Origin of Circadian rhythmicity

Review of Literature

Review of Literature Most organisms living in natural conditions express daily rhythm in their behaviour, physiology and biochemistry. Much of what organisms do is temporally organised with respect to the environmental day and night cycle. This observation, inspite of its simplicity, is central to circadian biology. The circadian system optimises an organism's physiological and behavioural adaptation to predictable diurnal or seasonal changes in the environment. This predictive homeostasis is essentially anticipatory, enabling the organisms to respond in advance to predictable variations in environmental stimuli (e.g., day length, temperature, food availability). In essence, the circadian system translates environmental information to the internal milieu via several sensory systems, integrates this information and then broadcast this signal to the rest of the body. Origin of Circadian rhythmicity The evolutionary history of circadian systems remains highly speculative. Pittendrigh and Daan (1976) speculated that light and dark cycle are the selective agents and these rhythms reflect effects of solar radiation on cellular processes such as gene induction and DNA replication. Edwards and Laval Martin (1984) correlated this with rhythmic gating of cell division and minimisation of the inhibitor effects of light during DNA replication. This is no doubt a very attractive hypothesis. But it is quite amazing to find the spontaneously recycling internal timer which functions even under constant conditions which is rarely seen in nature- after all organisms were only selected for their ability to show rhythmicity in a regularly cycling environment. Many other theories were also given to explain the origin of rhythmicity. Paieta (1982) proposed that the concentration of free oxygen is responsible. Kippert (1987) speculated that it is primarily not necessary to synchronise with the environment but there is the necessity of co-ordination of metabolism between largely autonomous compartments of unicellular organism which result in the evolution. The hypothesis proposed by different scientists, 7

Review of Literature suggested that the different principles might have contributed to the origin of circadian clock under different conditions. Oscillators The daily rhythms that are observed in different organisms in nature persist equally vigorously in the laboratory under constant conditions. This strongly suggests that the rhythms are generated endogenously. Another crucial and important fact that has been recognised is the deviation of the periodicities of different rhythms, expressed under constant conditions from 24 hours. This difference between the 24 hour-geophysical periodicity and the non 24 hour periodicity expressed by the biological rhythms, may be accepted as a proof of endogenicity of the latter. The sky lab experiments (Sulzman et al., 1984) proved beyond doubt that the rhythms are endogenous in nature. Implicit in this concept of endogenicity of the circadian rhythms is that the rhythms are driven from within the organism by sole pacemaker, a self sustained oscillator, that times the system. The effort to understand the physiology of the circadian system is invariably based on the conceptual model of the circadian system that consists of four functionally defined components see Fig.l. This model consists of ;..- Pacemaker(s) that generates primary timing cue..- Photoreceptors..- The coupling system one that controls the flow of information from the photoreceptors to the pacemaker(s) and..- Effector follower systems On the basis of this pacemaker may be broadly defined as those parts of the circadian system which confers upon the system the ability to persist in its rhythmicity without rhythmic environmental inputs. Experiments with fruitflies (Pittendrigh, 1954) 8,lizards (Hoffman, 1957) 8, chicken (Aschoff and Meyer-Lohman, 1954) 8, mice (Aschoff, 1960) 8, rats (Brown, 1952) 8 and humans (Martin-du pan, 1974; Miles et al., 1977) 8 raised under 8

Review of Literature continuous light or darkness have shown that animals need not expenence light-dark (LD) cycles to posses a circadian clock. ZEITGEBER ENTRAINMENT PATHWAY I Photic input I ' RHT CLOCK MECHANISM Pacemaker (SCN) COUPLING PATHWAYS Activity Arousal Sleep OUTPUT PATHWAYS ANDFEEDBACKLOOPS ~--~--~ CIRCADIAN RHYTHM Fig.l Schematic diagram of components of a generalised mammalian biological clock as a hierarchical system. Although protists and individual cells in metazoa probably contain or constitute clocks in their own right, it is probable that particular groups of cells or organs in a metazoan organisms have a particular clock function. The search for anatomical localisation of such driving oscillators occupy central and important position in the study of circadian systems. It should be stressed that the use of the term circadian clock does not mean that a single discrete clock is responsible all of an organisms daily rhythms. It is now well known fact that multicellular organisms are multioscillator in nature, that is more that one circadian clock may exist in an organism. Normally however all of an 9

Review of Literature organism's different overt rhythms exhibit a fixed phase relationship with one another. So, when multiple clocks exist within an individual organism, they must be coupled together in some manner. The anatomical correlates of the functionally defined circadian clock in different organisms are given in Table.l. It should be noted that the putative pacemakers mentioned in this table are not the only pacemaker these organisms. Although bio-rhythmicity can be considered as a fundamental property of a single cell, any analysis trying to elucidate the anatomical basis of rhythmicity in multicellular animals led to focusing the attention on the complex structure- the brain. At the present state of knowledge, it is impossible to have a clear cut anatomical model of the circadian oscillators because of the variety of structures involved though recent findings indicate that there are some master oscillators which control all other oscillators for example suprachiasmatic nucleus (SCN) of mammals. Properties of the Clock Each biological rhythm is composed of repeating units called Cycles. The time length required to complete an entire cycle in the rhythm is known as Period and is represented by "t (24 hours is the period in the case of a circadian rhythm). The magnitude of the change in activity during a cycle (the difference Time (hours) Fig.2: Phase response curve of circadian activity of a diurnal animal. 10

