CHRONOBIOLOGY INTERNATIONAL Vol. 20, No. 4, pp.

637655, 2003

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Hypothalamic Circadian Organization in Birds. I. Anatomy, Functional Morphology, and Terminology of the Suprachiasmatic Region
Roland Brandstatter* and Ute Abraham
Max-Planck-Research Centre for Ornithology, Andechs, Germany

ABSTRACT
In mammals, the master clock controlling circadian rhythmicity is located in the hypothalamic suprachiasmatic nuclei (SCN). Until now, no comparable structure has been unambiguously described in the brain of any nonmammalian vertebrate. In birds, early anatomical and lesioning studies described a SCN located in the anterior hypothalamus, but whether birds possess a nucleus equivalent to the mammalian SCN remained controversial. By reviewing the existing literature it became evident that confusion in delineation and nomenclature of hypothalamic cell groups may be one of the major reasons that no coherent picture of the avian hypothalamus exists. In this review, we attempt to clarify certain aspects of the organization of the avian hypothalamus by summarizing anatomical and functional studies and comparing them to immunocytochemical results from our laboratory. There is no single cell group in the avian hypothalamus that combines the morphological and neurochemical features of the mammalian SCN. Instead, certain aspects of anatomy and morphology suggest that at least two anatomically distinct cell groups, the SCN and the lateral hypothalamic nucleus (LHN), bear some of the characteristics of the mammalian SCN. Key Words: Circadian system; House sparrow (Passer domesticus); Lateral hypothalamic nucleus; Hypothalamus; Suprachiasmatic nucleus.

*Correspondence: Dr. Roland Brandstatter, Department of Biological Rhythms and Behaviour, Max Planck-Research Centre for Ornithology, Von-der-Tann-Strasse 7, D-82346 Andechs, Germany; E-mail: brandstaetter@erl.ornithol.mpg.de. 637
DOI: 10.1081=CBI-120023343 Copyright # 2003 by Marcel Dekker, Inc. 0742-0528 (Print); 1525-6073 (Online) www.dekker.com

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INTRODUCTION The hypothalamus of the vertebrate brain is a complex structure responsible for integrating innumerable endocrine, autonomic, and behavioral responses that are involved in regulating metabolism, homeostasis, and reproduction. Exploratory, ingestive, thermoregulatory, aggressive, defensive, sexual, and parental behaviors are all involved, and their expression is tied in a fundamental way to a circadian rhythm of activity and rest (Bjorklund et al., 1987). In mammals, this rhythmicity is controlled by a master clock acting as a circadian pacemaker that is located in the hypothalamic suprachiasmatic nucleus (SCN), a paired cell group adjacent to the third ventricle, just above the optic chiasm. It is characterized by specic molecular, morphological, and physiological features (for recent reviews, see Ibata et al., 1999; van Esseveldt et al., 2000): morphologically, the SCN can be divided into a small rostral and a large caudal part (van den Pol, 1980). The latter is composed of a ventrolateral part, termed core, and a dorsomedial part, termed shell. The core, which receives photic input from the retina (Moore, 1973), rhythmically expresses neuropeptides including vasoactive intestinal polypeptide (VIP) (Laemle et al., 1995; Shinohara et al., 1994) and gastrin-releasing peptide (Isobe and Muramatsu, 1995). Vasopressin is rhythmically produced in the shell (Earnest and Sladek, 1986; Isobe and Muramatsu, 1995; Tominaga et al., 1992; Yamase et al., 1991). Light pulses induce expression of immediate early response genes in the core of the SCN, depending on the phase of the circadian oscillator (Kornhauser et al., 1990; Rusak et al., 1990). The role of the SCN as a circadian pacemaker in mammals has been clearly demonstrated by lesions that abolish circadian rhythms at the whole-organism level (Moore and Eichler, 1972; Rusak and Zucker, 1979) as well as by transplantation of SCN tissue into the brains of SCN-lesioned mammals that restored rhythmicity (Lehman et al., 1987; Ralph et al., 1990). Until now, no comparable structure has been unambiguously identied in the brain of any nonmammalian vertebrate, and little is known about the exact localization and specic properties of the hypothalamic circadian oscillator in nonmammalian vertebrates. In this article, we shall emphasize insight into the organization of the anterior suprachiasmatic hypothalamus in birds, based on information from a variety of bird species gained over the last six decades by various research groups as well as recent investigations performed by the authors focusing on anatomical and morphological properties of the suprachiasmatic hypothalamus in songbirds, particularly that of the house sparrow (Passer domesticus).

