one hand, phase (Antle and Mistlberger, 2000). The shifts appear to occur in advances during the midsubjective day are blocked by about two thirds of the animals and it was observed that the unknown mechanism invoked by restraint, but one that is fewer the number of interventions needed to keep an animal apparently not directly related to the circadian clock; on the awake, the larger the phase shift to the sleep deprivation other hand, blockade of the dark-induced phase advance at procedure. Additionally, exposure to light (10 lx) during a 3 dusk may be the simple consequence of a restraint-induced h gentle handling interval effectively eliminated phase phase shift of equal magnitude but opposite direction (Rosen- advances. This observation suggests that the phase shifts wasser and Dwyer, 2002; Dwyer and Rosenwasser, 2000). consequent to “gentle handling” are not caused by the absence of sleep, but rather by a correlate. Such a correlate could be 8.4.3. Photic regulation of rhythmicity “stress” generated by the sleep deprivation procedure. One major function of the IGL is modulation of the circadian In a comprehensive series of experiments (Mistlberger et period during LL conditions (Pickard et al., 1987; Harrington al., 2003), several of the stress-related issues consequent to and Rusak, 1986). However, the visual system projects to a sleep deprivation were clarified. In particular, stress as variety of midbrain regions that connect with the IGL leaving reflected by elevated cortisol levels, is not associated with the possibility that photic information effecting the circadian phase advance shifts, nor are shifts elicited by restraint which clock acts indirectly through, rather than directly in, the IGL. also elevates cortisol (Mistlberger et al., 2003). Nor is sleep This perspective has been enhanced by the demonstration that deprivation caused by the “pedestal over water” method visual midbrain structures are involved in circadian rhythm associated with phase shifts. In contrast, mere access to a 1 response to a benzodiazepine and that phase shift magnitude m2 open field did cause phase shifts similar in magnitude to to a novel wheel stimulus is modulated by light (Marchant and those resulting from gentle handling. Phase shift magnitude Morin, 1999). IGL lesions reduce the circadian period length- was related to the amount of forward locomotion and those ening effect of LL by about 50%, but do not alter the normal animals classified as “shifters” engaged in considerably more period in DD (Morin and Pace, 2002). This result suggests the locomotion than the nonshifters. The conclusion of these IGL mediates the tonic effects of light. In contrast, large lesions studies is that some level of locomotion is indeed necessary to of the pretectum and tectum that eliminate rhythm response achieve phase advance shifts (Mistlberger et al., 2003). to benzodiazepine treatment (Marchant and Morin, 1999) have Observations that such shifts occur following the simple no effect on circadian period in LL (Morin and Pace, 2002). 38 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60

In mouse, IGL lesions lengthen the circadian period of DD- 9.1. Effects of serotonin-specific neurotoxins housed mice, but do not modify the period lengthening effect of LL (Pickard, 1994; Lewandowski and Usarek, 2002). In the rat, The sense of the literature is that, in hamsters, serotonin has IGL lesions do not alter the circadian period in DD nor do they an inhibitory effect on rhythm response to light which, in prevent LL-induced circadian rhythm disruption (Edelstein animals lacking median raphe serotonin neurons, is evident and Amir, 1999a). Period in LL before rhythm disruption was as a larger amplitude PRC, greater prevalence of, and shorter not assessed. Under skeleton photoperiod conditions (1 h of latency to, light-induced rhythm splitting and longer circadian light from ZT0-1 and ZT11-12, rats with IGL lesions generally periods under LL (Meyer-Bernstein and Morin, 1996; Morin and fail to entrain indicating the necessity of the IGL under these Blanchard, 1991; Bradbury et al., 1997). No effects on rhyth- conditions; Edelstein and Amir, 1999b). Equivalent studies micity are seen following destruction of only the dorsal raphe have not been done in other species. serotonergic cells. Most recently, evaluation of irradiance Just as NPY is able to attenuate light-induced phase shifts, response curves in animals with neurotoxic lesions of median effects of NPY on phase are also inhibited by light. Novel raphe serotonin neurons has shown similar sensitivities of wheel-induced phase advances are greatly attenuated by a lesioned and unlesioned controls to light, but a greater phase subsequent light pulse. Similarly, NPY-induced phase advan- response by lesioned animals to high intensity (probably ces are curtailed by a light pulse (Biello and Mrosovsky, 1995). saturating) light (Muscat et al., 2005). A