Review of Literature between the peaks and the troughs) is the Amplitude. Any specified, recognisable part or portion of a cycle is called a Phase and the curve representing the activity cycle with respect to time is known as Phase response curve (PRC), Fig. 2. Generally phase advances are plotted in positive direction along vertical axis and phase delays are plotted downward. Despite their endogenous nature, circadian rhythms are responsive to light and darkness. A circadian rhythm can to a certain extent be advanced or delayed (but it depends upon the past history of the animal) (Pittendrigh and Dann, 1974), depending on the phase at which light or darkness is presented. Such advances and delays are called Phase Shifts, for example in nocturnal Table 1 S.No. Organisms/Species I Area or Localisation 1. Scorpions Brain 2. Limulus Brain 3. Stick insect Protocerebrum 4. Cricket Optic lobes 5. Silk moth Cerebral lobes 6. Drosophila Brain 7. Beetle Eye 8. Aplysia Eye 9. Crab Eye stalk 10. Hagfish Ventromedial part of diencephalon 11. Fish Pineal 12. Bulla Eye 13. Frog Eye 14. Reptiles Pineal 15. Desert iguana SCN 16. Aves Pineal/SCN 17. Mammals SCN animals presenting light at the beginning of its active period induces a phase delay, whereas light at the end of the activity causes a phase advance. Light pulses during the resting phase of the animal do not effect the rhythm, even when 11

Review of Literature the animal is wakened. The time-dependent responsiveness of the circadian system is usually summarised in phase-response curve. When an animal is exposed to the natural light-dark cycle, light will advance or delay the circadian rhythm, depending on when the animal is exposed to the light. The results will be an advance for animal with a period >24 hours and a delay when this period is< 24 hours. Thus the overall result is that the endogenous rhythm adopts the environmental periodicity of precisely 24 hours. This process is known as entrainment. The agents responsible for entrainment can be photic and non photic. Though light is a primary entrainment agent or zeitgeber but besides light there are other non-photic agents. The idea that biological pacemakers can be reset by stimuli other than light goes back a long way (Halberg et al., 1954). The general findings were centred on synchronisation by periodic feeding or by social interaction. These findings suggested that non-photic events may be weak zeitgebers, secondary to light (Menaker and Eskin, 1966). Later social entrainment was thought to be more important than light (Aschoff, 1978; Wever, 1979). More recent work on clock-resetting by non-photic agents suggests nonphotic zeitgebers to be generally weak but under some conditions could be as large as those to the light pulses the phase response curve varies from species to species besides differing with the age of the individual (Pittendrigh and Daan, 1974). They showed that t shortened as the animal aged, later on they correlated the shortening oft with the testosterone level of the animal. By convention when a nocturnal animal becomes active before darkness the phase angle is positive. When it becomes active after dark, the phase angle is negative. The phase angles obtained depend on the relationship between the periodicity of the cycle of entraining stimuli (T) and the periodicity of the animal's free running rhythm (t ). If Tis longer than t, calling for delays, then there are large negative phase angels and the activity pulses fall in the late subjective night. If T is shorter than t, calling for advances, then phase angles are small and the pulses fall close to the time of activity onset. 12

Scorpion Pittendrigh and Daan (1976), concluded that after effects are generally written in the pages of the clock. After effects are slowly decaying changes in pacemaker period incurred by prior experience. This can last for 100 days. All these after effects can be attributed to light. Besides after effects there are transients, these are cycles of rapidly changing duration intervening between two steady states, each characterised by a nearly stable (Pittendrigh and Dann, 1976). Transients lasts for a few days only and thus are distinguished operationally from after effects which can persist for a few months. The concept of an endogenous clock that was first formulated by Johnson remained largely hypothetical for another 33 years. In 1972, two research groups independently identified a structure in the anterior hypothalamus of the rat that appeared essential for circadian rhythmicity and secretion and synthesis of several hormones (Moore and Eichler, 1972; Stephan and Zucker, 1972). The localisation of the 24 hour clock was guided by the expectation that a neuronal connection between the clock and the optic pathways could reasonably be expected because the circadian clock is susceptible to light. By selective lesioning along the optic pathways, the SCN were eventually found to be essential for the circadian activity rhythm. Consecutive lesion experiments have demonstrated that the SCN drive a great number of other circadian rhythms in rodents, such as sleep-wake cycle, the food intake rhythm and the rhythm in pineal melatonin and temperature. The identification of an endogenous clock in mammals boosted the identification of other possible candidates for clocks in other organisms, a list of possible clock is already given in Table 1. As our work is limited to Scorpion and SCN of rat, we are reviewing the concerned species only. Scorpion The scorpion Hetrometrus swammerdami, belongs to Phylum Arthropoda, class Arachnida, commonly they are known as Giant Rock Scorpion. These scorpions 13

Scorpion are found in tropical and subtropical region. Scorpions are generally hated and feared by common people. The scorpions were evolved during Silurian period and dominated the world since then. During the entire course of their evolution very little changes have taken place, hence they are also called living fossils. Due to unavailability of scorpions and their furious nature they are not extensively worked upon and the literature is limited. The most extensive area of research in scorpions is their toxins, their chemical nature and functional significance. The first study on neurosecretory cells and their rhythmic discharge was reported by Habibulla, 1970. The nature of neurosecretory cells in scorpions and other Arachnids was described, first by Hanstrom, 1923 (as cited by Habibulla, 1970). Later Gottlieb (1926 as cited by Habibulla, 1970) compared the protocerebral cells (neurosecretory cells) of the scorpion. These neurosecretory cells are present in the protocerebrum of Hetrometrus swammerdami. There are three sets of neurosecretory cells in the brain, which are situated as three sets in the protocerebrum and metamerically arranged in the subesophageal ganglion. The metameric arrangement of neurosecretory cells are different from other arthropodan members. Furthermore, the observations on the neurosecretory cells and secretions indicate an ultimate relationship to the day-to-day activities and metabolism of the animal. Diurnal rhythm of neurosecretory activity could be seen in these cells. When extracts of these neurosecretory cells at the time of their peak activity is injected to another scorpion, they start showing activity and induced locomotion is seen (Rao and Habibulla, 1973). Till late eighties the work on scorpion was centred around neurosecretory cells only. Fleissner, described the circadian clock of scorpion and predicted its location in eyes. Most scorpions possess two pairs of eye-groups commonly referred to as median eyes and lateral eyes. One pair of comparatively large ocelli in close apposition to each other is located in the middle of the prosoma, while two to five pairs (depending on the species) of small lateral eyes are found near its antero-lateral rim. Both eyes have in common the presence of retinula cells, pigment cells, arhabdomere cells and neurosecretory fibres. The visual retinula cells are the main cellular constituent of the retina. They bear a distal receptive 14