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The Circadian System of Birds In variance to mammals, birds have the capacity to perceive information about the photic environment by retinal, pineal, as well as by deep encephalic photoreceptors (Cassone and Menaker, 1984; Foster and Soni, 1998; Kojima and Fukada, 1999; Menaker and Tosini, 1995; Silver et al., 1988). Depending on the species, circadian pacemaking at the whole-organism level is organized by autonomous and anatomically distinct oscillators localized in the retina (Pierce et al., 1993; Underwood et al., 1990), the pineal gland (Binkley et al., 1978; Brandstatter et al., 2000; Gaston and Menaker, 1968; Zimmerman and Menaker, 1979), and the hypothalamus (Takahashi and Menaker, 1982). Several lines of evidence suggest that these components interact with each other to produce a stable

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circadian rhythmicity of the animal (for recent reviews, see Brandstatter, 2002; Gwinner and Brandstatter, 2001) [Fig. 1(A)]. Initial functional evidence for a hypothalamic circadian pacemaker in birds is mostly coming from lesioning studies. Takahashi and Menaker (1982) demonstrated that house sparrows bearing lesions of the anterior suprachiasmatic hypothalamus gradually lost their locomotor activity rhythms in constant conditions. Further indirect evidence was found in pigeons that had been both pinealectomized and blinded and still showed residual rhythmicity for a while after transfer to constant conditions before they became arrhythmic (Ebihara et al., 1987). Circadian activity rhythms of pinealectomized house sparrows did
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Figure 1. Diagram of the major components of the avian circadian pacemaking system and the anterior suprachiasmatic hypothalamus. Oscillatory components are indicated by clock symbols; photoreceptive structures are indicated by open arrows. Signal pathways are demonstrated by hatched arrows. ep encephalic photoreceptors. The diagram is not indicative for a certain species but summarizes information that has been obtained in chicken, house sparrow, pigeon, and Japanese quail. Circadian oscillators that may act as pacemakers are present in the pineal gland, the retina, and the hypothalamus. Oscillators may interact with each other by hormonal signals (hatched arrow from pineal to hypothalamus), neural signal pathways (hatched arrow from hypothalamus to pineal) or both (cross-hatched arrow from retina to hypothalamus). For detailed information see references (Gwinner and Brandstatter, 2001) and (Brandstatter, 2002). The insert (B) shows the anatomical position of the hypothalamic cell groups shown in C. The two cell groups that have been assumed to represent the functional equivalent to the mammalian hypothalamic oscillator are drawn in grey (right hemisphere) and indicated by grey arrows. GLV, ventro-lateral geniculate nucleus; LHRN=vSCN, lateral hypothalamic retinorecipient nucleus (also called visual SCN); LHy, lateral hypothalamic area; OC, optic chiasm; PON, preoptic nucleus; SCN, suprachiasmatic nucleus; V, third ventricle.

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not disappear immediately but damped out over a series of transient cycles following release into constant conditions (Gaston and Menaker, 1968; Zimmerman and Menaker, 1979), suggesting that there remained at least one damped oscillator after removal of the pineal gland. It is likely that the presence of a hypothalamic oscillator is a common feature of the avian circadian pacemaking system, since disruptive effects of hypothalamic lesions on the circadian rhythms of birds were found in all further species investigated until now, including the Java sparrow and the Japanese quail (Ebihara and Kawamura, 1981; Simpson and Follett, 1981). However, the exact localization and specic properties of the hypothalamic circadian oscillator in birds remained obscure. Several reasons may have contributed to the lack of detailed knowledge about the avian equivalent of the mammalian hypothalamic circadian oscillator, including incomplete and inconsistent anatomical descriptions of the avian hypothalamus, confusion in delineation as well as terminology of cell groups, and the lack of a molecular approach dening key genes and=or transcription factors of circadian oscillators.