glutamate agonist (NMDA), injected onto the SCN to mimic The drug of abuse, MDMA (‘ecstasy’), is rapidly toxic to the effect of photic input, also greatly reduces the phase serotonin terminals (Battaglia et al., 1991). Treatment of rats advance effects of NPY administered in the midsubjective day with this drug for several days reduces phase advances of the (Gamble et al., 2004). At CT20, NMDA injection onto the SCN rhythm in SCN firing rate by about 50% in response to 8-OH- induces phase advances (Mintz et al., 1999), but when given DPAT applied to a slice at ZT6 (Biello and Dafters, 2001). The simultaneously with NPY, small phase delays occur (Gamble effects of MDMA on rhythmicity have been extended to the et al., 2004). This result, in conjunction with the observation evaluation of its effects on hamsters in vivo. After drug that the effect occurs in the presence of TTX, suggests that injections of MDMA, phase advances to 8-OH-DPAT adminis- NPY is acting postsynaptically to induce phase shifts, rather as tered at CT8 were blocked. Light-induced phase delays were a presynaptic inhibitor of RHT function. convincingly reduced about 50% (Colbron et al., 2002), Glutamate receptor antagonists, known to modify SCN and although the effect is not consistent with the literature rhythm response to light (Abe et al., 1992; Rea et al., 1993b; showing that serotonin loss increases phase response to Vindlacheruvu et al., 1992), fail to alter light-induced FOS light. Damage to the serotonin terminals varies according to expression in the rat IGL (Edelstein and Amir, 1998). This is brain region, with some areas being completely unaffected by consistent with the likelihood that there appears to be little or the drug (Battaglia et al., 1991). In the above MDMA studies, it no glutamatergic innervation of the IGL (Fujiyama et al., 2003). is unclear as to which part of, or to what extent, the serotonin system has been damaged.

9. Serotonin and midbrain raphe contribution 9.2. Serotonin receptor function to circadian rhythm regulation A major development in the study of serotonergic effects on The 1994 review (Morin, 1994) fell victim to the typical problem circadian rhythm regulation has been the increased focus on of being partially out of date by the time it was published. No particular receptor types and their function. One critical topic was more affected by this than serotonin and its effect development has been the recognition that 8-OH-DPAT, long on circadian rhythmicity. In the year of publication, research thought to a 5HT1A-specific agonist and frequently used on the topic began to blossom and there have been several because of that specificity (see Morin, 1999), is actually specific major reviews published (Morin, 1999; Rea and Pickard, 2000a; to the 5HT1A, 5HT5A and 5HT7 receptors (Lovenberg et al., Mistlberger et al., 2000; Yannielli and Harrington, 2004). 1993; Sprouse et al., 2004a; and reviewed by Gannon, 2001). One of the more important changes in perspective There are now reports indicating that the SCN contains the concerning the role of serotonin has been a change of focus 5HT1A, 5HT1B, 5HT2A, 5HT2C, 5HT5A and 5HT7 receptor types away from the dorsal raphe nucleus, previously thought to be (Sprouse et al., 2004a,b; Belenky and Pickard, 2001; Duncan et the source of SCN innervation by serotonin fibers (Smale et al., al., 1999, 2000; Prosser et al., 1993; Oliver et al., 2000; Moyer and 1990; Azmitia and Segal, 1978). Anatomical studies, using three Kennaway, 1999). A useful review of the evidence favoring the separate methods (anterograde and retrograde tract tracing of presence and function of 5HT7 receptors in the SCN was raphe projections, and lesion studies designed to destroy published in 2001 (Gannon, 2001), but needs to be interpreted raphe projections), have unequivocally demonstrated that the in the context of subsequent information. median raphe sends mixed serotonergic and nonserotonergic The 5HT5A receptor has most recently been appreciated as projections to the SCN, and the dorsal raphe sends mixed a serotonin receptor type contributing to circadian rhythm serotonergic and nonserotonergic projections to the IGL (Moga regulation. It is distributed in the dorsal and median raphe, as and Moore, 1997; Meyer-Bernstein and Morin, 1996; Hay- well as in the SCN and IGL (Duncan et al., 2000). Thus, the Schmidt et al., 2003). This simple anatomy is complicated by 5HT1A, 5HT5A and 5HT7 receptors are found in similar places the presence of reciprocal serotonergic and nonserotonergic and respond to similar agonists/antagonists thereby increas- connections between dorsal and median raphe nuclei (Tischler ing the difficulty of discerning receptor-specific function. One and Morin, 2003). suggestion is that 8-OH-DPAT injection into the dorsal raphe BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 39 induces phase shifts via 5HT7 (but not 5HT5A) receptor ing upon