Scorpion segment characterised by rhabdomeral microvilli that interdigitate with microvilli of neighbouring cells to form a rhabdom. Since rhabdomeres extend around the entire periphery of the receptive segments of all retinula cells, the retina forms an interconnected rhabdomeral network (Schliwa and Fleissner, 1980). Visual cells of both the eyes, lateral as well as the median, display characteristic movements of pigment granules within their cell bodies. In the light adapted state, pigment granules accumulate in the distal portion of the cell close to the preretinal membrane and are more sparsely distributed in other regions of the cell (except the perinuclear sheath of pigment granules). In the dark adapted state, pigment granules gather near the nucleus, but are more sparsely distribute in the receptive segment. On the other hand when scorpions are maintained in constant darkness, retinula cells of the lateral eye show only weak circadian pigment movements. This is in marked contrast to median eyes where displacements of the secreting pigment are rather dramatic under these conditions (Fleissner, 1974). Arhabdomeric cells are present in retinula unit of median eye, they do not contribute to the structure of the rhabdom. It seems that they do not contribute to the circadian rhythmicity. The retinae of both, the larger and smaller lateral eyes, are supplied with neuroscretoy fibers. Essentially similar as the median eyes (Fleissner and Schliwa, 1977). They contain electron-opaque vesicles 80-100 nm in diameter, mitochondria and glycogen granules. Generally there is one filter per retinula cell. The efferent neurosecretory fibers in the optic nerve are found to convey the circadian signals (Fleissner and Fleissner, 1978; Fleissner,and Schilwa, 1977) and may represent essential components of this clock system. The optic nerve supplying the median eye of the scorpion contains only three types of axons:..- Axons of the visual cells..- Axons of the arhabdomeric cells..- Neurosecretory axons. The somata of fibers of the first and second types are located within the retina (Fleissner and Heinrichs, 1977). The circadian system of the scorpion is a bilaterally symmetrical unit and there is an internal synchronisation between the 15

Scorpion left and right sides (Fleissner, 1977). There is a reciprocal interaction between the two median eyes, such that contra lateral adaptation occurs (Fleissner, 1972). For example, when the left median eye is exposed to light while the right is in darkness, light adaptation can be observed in the right eye as well as in the left. In summary, these various fine structural characteristics strongly suggest that the importance and biological role of lateral eyes differs from that of median eyes. One possibility is that they function as light detectors for which a decrease _in activity would be advantageous. Furthermore, lateral eyes are light sensitive and can detect differences in brightness even at very low light intensities. Their sensitivity lies at least 1 log unit above that of the median eyes and unlike the latter, they do not show appreciable circadian variations in the sensitivity. Though for 1970 to 80 the position of the biological clock was considered to be existent in the eyes only but in late seventies the concept changed and Fleissner in 1987 gave a model that though the sensitivity of the median eyes of the scorpion displays clear cut circadian rhythm but it is controlled via the optic nerve by an oscillator located in the central nervous system. The efferent neurosecretory fibers (EMSF), that are presynaptic to the visual cells have been described as probable pathways for the circadian signal. The location and anatomy of clock of scorpion seems to be similar to that of cockroach (Periplaneta americana), where frontal Ganglion also shows circadian rhythmicity and acts as an effector follower system except that in scorpions median eyes also perceive the environment cell i.e. light (Pandey and Habibulla, 1982). Our work on scorpion is to locate the exact position of clock and to find out the basic neurochemical rhythmicity in it and establish a model system. 16.

Suprachiasmatic Nucleus Suprachiasmatic nucleus (SCN) In vertebrates, several behavioural, physiological, endocrine and biochemical processes display circadian rhythmicity, which are related to SCN. This hypothalamic nucleus with rhythm-generator properties is capable of integrating stimuli conveyed from different centres of the CNS in order to control the expression of circadian rhythms. One SCN has a volume of 0.068 mm3 and contains close to 8,000 neurons. Anteroventrally, SCN is bounded by the optic chiasma and posteroventrally by the supraoptic commissue. The fibers of the supraoptic commissue are closely juxtaposed with those of the optic chiasma. The third ventricle divides the paired SCN in their dorsal aspect. A cell free zone of about 60 Jl separates the medial SCN from the wall of the third ventricle. Dorsomedially, cells tend to smaller and more tightly bound then ventrolatrally; significantly more somatic appositions occur in the dorsomedial SCN than in other parts of the nucleus. Two neurons with an extended region of somatic apposition may have no intercellular specialisations. Chains of neurons with long regions of somata-somatic apposition are found in the dorsomedial SCN. The length of these chains is generally orientated in an antero-posterior direction. Interspersed with the neurons are astroglia. The astroglia have a rich cytoplasm. The nuclei of SCN glia and neurons have a large number of nucleoli. With Golgi impregnations a number of relatively simple dendritic arbors are found to exist. These include simple bipolar cell, curly bipolar cell, radial neuron, monopolar neuron and spinous cell. At the borders of the nucleus some dendrites may travel into the adjacent anterior hypothalamus. Similarly dendrites from neurons outside may enter into the nucleus boundaries. The SCN is a complex nucleus with recognisable subdivisions that contain neurons with several types of relatively simple dendritic arbors. Within any area of SCN, ultrastructural differences can be found between neighbouring cells, suggesting heterogeneous population of neurons. The demonstration of a direct retina-hypothalamic tract (RHT) from retina terminating in the hypothalamic SCN of the rat was reported as early as 1971 17