The Hypothalamus of Birds The avian hypothalamus forms the ventral portion of the diencephalon on either side of the third ventricle. It has been divided into a large number of more or less distinct cell groups and it is traversed by several major ber systems (Crosby and Showers, 1969). The parcellation of the hypothalamus as well as the delineation of cell groups has been highly variable in the different bird species investigated, and the terminology applied has differed greatly. A survey of nomenclatures used for the avian hypothalamus has been given by Kuenzel and van Tienhoven (1982). Following the Nomina Anatomica Avium (Breazile and Kuenzel, 1993), the hypothalamus of birds can be divided into three main portions the preoptic region, the medial (tuberal) region, and the caudal (mammillary) region, with 19 nuclei and two areas. However, we still lack a clear and generally applicable demarcation of cell groups as well as a uniform terminology that would facilitate comparison among species. Due to the rather high species-specic diversity of brain morphology, different nomenclatures used, section planes shown that are not always comparable, and inconsistency in the description of different cell groups, a uniform picture of the avian hypothalamus does not exist. This is exemplied by anatomical descriptions of the anterior hypothalamus in frequently used atlases of the avian brain (Fig. 2). Major problems for a comprehensive understanding of the avian hypothalamus are the rather high species-specic diversity of brain morphology as well as the fact that functionally distinct cell types do not always respect the boundaries of traditional cell groups delineated in Nissl stainings. Thus, functional studies and anatomical descriptions often are in variance to each other. This ambiguity is also true for those cell groups that have been associated with circadian function, i.e., that possibly represent the equivalent to the mammalian SCN. Additionally, the term SCN has become a synonym for a hypothalamic circadian oscillator or pacemaker and, thus, has sometimes been used to describe a structure that shows evidence for the presence of a rhythmic parameter rather than being used in its original anatomical meaning. Whether an anatomical equivalent to the mammalian SCN is present in all vertebrate groups and whether it is the site of a hypothalamic circadian oscillator in all species remains to be determined. Hence, when identifying the avian SCNas recently claimed by Yoshimura et al. (2001)it is not

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Figure 2. Diagrams of the anterior hypothalamus as shown in frequently used atlases of the avian brain. (A), (B): Schematic drawings of selected coronal sections through the anterior suprachiasmatic hypothalamus of the pigeon (A: level A 8.50; B: level A 7.75; redrawn from (Karten and Hodos, 1967). (C), (D) Schematic drawings of selected coronal sections through the anterior suprachiasmatic hypothalamus of the chicken (C: level A 9.0; D: level A 8.5; redrawn from (Nieuwenhuys et al., 1998), adapted from (van Tienhoven and Juhasz, 1962)). (E), (F): Schematic drawings of selected coronal sections through the anterior suprachiasmatic hypothalamus of the canary (E: level A.2.8; F: level 2.6; redrawn from (Stokes et al., 1974)). Scale bar 1 cm. Variability in the description of hypothalamic organization becomes evident from differences in delineation and nomenclature of cell groups. In the pigeon atlas, a supraoptic nucleus is described at the lateral angle of the ventricle (A) where the SCN is shown in the chicken atlas (C). Neither a supraoptic nor a SCN are shown in the atlas of the canary (E), possibly due to the lack of a section plane available frontal to the supraoptic decussation. The LHy is mentioned but not demarcated in the pigeon (B), delineated in the chicken (D) but not present at all in the atlas of the canary (F). AM, nucleus anterior medialis hypothalami; CO, chopt, chiasma opticum; LA, nucleus lateralis anterior thalami; LHy, nulhy, nucleus lateralis hypothalami; POM, nucleus preopticus medialis; PPM, nucleus preopticus paraventricularis magnocellularis; PVM, nucleus periventricularis magnocellularis; QF, tractus quintofrontalis; SO, nucleus supraopticus; TSM, TrSM, tractus septomesencephalicus; VLT, nucleus ventrolateralis thalami. t, fasciculus lateralis telencephali; nuhyma, nucleus medialis hypothalami anterioris; nuprmc, nucleus preopticus magnocellularis; nupvhy, nucleus paraventricularis hypothalami; nusc, nucleus suprachiasmaticus; pra, nucleus preopticus anterior. DS, dso, decussatio Supraoptica; DSD, decussatio supraoptica dorsalis; FDB, fasciculus diagonalis brocae; GLV, nucleus geniculatus lateralis, pars ventralis; POA, nucleus preopticus anterioris; TeO, tectum opticum; TrO, tractus opticus.