prior light exposure (Knoch et al., 2004). Hamsters activation (Duncan et al., 2004), whereas systemic injection exposed to LL for as little as 1 day can have very large phase of the drug induces phase shifts by activating the 5HT1A shifts in response to intracranial NPY given at CT6, a sleep receptor (Tominaga et al., 1992). The phase response of deprivation procedure at ZT18 or 8-OH-DPAT at ZT18 or ZT21 hamsters to dorsal raphe application of serotonin 5HT1A/5A/ (Aschoff type 2 test). LL exposure also eliminates the early 7 agonists diminishes with age (Duncan et al., 2004; Penev et night time peak in peri-SCN serotonin and reduces the overall al., 1995), and this may be the result of specific loss of available level to approximately the typical late afternoon levels seen 5HT7 receptors (Duncan et al., 1999). under LD conditions (Knoch et al., 2004). In the rat, the 5HT2C receptor may contribute to the The 5HT1B receptor has received substantial attention with regulation of circadian rhythm phase. The receptor type is respect to its role in rhythm regulation. The receptor is present in the SCN (Moyer and Kennaway, 1999). Agonists of identifiable in the SCN, specifically on terminals of the RHT the 5HT2C receptor are able to induce phase delays in rats and (Belenky and Pickard, 2001; Pickard et al., 1999) and there is an induction of FOS in SCN neurons at CT18, but not at CT6 approximately 35% reduction in 5HT1B binding sites in the (Kennaway and Moyer, 1998), and generally mimic the effects SCN after bilateral enucleation (Pickard et al., 1996; Manrique of light. The suggestion has been made (Kennaway and Moyer, et al., 1999). 5HT1B receptor agonists markedly attenuate light- 1998) that previous observations of quipazine-induced phase induced phase shifts and the induction of FOS in the SCN, shifts in rat (Prosser et al., 1993; Kennaway and Moyer, 1998) although cells in a central region that appears to include the may have been the result of that drug's affinity for the 5HT2 CALB neurons continue to express FOS (Pickard et al., 1996, class of receptors. Kennaway and Moyer (1998) suggest that the 1999; Pickard and Rea, 1997; Shimazoe et al., 2004). Light- literature is ambiguous with respect to the certainty that the induced suppression of nocturnal melatonin is also alleviated rat SCN contains 5HT1A and 5HT7 receptors. Further, they by treatment with 5HT1B agonists (Rea and Pickard, 2000b). suggest the possibility that light may act through the known Infusion of receptor agonist directly onto the SCN eliminated retina-dorsal raphe projection in the rat (not evident in the phase response to light (Pickard et al., 1996). hamster; see Section 4.2) and it is the modulation of the 5HT2C Use of the 5HT1B receptor knockout mouse has yielded receptors on this pathway that results in light-like phase substantial additional data. Phase shifts elicited by light in this response and FOS induction (Kennaway and Moyer, 1998). strain are not modified by 5HT1B receptor agonists. Electrical Consistent with this view is the observation that reduction of stimulation of the optic nerve or glutamate application to the serotonin in rats by pCPA injection attenuates light-induced bath elicit excitatory postsynaptic currents (EPSCs) in SCN FOS (Moyer and Kennaway, 2000), suggesting that normal neurons recorded in a slice preparation. Agonists for the serotonin innervation is necessary for light-induced FOS. 5HT1B receptor greatly reduce EPSCs in the wild-type mouse, Agonist activation of 5HT2A/C receptors induces substantially but have no effect in the 5HT1B receptor KO mouse (Smith et (but not completely) light-like FOS expression in the rat SCN al., 2001; Pickard et al., 1999). Although the circadian period of (Kennaway et al., 2001). The investigators suggest that, in the wild-type mice does not differ from that of the 5HT1B KO mice, rat, but probably not in the mouse or hamster, there are two the period is longer in LL consistent with the view that this pathways by which light can modulate SCN rhythmicity. One strain lacks inhibitory control of retinal input to the circadian of these is the RHT, while the other involves the retinal visual system (Sollars et al., 2002). Most recently, bicuculline- projection to the dorsal raphe which, it is hypothesized, sends sensitive miniature inhibitory currents were recorded on SCN a direct serotonergic projection to the SCN that effects (which are predominantly GABAergic; Moore and Speh, 1993; glutamate release at that location (Kennaway et al., 2001). A Morin and Blanchard, 2001). Frequency of the inhibitory cur- dorsal raphe projection to the SCN is probably absent in the rat rents is reduced by a 5HT1B receptor agonist, but the agonist (Moga and Moore, 1997), as it is in hamster, but there is had no effect on the currents in SCN neurons of 5HT1B KO mice abundant dorsal raphe innervation of adjacent hypothalamus (Bramley et al., 2005). The authors