Suprachiasmatic Nucleus (Moore et al., 1971; Moore and Lenn, 1972). The function of the RHT terminating in the SCN is entrainment. Transecting all visual pathways beyond the optic chiasma results in a loss of visual responses to light and visual reflexes without affecting entrainment (Moore and Eichler 1972; Moore and Klein, 1974). Transection of the RHT is sufficient to mediate entrainment. Recently the RHT is found to project beyond the SCN (Johnson et al., 1988; Levine et al., 1991). The function of these extra-scn projections is unknown but these projections are very extensive and overlap much of the SCN output. Besides retina, SCN receives inputs from other regions also (Moga and Moore, 1996). These include inputs from mid-brain raphe serotonin neurons, other hypothalamic areas and the intergeniculate leaflet of the thalamus. The latter is particularly important as it is believed to be involved in entrainment (Janik and Mrosovsky, 1994; Moore and Card, 1994). The RHT appears to have two components (Speh and Moore, 1993). The first is the projection to the SCN and adjacent anterior hypothalamic areas. The second is the projection to the lateral hypothalamic area. The latter appears to be anatomically unrelated to the more medial projections; it is predominantly contralateral (Johnson et al., 1988), develops before the medial projections (Speh and Moore, 1993) and originates from a subset of retinal ganglion cells (RGCs) that are nearly confined to the outer temporal quadrant of the retina (Leak and Moore, 1994). The RHT projections to the medial hypothalamus appear to be a component of projections to the SCN. The RHT projection from the retina to the SCN terminate in the ventral portion of the nucleus where predominant cell populations are those characterised by VIP or GRP content (van den Pol and Tsujimoto, 1985). New findings indicate that a circadian rhythm is also maintained in the retina; the portion of retina which show this rhythm is known as circadian retina. It seems that there may be photoreceptors, probably a class of cones, that are dedicated to the circadian timing system and do not participate in other visual functions. The intergeniculate leaflet (IGL) is a component of the lateral-geniculate complex of the thalamus, derived from the ventral thalamic diencephalic cell 18

Suprachiasmatic Nucleus column. It was identified as a distinct subdivision of the lateral geniculate by Hickey and Spear (1976) in the rat on the basis of its overlapping bilateral retinal input, a pattern of retinal afferents not found in other lateral geniculate subdivisions (Moore and Card, 1994). The IGL contains a population of neuropeptide Y (NPY)- containing neurons (Card and Moore, 1984) which project to the SCN in a pattern that overlaps the direct retinal afferents (Card and Moore, 1982, 1989). The IGL is comprised of about 2,000 neurons in the rat (Moore and Card, 1994). Of these, about 1250 cells are enkephalin-containing and project to the contralateral IGL. Approximately 650 cells are NPY and project to the SCN. All neurons in the IGL appear to be GABA containing (Moore and Speh, 1993; Moore and Card, 1994). The dorsal raphe (DR) is the most prominent member of the brainstem serotonergic nuclei. It is located in the ventral part of the periaqueductal gray matter of the midbrain, but its caudal section extends well into the peri ventricular gray matter of the rostral pons. It demonstrates a highly characteristic bilateral symmetry, and in cross sections of the midbrain the appearance of the nucleus creates the outline of a fountain (Tillet, 1987)1. The DR nucleus is composed of several subregions which can be distinguished not only because of their different cell densities and differential cell morphology but also of their differential projections. The serotonergic cells in this subnucleus are arranged in two parallel streams of cells located just lateral to the midline in the floor of fourth ventricle. The number of serotonergic neurons is the largest in DR of all raphe nuclei, 24,250 ± 1,6001 in the cat, and 165,000 ± 34,0001 in the humans. The SCN has two major subdivisions, considered as core and shell. This division is done on the basis of functional distinctions. The two regions also have different neurons. The neurons in the dorsal SCN, the SCN shell, are small with rather sparse dendritic arbors whereas those in the ventral SCN, the SCN core, are larger with more extensive dendritic arbors (van den Ppl, 1980). Another characteristic of shell neurons, particularly those in the dorsal and lateral parts of 1 Anatomy of the Serotonergic System; Tork,l., in The Neuropharmacology of Serotonin, Annal NY. Acad. Sci. (1990) 19

Suprachiasmatic Nucleus the nuclei, is that they tend to have dendrites that extend beyond the cytoarchitectonic border of the nucleus. Similarly, it has been known that the core SCN in the rat is characterised by a population of VIP neurons, where as the shell is characterised by VP neurons (Speh and Moore, 1994). Besides this, input from visual nuclei ends in the SCN core suggesting that this is the location of the pacemaker (Gillette et al., 1993). Neurochemicals and Neuropeptides involved in Circadian rhythm generation control and entrainment Though evolutionary forces have selected different neurochemicals in different species and stages of evolution, many of them have a common origin and function. Their functional details and properties are immense and it would not be justifiable to discuss them at this level as they are beyond the demands of the topic. Given below is an account of the neurochemicals which are established in the working of the mammalian system, specifically rat. As we have already mentioned that SCN is an established centre for the control of biological clock in mammals, we are reviewing its neurochemicals as a model system. NEUROCHEMICALS OF SCN On the basis of immunohistology and in situ hybridisation studies, van den Pol and Tsujimoto (1985) divided the neurotransmitters of the SCN into the following categories on the basis of their presence and localisation:..- Neurotransmitters of endogenous origin: There are various neurotransmitters which are intrinsic to SCN; these include, Vasopressin, N europhysin, Somatostatin (SS), Bombesin, Gastrin releasing peptide (GRP) and Gamma-aminobutyrate (GABA)...- Neurotransmitters of extrinsic origin: Some neurotransmitters are found in large concentration in SCN as compared to any other region but their synthesis occurs elsewhere; these include, 20