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sufcient to identify the site of circadian oscillations but to characterize a dened cell group showing anatomical and=or morphological features justifying the term suprachiasmatic nucleus. On the other hand, identifying the site of a hypothalamic oscillator that may act as a pacemaker requires the characterization of oscillatory components, either molecular or physiological, that may be attributed to circadian function, independent of whether these oscillations are present in the SCN or any other structure. Thus, whether nonmammalian vertebrates do have a SCN and whether this cell group then contains a circadian oscillator can only be answered when anatomical and functional studies are combined, preferably in the same species, and then compared with different species. It is important to accept that birds and mammals had a long parallel evolution and that specic properties of the mammalian brain are not necessarily present in birds or other vertebrates, particularly when regarding the high plasticity of the vertebrate brain and the enormous diversity of brain morphology within and among vertebrate groups.

Anatomical and Morphological Features of the Suprachiasmatic Hypothalamus in Birds The hypothalamus of birds has been investigated in a large number of studies, many of them either focusing on general anatomy, the neurosecretory hypothalamo-hypophyseal system, retino-hypothalamic projections, or the distribution of various neurotransmitters and neuropeptides. Early anatomical studies described a paired cell group adjacent to the base of the third ventricle directly above the optic chiasm, termed SCN because of its anatomical similarity to the mammalian SCN (Crosby and Showers, 1969; van Tienhoven and Juhasz, 1962). Later experimental lesioning studies suggested that this cell group might indeed represent the functionally equivalent structure to the mammalian SCN (Ebihara and Kawamura, 1981; Simpson et al., 1981; Takahashi and Menaker, 1982). However, a delineation of this cell group and a clear description of its rostro-caudal extension as well as its cytoarchitectonic properties in different bird species are still not available. This cell group has been described at various levels of the rostro-caudal extension of the hypothalamus, sometimes even as far caudal as at the height of the supraoptic decussation. This cell group has not only been termed SCN (Abraham et al., 2002; Bons, 1980; Brandstatter et al., 2001; Ebihara and Kawamura, 1981; Hartwig, 1974; Takahashi and Menaker, 1982) but also supraoptic nucleus (Ebihara et al., 1987), medial hypothalamic nucleus (Norgren and Silver, 1989), medial hypothalamic retinorecipient nucleus (Shimizu et al., 1994), and medial SCN (King and Follett, 1997). Another cell group located in the more lateral suprachiasmatic hypothalamus has also been claimed to possibly represent the equivalent to the mammalian SCN and to be the site of the avian hypothalamic circadian oscillator. This assumption was primarily based on the presence of retinal projections (see below), the presence of melatonin binding sites (Cassone and Brooks, 1991), and presumed rhythms of metabolic activity (Cassone, 1988; Lu and Cassone, 1993). This cell group has been called zone 1 to 3 (Meier, 1973), supraoptic nucleus (Ebihara and Kawamura, 1981), SCN (Cooper et al., 1983), lateral hypothalamus (Ehrlich and Mark, 1984), visual SCN (vSCN) (Cassone and Moore, 1987; King and Follett, 1997) or lateral hypothalamic retinorecipient nucleus (LHRN) (Norgren and Silver, 1989; Shimizu et al., 1994) [Figs. 1(B), (C)].

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The analysis of coronal sections of the house sparrow hypothalamus by utilizing traditional Nissl-staining methods as compared to immunocytochemistry with an antibody against a neuron-specic nuclear protein revealed that neuron-specic staining is a helpful tool to identify and demarcate cellular aggregations (Fig. 3). A description of the gross morphology of neuronal aggregations in the house sparrow hypothalamus is given in Fig. 4. A clearly demarcated SCN can rst be observed at the height of the anterior portion of the preoptic nucleus as small aggregations of nerve cells at each lateral angle of the third ventricle interconnected by a small subventricular queue of neurons. Thus, together with the anterior preoptic nucleus and the anteroventral preoptic nucleus, the SCN is the most rostral cellular aggregation that can be found in the house sparrow hypothalamus as well as in other songbirds (Fig. 4). The morphological appearance of the SCN changes throughout its longitudinal extension by increasing in diameter until about half of its total length and decreasing in diameter towards its caudal end. The caudal SCN becomes less clearly distinguishable from surrounding neurons until the nucleus is nally no longer demarcated in coronal sections, whereas horizontal sections allow a very clear demarcation of this cell group (Fig. 5). Lateral to the SCN, an aggregation of neurons is present at the dorsal border of the optic chiasm partly extending into it. Along its rostro-caudal extension this cell group increases in size and extends into the lateral hypothalamic area. A detailed