suggest that abundant (Moga and Moore, 1997; Vertes, 1991) and peri-SCN neurons 5HT1B receptors are present presynaptically on GABA term- modulated by serotonin from the dorsal raphe may provide inals, and thereby modulate GABA release within the SCN. glutamate signaling to the SCN. Involvement of the 5HT2C receptor in rat rhythm regulation is further supported by the 9.3. Regulation and consequences of serotonin release observation that agonists specific for this type of receptor induce expression of FOS, Per1 and Per2 in the SCN. This effect Extracellular serotonin increases abruptly from daytime levels was more robust at ZT16 than ZT22 (Varcoe et al., 2003). to higher night time levels in both the peri-SCN and peri-IGL The issue of species differences has been addressed by region (Dudley et al., 1998; Grossman et al., 2004). Similarly, direct comparison of mouse and hamster responses to a extracellular serotonin increases in both the SCN and IGL in variety of 5HT1A, 1B, 2 or 7 agonists. The results are complex response to certain nonphotic stimuli (Dudley et al., 1998; with differing phase and spontaneous Fos-induction Grossman et al., 2000, 2004). Serotonin availability in these responses in each species, plus differing effects on light- nuclei is also augmented by electrical stimulation of the dorsal induced phase shifts and Fos-induction. In addition, response raphe nucleus (Grossman et al., 2004; Dudley et al., 1999; Glass has also been shown to be related to whether the drug was et al., 2000b, 2003b). injected centrally (SCN) or administered peripherally (Antle et Despite these effects on serotonin release, it is not yet al., 2003b). One aspect of the complexity of serotonergic known to what extent the stimulation-induced transmitter activity relative to the effects of light is the demonstration increase in either the SCN or IGL contributes to circadian that phase response to agonists can vary enormously depend- rhythm phase control. Studies involving electrical stimulation 40 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 of the dorsal or median raphe in which serotonin neurons have been destroyed have not been performed. Arguing against a normal effect of serotonin on rhythm phase control is the observation that treatment of hamsters with a 5HT1A receptor antagonist elevates serotonin release in the peri-SCN region to levels comparable to those seen during novel wheel access or raphe electrical stimulation without augmenting phase shifts in response to novel wheel activity (Antle et al., 2000). Nor do systemic or SCN injections of 5HT1/2, 5HT1A and 5HT2/7 receptor antagonists alter novel wheel-induced phase shifts (Antle et al., 1998). Consistent with those results is the observation that the serotonergic median raphe projections appear to be essential for triazolam-induced phase shifts, but not for those induced by novel wheel activity (Meyer-Bernstein and Morin, 1998). It is possible that the electrical stimulation activates nonserotonergic raphe cells projecting to the SCN or IGL (Meyer-Bernstein and Morin, 1996). The most directly related data on the topic come from the mouse in which a serotonin- specific neurotoxin was injected intracranially into the region of the SCN (Marchant et al., 1997; Edgar et al., 1997) and, in Fig. 5 – Diagrammatic conceptualization of the functional combination with IGL lesions, block activity-related phase serotonergic circuitry governing circadian rhythm regulation. control in the mouse. The suggestion is that the serotonergic Based on an unpublished update provided courtesy of Dr. J.D. projections to the SCN destroyed by the neurotoxin are Glass, modified from the previous version (Glass et al., necessary for such phase control. However, in the experiments, 2003b). The presumptive intra-IGL GABA connection is based serotonin depletion occurred widely within the diencephalon upon unpublished work from the Glass laboratory. There are and localization to the SCN cannot be assured. In the hamster, data for (Glass et al., 2003b) and against (Meyer-Bernstein and small injections of neurotoxin that destroyed nearly all Morin, 1998) the likelihood that behavioral input induces serotonergic innervation to the SCN without damaging most phase shifts by acting through the serotonin neurons of the of the surrounding hypothalamus had very little effect on dorsal raphe nucleus. circadian rhythmicity compared to the consequences of loss of allmedianrapheprojections (Meyer-BernsteinandMorin,1996; Meyer-Bernstein et al., 1997; Bobrzynska et al., 1996). There are occurs when the drug/light combination occurs at CT14. The two implications of these studies. One is that median raphe reason for this is not clear, although it may result from projections to non-SCN targets (also destroyed by intraraphe cancellation of light-induced phase delays by drug-induced neurotoxin injection) may contribute to the regulation of phase advances when each is administered at CT14. At CT19, hamster circadian rhythmicity. The other is that the dense BMY 7378 alone induces small (1 h) phase advances, but the serotonergic projection to the SCN (destroyed by the intra-SCN CT19 combination of light and drug yields shifts more than neurotoxin) may be minimally involved in such regulation. 