Suprachiasmatic Nucleus Serotonin (5-HT) from Raphe nuclei, Neuropeptide Y (NPY) from the IGL, Histamine from the tuberomammlliary nucleus and Substance P (SP) from the retinohypothalamic tract...- Neurotransmitters of dual origin: These neurotransmitters are synthesised in situ, besides this they are also imported from outside, examples for this kind includes Dopamine (DA), Norepinephrine (NE) and Epinephrine. Besides these three types, there are other neurotransmitters such as Luteinizing hormone releasing hormone (LHRH), Prolactin, Thyrotrophin, Neurotensin and Peptide histidine isoleucine (PHI) present in minimal quantities but their origins are not known. Neuropeptides involved in circadian rhythm generation and "'-. control \:: A number of neuropeptides are involved in circadian rhythm generation and f". control by acting as a neurotransmitter and/or neuromodulator. There are \ ~ numerous studies which showed that the SCN contains a variety of peptides (van r-- den Pol and Tsujimoto, 1985; Watts and Swanson 1987; Watts et al., 1987, Daikoku et al., 1992). In the dorsomedial (DM) sub-division of the SCN, neurons with immunoreactivity for arginine vasopressin (A VP) or somatostatin (SS) are present. On the other hand, the ventrolateral (VL) subdivision of the SCN contains a large number of vasoactive intestinal polypeptide (VIP) and gastrin releasing peptide (GRP) synthesising neurons. In this area, substance Pis also localised (Daikoku et al., 1992; Kalsbeek et al., 1993; Inouye and Shibata, 1994), where a small fraction of retinal ganglion fibers innervate. Besides this, there are other neuropeptides present in minute quantities which include neurophysin, bombesin, neuropeptide Y, PHI, LHRH, prolactin, thyrotropin and neurotensin. Out of all these neuropeptides A VP, SS, VIP and GRP are of greater interest. 21

Suprachiasmatic Nucleus Vasopressin Vasopressin is one of the important neuropeptides of the SCN. Its contents increases to the maximum level at about CT4, then gradually decreases and stays low during the night or subjective night. Circadian profiles of A VP contents over a day appeared similar in rats kept under LD and DD conditions. The AVP mrna level in the SCN also showed a circadian rhythm under DD conditions (Cagampang et al., 1994). The AVP mrna profile has a peak at CT 8 and a trough around CT 20 and remained unaltered when animals were exposed to light. The rhythm does not alter under DD conditions which shows that it is an endogenous rhythm, which is not driven by external light/dark cycles, but it is regulated by the pacemaker located in the nucleus. The concentration of A VP in the cerebrospinal fluid (CSF) in rats has been shown to have diurnal rhythmicity in LD conditions (Schwartz et al., 1983). A VP level in the CSF reaches peak values at ZT 4 and starts to decrease at ZT 6 before reaching the lowest level at ZT 10. This rhythm is maintained even in the absence of periodic environmental lighting. Schwartz and Reppert (1985) using SCN lesions demonstrated that A VP rhythms found in the CSF are derived from A VP released from the SCN neurons. Gillette and Reppert (1987) further showed that SCN slices kept in vitro are capable of releasing A VP into the perfusate in circadian fashion similar to that found for the A VP rhythm of the CSF. The presence of an endogenous A VP rhythm in the SCN under constant conditions indicates that the peptides contained in the neurons located in the dorsomedial region play a role in signal mediation through the output pathway from the circadian pacemaker. This is also consistent with other lines of evidence. Some A VP synthesising neurons project to areas outside the SCN (Sofroniew and Weindl, 1982; van den Pol, 1991). Furthermore, no appreciable changes in the phase of hamster locomotor activities were observed after administration of A VP (Albers, 1984) and genetically A VP deficient rats exhibit almost normal rhythms in behaviour (Peterson et al., 1980; Groblewski et al., 1981). Besides this, timing of the A VP mrna peak almost coincides with the peak time of electrical or 22

Suprachiasmatic Nucleus metabolic activity of the SCN which further supports the role of AVP as a signal mediator. Somatostatin Somatostatin (SS) is another major neuropeptide found in higher concentration in SCN. It follows a similar time course to that of AVP. Somatostain contents are found to be at its highest level at about CT 4 in animals both under LD and DD conditions (Shinohara et al., 1991; Fukuhara et al., 1993). Elimination of environmental light/dark cycles do not induce a significant change in the circadian profile of SS level in the SCN. It has been shown that SS rhythm is also endogenous which is not driven by external light/dark cycles, but regulated by the pacemaker located in this nucleus. The cellular content of SS in the SCN is at maximum around CT 4. The rhythm of this peptide persists even when the animals are kept under DD conditions for two weeks (Fukuhara et al., 1993) Although A VP and SS contents in the SCN seem to have similar rhythmic patterns, SS could possibly possess a different function from A VP with respect to the circadian mechanism of the SCN. The cell bodies of the SS containing neurons are most prevalent along the border between the dorsomedial and ventrolateral sub-divisions of the SCN and the innervation fields of the SS neurons are mostly confined within these portions (Card et al., 1988; Daikoku et al., 1992) while many A VP fibers project out of the SCN. The SS level in the cerebrospinal fluid was reported to be higher during the night than during the day (Arnold et al., 1982; Berelowitz et al., 1981), which is out of phase with the SS rhythm in the SCN and the A VP rhythm in the CSF (Reppert et al., 1987). These results, particularly the observation that most SS neurons do not project outside the SCN confirmed that unlike A VP, the major function of SS is not the mediation of output information to the rest of the brain. Furthermore, SS administration on to the SCN in the slice preparation induces a phase shift whose dependence on the phase of the pacemaker is similar to that of the response to light (Hamada et al., 1993). This result, together with the other findings indicates the possibility that SS plays a 23