Figure 3. Coronal sections through the anterior suprachiasmatic hypothalamus of the house sparrow. Comparable section planes Nissl stained with cresyl violet (A) or immunocytochemically processed to label cells that are immunoreactive for a neuron-specic nuclear protein (B; for details see Abraham et al., 2002) demonstrate that neuron-specic immunocytochemistry allows a better recognition of cellular aggregations that can hardly be demarcated in Nissl stainings. Scale bar 200 mm.

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Figure 4. Gross morphology of neuronal aggregations in the suprachiasmatic hypothalamus of songbirds. Coronal sections through the suprachiasmatic hypothalamus of the house sparrow from rostral (A) to caudal (E). Left: Micrographs of coronal sections immunocytochemically processed to label cells immunoreactive for a neuron-specic nuclear protein (for details see Abraham et al., 2002). Right: Digitally averaged images to enhance stained areas as a tool to visualise areas of aggregated neurons. AV, anteroventral preoptic nucleus; GLV, ventrolateral geniculate nucleus; LHN, lateral hypothalamic nucleus; PONa, anterior preoptic nucleus; PONm, medial preoptic nucleus; PPN, periventricular preoptic nucleus; SCN, suprachiasmatic nucleus; TEL, telencephalon. Asterisks indicate neuronal aggregations that do not respect the boundaries of traditional cell groups. The presence of a SCN in other songbird species is indicated by coronal sections through the anterior hypothalamus of the stonechat (F), the blackbird (G), and the zebranch (H). Scale bar 200 mm.

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Figure 5. Demarcation of the SCN in horizontal sections through the suprachiasmatic hypothalamus of the house sparrow. Horizontal sections from ventral (A) to dorsal (G) demonstrate the presence of a clearly demarcated SCN. It is located in the most anterior part of the hypothalamus extending to about the height of the rostral border of the supraoptic decussation. It is laterally anked (C, D) or overlapped (E, F) by magnocellular neurons (asterisks) that are present throughout the anterior hypothalamus but do not respect the boundaries of the traditional cell groups. Scale bar 200 mm. The rostro-caudal axis within the sections is indicated in (A). DSO supraoptic decussation; LHN lateral hypothalamic nucleus; r, rostral; c, caudal; OC, optic chiasm; PPN, periventricular preoptic nucleus; TEL, telencephalon.

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morphological analysis revealed that this cell group may be subdivided into two parts, a ventral and a dorsal one. The ventral portion of this cell group is characterized by a dense aggregation of neurons along the dorsal border of the optic chiasm, whereas a more diffuse aggregation of neurons extends dorsally into the lateral hypothalamic area. The ventral portion, representing the previously described LHRN (Norgren and Silver, 1989) or vSCN (Cassone and Moore, 1987), does not appear to be anatomically distinct from the dorsal part. Because of its anatomical localization and its extension into the lateral hypothalamic area, this cell group has recently been termed lateral hypothalamic nucleus (LHN) (Abraham et al., 2002). From rostral to caudal, neuronal aggregations that do not respect the anatomical borders of the identied cell groups are present in the preoptic, suprachiasmatic, and lateral hypothalamus, indicating that functionally distinct cell populations may overlap in the hypothalamus. These neurons sometimes complicate a clear delineation of cell groups or may even result in misinterpretation of the borders of cell groups. This exemplies that anatomical investigations allow only a limited view on how complex neural systems such as the hypothalamus are organized. However, aggregation of cells or even neurons is without any doubt an indicator of a certain functional relationship but does not prove that cells are more functionally coupled than less densely aggregated or surrounding cells and, thus, the investigation of functional features is a prerequisite for a more comprehensive understanding of the avian hypothalamic organization.