300% larger than those induced by light alone (light-induced One perplexing issue has been the fact that electrical shifts are about 1.5 h; potentiated shifts are about 5 h; Gannon, stimulation of the dorsal or the median raphe nuclei yields 2003). This phenomenon is similar to what has been obtained similar release of serotonin in the SCN (Dudley et al., 1999) and with other drugs with different effects on phase response in stimulation of either nucleus during the subjective day elicits the absence of light (Gannon, 2003; Rea et al., 1995; Moriya et equivalent magnitude phase advances (Meyer-Bernstein and al., 1998). For example, NAN-190 administered alone at CT14 or Morin, 1999). A model has been developed (Glass et al., 2003b) CT19-20 has essentially no effect on rhythm phase (Rea et al., that provides a reasonable reconciliation of behavioral, 1995; Moriya et al., 1996), but potentiates both phase advances pharmacological and anatomical data (Fig. 5; see Glass et al., and delays when administered at those times in combination 2003b for an extensive explanation and presentation of the with light (Rea et al., 1995). One interpretation (Gannon, 2003) data implicating GABA). The similar effectiveness of stimula- is that the drugs, which have mixed agonist/antagonist tion occurring in either dorsal or median raphe may be properties, facilitate light-induced phase shifts by acting as possible because of the serotonergic–nonserotonergic inter- agonists on presynaptic 5HT1A receptors of median raphe connection between the two nuclei (Tischler and Morin, 2003). serotonin cell dendrites to inhibit release of serotonin in the SCN. Elsewhere in the brain, the drugs act on postsynaptic 9.4. Serotonergic potentiation of light-induced phase shifts 5HT1A receptors which are, presumably, antagonized by the drugs. WAY 100635, a 5HT1A antagonist, has no effect on It is generally the case that a manipulation during the light-induced phase advances (Smart and Biello, 2001), and it subjective night that modifies phase advances will also is thought that the particular combination of raphe cell modify phase delays. Such is not true for the drug BMY 7378. autoreceptor activation plus the antagonism of SCN postsyn- Administered at CT19, this drug potentiates the phase shifting aptic receptors is necessary for the large light-induced phase effect of light given shortly thereafter. However, no such effect advances potentiated by the mixed agonist/antagonists BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 41

(Gannon, 2003). If this hypothesis is correct, then it may be possible to mimic the effect of the mixed agonist/antagonist drugs by concomitant injection of a 5HT1A antagonist and a 5HT1A agonist known to act on raphe autoreceptors. The complex control of serotonergic involvement in rhythm regulation is further suggested by the observation that depletion of brain serotonin (88% lower in the peri-SCN region) by systemic pCPA treatment greatly augments phase shifts in response to SCN application of serotonin or the 5HT1A/7 receptor agonist, 8-OH-DPAT (shifts are about 100 min with pCPA treatment vs. 30 min without) (Ehlen et al., 2001). The results suggest that a necessary condition for normal phase response to agonists is lack of activity at the postsynaptic receptors. Short-term changes in postsynaptic receptor number or sensitivity to endogenous or exogenous agonists may be critical to their effect on rhythm phase (see Ehlen et al., 2001 for a discussion). In animals pretreated with pCPA, phase advances resulting from 8-OH-DPAT infusion onto the SCN at CT6 were reduced by about 68% in animals treated with 5HT7 receptor antagonists (Ehlen et al., 2001). On the one hand, the result supports the view that 5HT7 receptors mediate the effect of serotonin agonists applied to the SCN. On the other, there is a significant residual phase shift effect of 8-OH-DPAT that may be attributable to its affinity for the 5HT1A receptor. It is also interesting that light stimulation simultaneous with 8-OH- DPAT (i.e., CT6) in pCPA treated animals completely blocks the phase shifting effect of the 5HT1A/7 agonist (Ehlen et al., 2001). The in vivo result is consistent with observations that glutamate application to the SCN slice preparation, which presumably mimics the excitatory effect of light exposure, blocks the phase shifting effect of NPY or serotonin (Biello et al., 1997a; Prosser, 1998). The presumption is that the action of serotonin takes place in the SCN, although there is evidence for IGL involvement in the response to 8-OH-DPAT as well (Challet et al., 1998b).