Suprachiasmatic Nucleus role on the input side of the rhythm generating mechanism or on the feedback loop of the pacemaker. Substance P Substance P (SP) belongs to the tachykinin family of peptides with rapid stimulant actions on vascular and extravascular smooth muscle. The peptide in this group share a conserved C-terminal amino acid sequence: PHE-X-GLY-LEU-MET -NH2 which is the biological active domain. The amino acid sequence of SP is: H-ARG-PRO-LYS-PRO-GLN-PHE-PHE-GLY-LEU-MET -NH2 Substance P is found throughout the nervous system of various organisms and has a wide spread phylogenetic distribution (von Euler and Pernow, 1977). SP is one of the important mammalian neuropeptides that can act synaptically or hormonally to regulate a spectrum of cell and tissue functions (von Euler and Pernow, 1977). It has been implicated in pain physiology (Haigler, 1987), regulation of the cardiovascular system (Bayorh and Feuerstein, 1985) as well as modulation of other neurotransmitters (Olpe et al., 1987). It is involved in the modulation of levels of serotonin in vertebrates (Holgmgren et al., 1985). It has also been shown to be involved as a neuromodulator in photosensitivity rhythms in Limulus eye (Mancillas and Brown, 1984; Mancillas and Selverston, 1984, 1986). In SCN, SP like immunoreactive fibers from the retina were suggested to be a part of the RHT. It is localised in ventral region. With ocular enucleation its concentration decreases, while with the long light exposure its concentration increases (Takatsuji et al., 1991; Takastuji and Tohyama, 1993). However, another study contradicts these results (Otori et al., 1993). There is a contradiction in the actual site of presence of SP, as its receptors are localised in the dorsal and dorsolateral border of the SCN. Many recent findings do indicate that SP is involved in conveying light information to induce Fos protein in the hamster SCN. Besides this SP receptors are also found in retinal fibers. The 24

Suprachiasmatic Nucleus topographical studies on the neurons expressing the SP receptor in the SCN indicates that their dendrites extend towards the retina-recipient part of the nucleus, where they can be modulated by overlapping inputs from the intergeniculate leaflet and the raphe (Mick et al., 1992). Furthermore, the protein synthesis inhibitors block the SP induced phase shift of the circadian rhythm of neuronal activity in the rat SCN. The role of SP as a neuromodulator has been discussed separately. Neuropeptide Y Neuropeptide Y (NPY) 1s a 36- amino acid peptide, found in cells of the intergeniculate leaflet (IGL). Cells from this region project to the SCN via the geniculohypothalamic tract (Card and Moore, 1982; 1989; Moore et al., 1984; Morin et al., 1992; Harrington et al., 1985). In general, the lesions of the IGL do not produce a significant effect on locomotor activity rhythm. Only the rate of reentrainment to the displaced LD cycle became slower in rats with IGL lesions, and the advance position of the PRC to light pulses is concomitantly reduced in these animals (Pickard et al., 1987; Harrington and Rusak, 1988). It is now established that entrainment is not a function ofigl. The SCN contains one of the highest concentrations of NPY in the mammalian CNS (Allen et al., 1983; Colwell et al., 1985; Pelletier, 1990). Most SCN neurons are GABAergic (van den Pol and Tsujimoto, 1985; Okamura et al., 1989; Moore and Speh, 1993), and GABA is found in 50% of all presynaptic axons in the SCN (Dacavel and van den Pol, 1990). GABA may be co-localised with NPY in some SCN axon terminals (Francois-Bellan et al., 1990). NPY administration into SCN (Albers and Ferris, 1984; Shibata and Moore, 1993) and chemical stimulation of the geniculohypothalamic tract phase shifts the circadian rhythms in a phase dependent manner; moreover its dependence on the circadian time was similar to that for dark pulses. Bath application of NPY resulted in suppression of electrical activity of SCN neurons (Albers et al., 1990) while focal application of NPY induced excitatory responses (Mason et al., 1987). The PRCs of these 25

Suprachiasmatic Nucleus manipulations have large phase advances in the subjective day and smaller phase delays in the subjective night. The curves are broadly similar in shape to those of induced wheel running and social interaction in hamsters (Reebs and Mrosovsky, 1989; Mrosovsky et al., 1992). These advances were observed in response to NPY administration during the period between CT3 and CT9 (Shibata and Moore, 1993). NPY concentrations in the SCN showed a broad single peak at around CT12 (Shinohara et al., 1993), this indicates that the NPY level in the SCN is under the control of the circadian pacemaker, probably due to a rhythmic change of IGL neurons. When animals were kept in LD regimen, two peaks appeared quite distinct from that observed under DD conditions. A peak that is found 2 hours (CT2) after the light is on is induced by the dark to light transition and the other peak found 2 hours (CT14) after lights are off is the result of the transition from light to dark. Vasoactive Intestinal Peptide Vasoactive Intestinal Peptide (VIP) is a 28 amino acid peptide originally isolated from porcine intestine. It belongs to the glucagon/secretin family of peptides and is closely related to peptide histidine isoleucine (PHI) which is derived from the same VIP precursor protein. VIP is widely distributed throughout the central nervous system (CNS) and peripheral nervous system, and is found in brain, spinal cord, neurons of the gastrointestinal tract, sensory epithelium, exocrine glands and non-neuronal tissue such as mast cells and leukocytes. In the CNS, VIP is thought to act as a neurotransmitter or neuromodulator (Sundelr et al., 1988). VIP immunoreactive neurons are densely localised in the hypothalamic nuclei including the paraventricular nucleus (PVN) and the median eminence (ME). In addition, VIP has potent hypotensive and vasodilatory action and is present in high concentrations in the hypothalamo-hypophysial portal system, shows trophic and mitogenic activity on neural tissue during embryonic development, and inhibits the growth and mitosis of certain tumours. The actions of VIP are mediated by two different receptor subtypes, a low-affinity receptor 26