Retinal Projections to the Avian Hypothalamus Based on the fact that retinohypothalamic transmission of photic information to the SCN is a crucial step for the entrainment of the hypothalamic pacemaker in mammals, tract-tracing techniques were used in a number of studies in birds in an attempt to localize the avian hypothalamic oscillator. Dense arborizations of retinal bers were found in the SCN of the pigeon (Shimizu et al., 1994), whereas retinal input to the SCN in the same species has not been found (Cooper et al., 1983; Meier, 1973). Such inconsistent results were also obtained in house sparrow, quail, and starling, where either retinal input was detected (Hartwig, 1974; Norgren and Silver, 1989), or no retinal bers could be found in the SCN (Cassone and Moore, 1987; King and Follett et al., 1997). Consistently, a substantial number of retinal projections were found in the ventral portion of the LHN in all species investigated, but no uniform nomenclature was used for this terminal eld of retinal projections (Cassone and Moore, 1987; Ebihara and Kawamura, 1981; Ebihara et al., 1987; Ehrlich and Mark, 1984; Hartwig, 1974; King and Follett, 1997; Meier, 1973; Norgren and Silver, 1989; Shimizu et al., 1994). However, retinal input is not necessarily a unique feature of a hypothalamic oscillator, since, in contrast to mammals, light input to the hypothalamus of birds is not only mediated by retinal projections. There is convincing evidence that photoreceptors located within the brain are able to synchronize circadian oscillations in birds. Thus, retinal input to the suprachiasmatic hypothalamus may, at least in most species, be of less functional signicance for circadian pacemaking in birds than in mammals. A more useful tool to elucidate which cell groups in the avian hypothalamus respond to photic stimulation is the investigation of immediate early response gene (IEG) expression. Only few such studies were performed in birds until now. They demonstrated IEG expression following a light stimulus or in response to visual stimulation in the ventral part of the LHN but not in the

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SCN of chicken, starling, and quail. No differences were found when IEGs were induced during either day or night (King and Follett, 1997; Wallman et al., 1994). In the house sparrow hypothalamus, c-fos immunoreactive cells could be detected in and close to the most rostral portion of the SCN as well as in the preoptic nucleus, the lateral hypothalamic nucleus, and in the ventro-lateral geniculate nucleus of birds following exposure to a one-hour light pulse during the night (Fig. 6). These preliminary data indicate that the light signal is rapidly spread over the anterior hypothalamus of the house sparrow, including regions that receive prominent retinal input such as the LHN and the ventro-lateral geniculate nucleus, but also regions that receive no or only little direct retinal input such as the preoptic nucleus and the SCN.

Neurochemical Properties of the Hypothalamus in Birds The hypothalamic distribution of neurotransmitters and neuropeptides has been investigated in a variety of bird species. In the following paragraph, particular attention will be drawn to vasotocin (VT), the avian equivalent to vasopressin, and VIP, because both neuropeptides have been associated with circadian function in the mammalian SCN (reviewed in Abrahamson and Moore, 2001; van Esseveldt et al., 2000). In various bird species, VT-immunoreactive cells were found in the anterior and medial preoptic nucleus as well as in the periventricular preoptic nucleus, in the paraventricular nucleus and in the lateral hypothalamus [e.g., Japanese quail (Bons, 1980; Panzica, 1985), house sparrow (Cassone and Moore, 1987), chicken (Panzica, 1985), canary (Kiss et al., 1987), zebra nch (Voorhuis and De Kloet, 1992), ring dove (Norgren and Silver, 1990)]. The distribution of vasotocinergic cells in the avian hypothalamus mostly resembles the neurosecretory hypothalamo-hypophyseal system of mammals (Bjorklund et al., 1987) and appears to be highly similar to the different avian species studied. In a few studies, vasotocinergic cells were described in the SCN (e.g., Bons, 1980; Panzica, 1985). However, the population of vasotocinergic cells in the periventricular preoptic nucleus found in most of the other studies is localized in very close vicinity to the SCN, and whether vasotocinergic cells are indeed present in the SCN of birds remains to be established. In most studies VT-immunoreactivity was not found in the SCN, whereas the LHN contains a prominent population of vasotocinergic cells in all birds studied. Vasoactive intestinal polypeptide has been demonstrated in the hypothalamus of a number of bird species, varying considerably not only among species but also within the same species. This might be partly due to the fact that some authors used colchicintreatment, which has been shown to increase VIP-immunoreactivity (Cloues et al., 1990; Norgren and Silver, 1990; Yamada and Mikami, 1982). In the chicken, Kuenzel and Blahser (1994) found VIPergic bers in the periventricular region of the preoptic hypothalamus and VIPergic cells in the latero-caudal hypothalamus as well as in the paraventricular and preoptic nucleus, whereas Esposito et al. (1993) demonstrated few VIPergic perikarya in the SCN, but no VIP-immunoreactivity in the lateral hypothalamus. Yamada and Mikami (1982), Aste et al. (1995) and Teruyama and Beck (2001) localized VIPergic cells and bers scattered throughout the periventricular preoptic region and in the paraventricular nucleus of the Japanese quail. The latter two studies also mentioned VIPergic neurons in the lateral hypothalamus. The VIPergic system in the hypothalamus of columbiform birds is characterized by a varying number of cells and bers in the