10. Failure to re-entrain

It is axiomatic that an entrained animal will re-entrain to a shifted light–dark cycle. In this era of transgenic animals specifically designed to show abnormal behavioral traits that will allow novel probing of the way things usually work, it is ironic that one of the more provocative developments during the last decade has been the discovery that a standard Fig. 6 – Failure to re-entrain to a shifted photoperiod by laboratory rodent, the Siberian hamster, is a model for Siberian hamsters. The initial photoperiod was LD16:8 (white failure to re-entrain. The initial observation occurred in both and black bars at the top of each record; lights off = ZT12) young and old animals entrained initially to an LD 16:8 which, on the day of the arrowhead, was changed. On that photoperiod, then subjected to a 5 h delay of the photope- day at ZT17, the lights were turned on for 2 h. The next day, riod. About 90% of animals had free-running locomotor and the LD16:8 photoperiod was delayed by 3 h. Shading areas body temperature rhythms for many weeks after the shift or indicate the times of lights off under the modified became arrhythmic (Ruby et al., 1996, 1997)(Fig. 6). photoperiod. This combination of light manipulations Melatonin treatment, known to modify the phase angle of prevents re-entrainment in most animals. The expected entrainment in Siberian hamsters (Margraf and Lynch, 1993), re-entrainment (A) sometimes follows the phase shift, but is reduced the percentage of animals failing to re-entrain to a less likely to occur than the lack of re-entrainment (B, C). One shifted photoperiod (Ruby et al., 1997). Analysis of melatonin case (C) demonstrates neither re-entrainment nor a clear levels has shown that animals subjected to a 5 h phase delay free-running circadian rhythm, and a significant percentage have very low circulating melatonin at a time that it is of animals become arrhythmic. Unpublished data courtesy of expected to be high (Ruby et al., 2000), suggesting that the Dr. N.F. Ruby. 42 BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 absence of melatonin may contribute to the inability to A second major reason for studying masking concerns the achieve a stable phase angle of entrainment after the shift. anatomy of the visual system. Light-induced masking is a Siberian hamsters re-entrain properly to 1 or 3 h delays of visual function, but the visual system anatomy contributing to photoperiod phase, and to 1 h advances, but much less well to 3 it has not been satisfactorily elucidated. h advances. Generally, these animals do not re-entrain prop- Normal mice generate a characteristic irradiance response erly following 5 h advance or delay shifts in the photoperiod, curve when tested for light-induced masking of running wheel although the effect is modifiable by the exact manner in which revolutions. Through a middle range of intensities, the the photoperiod is shifted (Ruby et al., 1998, 2004). If Siberian stronger the stimulus, the greater the extent of negative hamsters are exposed to a 5 h phase delay in the photoperiod, masking. With very dim stimuli, activity level actually then immediately placed in DD for 14 days, all animals will re- increases slightly (positive masking; see Mrosovsky, 1999; entrain when the shifted photoperiod is resumed. If DD Redlin and Mrosovsky, 1999a). At the level of the retina, both exposure is delayed for several days, re-entrainment probabil- classical and melanopsin photoreceptors have been shown to ity is reduced to less than 50% (Ruby et al., 2002b). contribute to masking (Hattar et al., 2003; Panda et al., 2003). The Siberian hamster is noted for the existence of Melanopsin cells influence masking responses to the higher individuals in the general population that fail to undergo go- light intensities (Mrosovsky and Hattar, 2003). Loss of rod nadal regression when exposed to a short day photoperiod and function in mice eliminates the positive masking seen at dim breeding has selected this trait in a strain of animals identified illumination levels, but for some strains, there may also be a as ‘nonresponders.’ Photoperiodic time measurement for re- significant reduction in the masking response to higher production control relies on the use of a circadian clock (Elliott intensities (Mrosovsky et al., 1999, 2000). et al., 1972; Rusak and Morin, 1976; Stetson and Watson- The analysis of visual projections contributing to negative Whitmyre, 1976). Comparison of the responder and nonre- masking remains incomplete. Mrosovsky and colleagues have sponder strains with the phase shift paradigm has shown that evaluated contributions of the visual cortex, superior collicu- they also have the traits of re-entrainment and nonre- lus, lateral geniculate and SCN without obtaining data entrainment, respectively (Ruby et al., 2004). demonstrating that one of these areas is necessary for negative Most recently, Siberian hamsters that fail to re-entrain to a 5 masking. In fact, the most common effect of lesions is actually h photoperiod shift have been exposed to light to induce SCN increased masking. This is true for lesions of the visual cortex, gene activity. In animals that re-entrain, Fos and Per1 gene superior colliculus, dorsal lateral geniculate and IGL, although activity is induced, as expected, in SCN neurons. In contrast, it has not yet been possible to distinguish the effects of gene induction by light apparently does not occur in animals dorsolateral geniculate lesions from lesions aimed at the IGL that fail to re-entrain (Barakat et al., 2004). This is conceivably (Edelstein et al., 2001; Redlin et al., 1999, 2003). Lesions of the related to the absence of light-induced gene expression in SCN render animals arrhythmic and masking experiments golden hamsters showing split rhythms in LL (De la Iglesia et al., must, of necessity, be conducted differently from those in 2000). The collective results of these studies indicate that the which animals have normal circadian locomotor rhythms. lighting conditions dictate subsequent response of the Siberian When SCN lesioned animals are given access to running hamster circadian system to light. The most extreme con- wheels in a 3.5 h light:3.5 h dark schedule, about 90% of their clusion is that a consequence of the 5 h phase shift renders the wheel revolutions occur during the dark intervals and the circadian rhythm system unresponsive to the effects of light. same animals show a strong preference for darkened vs. a lighted chamber (Redlin and Mrosovsky, 1999b). These results indicate that negative masking is not abolished by SCN lesions. 11. Masking By inference, it would also appear that retinal projections passing through the SCN to the subparaventricular zone and Mrosovsky (1999) is synonymous with masking and has dorsomedial hypothalamus are not necessary for masking provided a substantial review of the topic. Masking is because they would also be destroyed by the lesions. However, important to the study of circadian rhythms for several these conclusions must be tempered by a recent report reasons. One is purely technical to the extent that, under a indicating loss of negative masking in SCN-lesioned hamsters variety of circumstances in which there are questions (Li et al., 2005). This study did not address possible masking concerning whether the evident rhythm is imposed by the functions of retinal projections destroyed while passing environment or is from endogenous sources, tests must be through and around the SCN to other hypothalamic sites. conducted to obtain an explicit answer. For example, mice Despite the absence of a demonstration that a particular lacking the genes coding for cryptochrome1 (Cry1) and structure is necessary for masking, the Mrosovsky work cryptochrome2 (Cry2) show nocturnal locomotor activity collectively supports the view that two visual processes under LD conditions, but are arrhythmic under DD (Albus govern the behavior. One, involving thalamus, midbrain and et al., 2002; Van der Horst et al., 1999). The transfer into DD is cortical visual structures, provides a normal inhibitory effect the test of persistent rhythmicity with the same initial phase on the magnitude of the masking response to light. The other, as seen under LD conditions. In the case of the Cry1/2 KO presumably in one of the two dozen or so other retinorecipient mice, the result of the transfer demonstrated that the brain regions (Morin and Blanchard, 1999; Horowitz et al., absence of wheel running during the light portion of the 2004) not yet tested, or in the SCN (Li et al., 2005), actually previous daily photoperiod occurred because the running controls the negative masking response to light. was strongly suppressed by the negative masking effects of As indicated above, the rhythmicity of Cry1 and Cry2 double light. KO mice under LD conditions has been explained as a BRAIN RESEARCH REVIEWS 51 (2006) 1– 60 43 phenomenon of negative masking because these mice are ar- types and already there is typical scientific confusion with one rhythmic under DD conditions (Albus et al., 2002; Van der Horst method showing an unexpected absence of rhythmicity in et al., 1999). This issue has also been tested in the standard certain cells (Hamada et al., 2001), while another method fashion (1 h light pulse at various levels of illumination). The demonstrates an unexpected form of rhythmicity in the same double KO animals show masking which is no different from type of cell (Hamada et al., 2003). This topic is rich and likely to the