Suprachiasmatic Nucleus (nm range) and a high-affinity receptor (pm range). The low-affinity VIP receptor is coupled to the adenylate cyclase system. Stimulation of VIP low-affinity receptors in many tissues and in different areas of the brain (i.e. cortex, hypothalamus, striatum and hippocampus) activates adenylate cyclase leading to an increase in c-amp. VIP was identified in SCN in 1981 (Card et al., 1981), it is localised in the ventro-lateral subdivision of the SCN. Anatomically, VIP neurons are located at a potentially pivotal position where information from various sources outside the SCN converges (Hisano et al., 1988; Ibata et al., 1989; Francois Bellan and Bosler, 1992). The fibers from other areas like RHT, GHT and DR afferents make synapses with VIP neurons. In turn, VIP neurons make synapses on VIP and A VP cells within the SCN and send their efferent fibers out of the SCN boundary (Bosler and Beaudet, 1985; Maegawa et al., 1987; Hisano et al., 1988 ; Ibata et al., 1993 and Tanaka et al., 1993). In different conditions fluctuations in the VIP levels has been observed, VIP levels show diurnal variation with respect to LD cycle, they decrease during the light period of LD cycle and increase during the dark period under LD conditions (Albers et al., 1987; Takahashi et al., 1989; Morin et al., 1991). Eye enucleation also elevates VIP level in the SCN (Okamoto et al., 1990). In situ and Northern hybridisation studies on VIP mrna have shown that diurnal variation of VIP even exist at the mrna level encoding VIP/PHI (Gazes et al., 1989; Stopa et al., 1989; Albers et al., 1990b; Okamoto 'et al., 1991; Zoeller et al., 1991). VIP mrna also decreases during the day and increases during the night. While VIP immunoreactivity and VIP mrna exhibits distinct diurnal variations under LD conditions, VIP binding activity in the SCN was found to be stable throughout an LD cycle (Robinson and Fuchs, 1993). This suggests that VIP receptor sensitivity remains constant during the day and circadian regulation involving VIP is carried out at the presynaptic level. The notion that VIP mediates light information has been contradicted by various findings. Albers et al. (1991) observed no significant effects on the phase of locomotor activity rhythm and electrical activity of the SCN in the brain slices 27

Suprachiasmatic Nucleus upon microinjection of VIP in SCN. Also, chronic infusion of VIP does not change circadian pattern of sleep-waking rhythm (Kruisbank et al., 1987). Many recent findings however suggest that VIP is important at the time of entrainment. Although, the level of VIP always decreases in response to light, the rate of decrease in VIP content after light exposure depends on the time of the day. Light at the early subjective night is less effective in reducing VIP level in the SCN, while light at the late subjective night is more effective (Shinohara et al., 1994). On the basis of these findings, VIP is regarded as a part of the timekeeping mechanism that mainly deals with the light response or entrainment to environmental lighting conditions in the SCN. VIP may include both input and output control of the circadian pacemaker, with relevance to entrainment to external lighting conditions. Furthermore, transplantation studies suggested that the presence of VIP neurons in the graft is most highly correlated with the recovery of the circadian rhythm of host animals (Lehman et al., 1987; Griffioen, 1992; Moore and Card, 1993) thereby confirming the role of VIP in the input pathway and entrainment. In addition, serotonin fibres are also known to synapse on VIP cells in the SCN (Kiss et al., 1984; Bosler and Beaudet 1985; Maegawa et al., 1987). In a recent in situ hybridisation study Roca et al.(1993) observed that 5- HT receptor mrna is expressed in both the ventro-lateral and dorsa-medial subdivisions of the SCN but depletion of 5-HT was also reported to reduce the level of VIP in the SCN (Kawakami et al., 1985; Kawakami, 1986). VIP has also been found to be co-localised with other two peptides, viz., PHI and GRP and it has been shown that a cocktail composed of VIP, PHI and GRP mixture (1:1:1) does induce a phase shift of overt rhythm and a change in electrical activity of the SCN (Albers et al., 1991; Peters et al., 1994). The co-localisation of VIP and GRP is predominant in the ventro-lateral region of the SCN and considered to be important for the entrainment of circadian rhythm (Okamura et al., 1986). The axons of VIP and/or GRP-containing neurons from local circuit interacts within the SCN and project to the hypothalamus and extrahypothalamus (ven den Pol and Tsujimoto, 1985). Besides this, VIP containing neurons form 28

Suprachiasmatic Nucleus synapses with arg-vasopressin (A VP) containing neurons localised in the dorsomedial area of the SCN. These neurons pass circadian oscillating signals from the SCN to the hypothalamus and extrahypothalamus (Watts et al., 1987). AVP expression itself shows free running rhythmicity in the absence of timing cues (Tominaga et al., 1992). The level of VIP is higher in the dark period under the light-dark cycle, while that of AVP and GRP are higher during the light period (Shinohara et al., 1993; Okamura and lbata, 1994). Very recently it has been found that if rats are kept under dim light then VIP content increases from 4 to 8 hours but returns to the baseline at 12 to 16 hour and then again increases until 36 hours after the light is switched off (dim light) but GRP showed no significant changes (lsobe and Nishino, 1996). Monoamines involved in control of circadian rhythm Monoamines like dopamine, melatonin, serotonin and its metabolites like 5- HTP and 5-HIAA are very important classical neurotransmitters involved 1n the circadian system. Among these monoamines, serotonin and its metabolites are important constituents of clock in invertebrates, these are known to control and regulate the clock. Serotonin For the last twenty years, serotonin, 5-hydroxytryptamine (5-HT), was the thrust area of our laboratory. The role of serotonin and its metabolites in circadian clock of cockroach was extensively worked upon in our laboratory and it has been established that the ratio of 5-HT/5-HIAA is an important factor which coordinates the clock (Pandey and Habibulla, 1980, 1982, 1983), This has been supported by various studies on mammalian circadian system (Glass et al., 1993; ' Poncet et al., 1993). 5-HT is an important biogenic amine and is one of the few amines found in both the plant and the animal world. It was detected for the first time in the blood serum and used to increase the tonicity of the endothelial walls of blood vessels, hence it was named as serotonin. It belongs 29

Suprachiasmatic Nucleus to the family of aromatic amino acids. Chemically, it is a decarboxylated tryptophan which is hydroxylated at fifth position of indole ring. 5-HT is found in high concentrations in the nervous systems of annelids, molluscs, crustaceans and arachnids, while is present in low concentrations in arthropods, echinoderms and tunicates.. The quantitative distribution of 5-HT in various parts of brain is quite similar in all classes of the vertebrates. Highest levels have been found in mid-brain and hypothalamus and lowest in the cerebellar tissue. Surprisingly, it is found in highest concentration in SCN as compared to other CNS areas. The neurons containing serotonin are localised mainly in raphe nuclei in brain stem of rat. From the cells located primarily in the dorsal and the median raphe nuclei there is a dense serotonergic projection to the SCN (Fuxe, 1965; Moore et al., 1978; Kiss et al., 1984; Hisano et al., 1988). Though its functional significance is still unclear, raphe lesions are known to be associated with an increase in the level of daytime activity (Block and Zucker, 1976). Besides this, the effect of brain serotonin by 5, 7 -dihydroxytryptamine ( 5, 7-D HT) on hamster circadian rhythm indicated that serotonergic system modulates the phasic actions of light (Morin and Blanchard, 1991). Moreover, monoamine oxidase inhibitors, such as clorgyline and tricyclic antidepressants which increase brain serotonin level, also showed the rate of re-entrainment to a shifted LD cycle, lengthened the free-running period under constant conditions and increased the magnitude of phase delays induced by a light pulse interrupts in darkness (Duncan et al., 1988). 5-HT agonist also give similar results, a 5- HT1A receptor agonist 8-0H DPAT, produces phase-shifts of wheel-running rhythm in hamsters similar to dark pulses (Prosser et al., 1990; Tominaga et al., 1992). On the other hand, a non-specific 5-HT receptor agonist, produces a phase shift in the circadian rhythm of neuronal activity in the SCN slices in vitro (Medanic and Gillette, 1992; Shibata et al., 1992). Application of 5-HT inhibits SCN unit activity in the brain slice and the field potentials within the SCN elicited by stimulation of the optic nerve fibers in vitro (Shibata et al., 1983a; Liou et al., 1986b). Thus activation of 5-HT neurons exerts inhibitory 30

Suprachiasmatic Nucleus on neuronal activity of the SCN. The study of 5-HT receptor gene in the SCN revealed that 5-HTic receptor mrna exhibit intense hybridisation in the SCN, while 5-HTih receptor mrna displays a weaker signal (Roca et al., 1993). Predominant 5-HTic receptors were found in both the VL and DM subdivisions of the SCN. Lovenberg et al. (1993) using sensitive emulsion autoradiograms demonstrated low levels of 5-HTib and 5-HT2 receptor mrnas. This result does not support the view that a major role is played by post synaptic 5-HTia receptors in phase shifting of the circadian rhythm of the SCN. Distinct circadian variations are present in the serotonin content of SCN tissue micropunched from rats kept under LD conditions. Serotonin concentration shows a peak at ZT 3-5 during the light period and remains low during the dark period of rats (Cagampang and Inouye, 1994). On the other hand, serotonin metabolites and serotonin uptake display maximum levels during dark period (Faradji et al., 1983; Semba et al., 1984; Glass et al., 1992). From this it has been predicted that serotonin concentration is high during the day because serotonergic activity is low in that period of the LD cycles. When animals are transferred to DD conditions, peaks of serotonin concentration in the SCN move to a time point during the subjective night in two cycles (Cagampang and Inouye, 1994). The serotonergic modulation of light information is confirmed by the quipazine administration studies, which showed a reduced c-fos expression which was normally induced by a light pulse and, at the same time, extracellular glutamate concentration in the SCN (Selim et al., 1993). Melatonin Melatonin, 5-Methyl-N-acetyltryptamine, IS an evolutionarily highly conserved molecule, present in organisms as different as algae and humans. In all organisms which have been studied, melatonin is almost exclusively synthesised during the night and it mediates information concerning the temporal position and duration of darkness (Poeggeler, 1983). 31

Suprachiasmatic Nucleus Identified in 1958 by a dermatologist Lerner, as the skin-lightening substance which influences the aggregation of the indole pigment melanin, this substance has been identified in most types of cells. On endogenous injection, melatonin can enter any type of cell. It is now accepted that exogenous melatonin is able to reset most rhythms in vertebrates and to modify circadian rhythmic activities in plants and unicellular organisms. Although still controversial, in humans, melatonin may be involved in sleep regulation, affective disorders, ageing and cancer, besides an unknown (yet to prove with substantial experiments though discrete findings are there) function in cell division, intracellular cell movements and intracellular signalling (King and Tay, 1993). The pineal gland, a part of the brain derived from the caudal portion of the embryonic dorsal diencephalic cell column, epithalamus, is a well known source of melatonin. Melatonin is.synthesised from serotonin in a two-step process in which serotonin is initially N-acetylated by the enzyme, arylalkylamine N acetyltransferase (NAT), and later N-acetyl serotonin is converted to melatonin by the enzyme, hydroxyindole-0-methyltransferase (HIOMT) (Axelrod, 1974). This process is regulated by the sympathetic innervation of the pineal, primarily through a J3-adrenergic receptor mediated regulation of NAT activity (Klein et al., 1971; Klein and Moore, 1979). During the day time, sympathetic activity, NAT activity and melatonin production are low, while the reverse is true during the night. Reiter (1991) termed this phenomenon as "the chemical expression of darkness. This rhythm in pineal melatonin production is a true circadian rhythm and, as such, is not dependent on the light-dark cycle. Moore (1996) reported that this rhythm depends upon the SCN and the entrainment pathways. Melatonin levels are low during the day, begin to increase shortly after lights are off, reach peak levels at midnight and then decrease in the late night to reach daytime levels shortly before onset of light. The role of light in regulating melatonin production is shown as shifts in the light-dark cycle which produces phase shifts in the melatonin rhythms in rodents and man (Shanahan and Czeisler, 1991). In rodents and man, the duration of melatonin peak is a function of the length of the light portion of the light-dark cycle (Wehr, 1991). Besides this, a pulse of light at night 32