wild-type controls, i.e., they are not more sensitive to the contribute substantially to such issues as the bimodal rhyth- activity suppressing effects of light than the wild-type animals micity observed in horizontal slices through the hamster SCN, (Mrosovsky, 2001). These mice show bouts of “predark” function-specific rhythm regulation and the general problem locomotion the magnitude of which may be related to the of specifying which cells are pacemakers. prevailing light intensity. These animals may have an altered Research focused on the IGL component of the circadian phase angle of entrainment that requires specific plotting visual system has refined the view that it contributes to both methods to discern (see Fig. 3 in Mrosovsky, 2001). photic and nonphotic regulation of rhythmicity. However, the research on IGL connectivity has pointed toward a function for this nucleus that may extend well beyond its role in circadian 12. The future rhythm regulation. In particular, the IGL appears to have widespread, bilateral and often reciprocal connections with Circadian rhythmicity is a pervasive feature of mammalian brain regions involved in the regulation of sleep, visuomotor physiology and behavior. Thus, it should not be surprising that and vestibular system activities. The nature of the functional discoveries concerning regulation of the circadian visual relationships among these systems remains to be seen, as is system have importance with respect to understanding the the circadian system role of the IGL with respect to their workings of the brain and body. In this context, the discovery regulation. of melanopsin as a photoreceptive molecule in a subset of Theserotonergicsysteminfluenceonrhythmregulationhas ganglion cells is one of the great strides forward in the study of been substantially elucidated, but large parts of that influence the circadian visual system during the last decade. However, remain a mystery. One characteristic, in particular, is compel- as is already being demonstrated (Dacey et al., 2005), it is ling and not yet understood. Certain manipulations of the unlikely that the importance of these ganglion cells lies serotonergic system contribute to very large, rapid phase shifts exclusively with photic regulation of circadian rhythmicity. to photic stimuli. How the circadian clock can overcome the The melanopsin cells of the retina are likely to project widely inertia of a stable, entrained system is not understood, but the in the visual system and influence most, if not all, retinor- presence of such large shifts gives hope that there may be ecipient regions. forthcoming manipulations that can reliably overcome the It is clear that the visual regulation of circadian rhythms detrimental effects of jet lag or modify the severity of timing is influenced by the classical visual photoreceptors as well disorders such as advanced sleep phase syndrome. as the intrinsically photoreceptive ganglion cells. There is an The last decade of research on the circadian visual system opportunity for the circadian rhythm field to contribute to has been extremely exciting and productive. The current understanding of retinal function by establishing the ana- decade will see this trend continue unabated with a much tomical and functional relationships between rods, cones enhanced recognition of the contributions made by investi- and melanopsin cells with respect to rhythm response to gators in the field of circadian visual system research to the light. understanding of general brain function and the normal The most obvious influence of circadian rhythm research physiology of bodily systems. on the understanding of normal bodily function may be derived from the large amount of research (most of which has not been considered by this review) concerned with the Acknowledgments molecular regulation of clock-like activity in the SCN. These studies have discovered a new window into the exploration of Supported by NIH grants NS22168 and MH64471 and National mammalian physiology through the demonstration of oscil- Space Biomedical Research Institute grant HPF0027 via lating clock genes in peripheral tissues. While not necessarily Cooperative Agreement NCC9-58 with the National Aeronau- circadian oscillations themselves (but see Yoo et al., 2004 to tics and Space Administration (to LPM) and NIH grants the contrary), under normal conditions, the genes do oscillate NS36607 and NS40782 (to CNA). with many of the same characteristics as those in the SCN (Yagita et al., 2001). The widespread presence of oscillating REFERENCES presumptive clock genes undoubtedly has enormous ramifi- cations for the normal functioning of organs as well as in disease states. Abe, H., Rusak, B., 1992. 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