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Figure 6. Functional features of the anterior suprachiasmatic hypothalamus of the house sparrow. Coronal sections from rostral (A) to caudal (C) demonstrating c-fos immunoreactive cells in the rostral SCN as well as lateral to it (black arrows), the LHN, the preoptic nucleus (black arrowheads), and the GLV following light exposure during night. TEL, telencephalon. Immunoreactivity for VT (D), somatostatin (E), and growth-hormone (F) indicate similar functional properties of the ventral (LHNv) and dorsal (LHNd) portions of the lateral hypothalamic nucleus, suggesting a certain demarcation of this cell group. Scale bars 200 mm.

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periventricular preoptic region, the paraventricular nucleus, and the lateral hypothalamus [ring dove (Cloues et al., 1990; Norgren and Silver, 1990), collared dove (den Boer-Visser and Dubbeldam, 2002), pigeon (Hof et al., 1991)]. Norgren and Silver (1990) found VIP-ir cell populations directly above the SCN and dorsomedial from the ventral portion of the LHN. Similar results were obtained in the house sparrow, where a population of small VIPergic cells was found medial to the ventral portion of the LHN (Cassone and Moore, 1987). Taken together, neither the avian SCN nor the LHN nor both combine anatomical, morphological, or functional features of the mammalian SCN. Thus, the ndings of existing literature as compared to our own investigations can be summarized as follows: 1. A SCN has been described in many but not all studies investigating the avian hypothalamus. If present, it has been variably termed by different investigators. In the house sparrow, a clearly demarcated SCN is present that can be well delineated according to neuron-specic staining in coronal and horizontal sections (Figs. 35). This nucleus can also be found in various other species, including zebranch, blackbird, and stonechat (Fig. 4). The reason for the lack of a consistent description of the SCN may be that traditional Nissl stainings do not allow a very good distinction of hypothalamic cell groups. Additionally, the SCN might have been overlooked in some studies because, in variance to mammals, it is located in the most anterior part of the hypothalamus and, thus, possibly represents the anatomical equivalent to the rostral portion of the mammalian SCN (van den Pol, 1980) or suprachiasmatic preoptic nucleus (Bjorklund et al., 1987) rather than the caudal portion of the mammalian SCN. 2. Lateral to the SCN, a morphologically heterogenous cell group is present that consists of a ventral portion, previously described as LHRN (Norgren and Silver, 1989) or vSCN (Cassone and Moore, 1987), and a dorsal part extending into the lateral hypothalamic area. Immediate early response gene expression and the distribution of VT, somatostatin, and growth hormone (Fig. 6) as well as the expression of the putative clock gene per2 (Abraham et al., 2003) do not support a separation into distinct cell groups, but rather indicate that the dorsal and the ventral part are indeed a functionally coupled cell group. This cell group is anatomically distinct from the SCN and, until now, no neural connections between the SCN and the LHN could be found in any avian species. Thus, the term vSCN, based on the presence of retinal input, melatonin receptors, and rhythmic metabolic activity (Cassone, 1988; Cassone and Moore, 1987; Cassone and Brooks, 1991; Lu and Cassone, 1993; King and Follett, 1997), should be avoided in future studies. Neither is this cell group a part of the SCN nor is retinohypothalamic input or the presence of melatonin binding sites a valuable justication for the term SCN. The vSCN is part of a much larger lateral hypothalamic cell group that extends into the lateral hypothalamic area and that shows more similarities to the mammalian supraoptic nucleus than to the SCN (Oksche et al., 1974). 3. The SCN and the LHN are anked and=or overlapped by neurons that stain positively for VT and some other neuropeptides and that appear to be part of the neurosecretory hypothalamo-hypophyseal system. Whether there exists any functional relation between this system and circadian oscillations in the SCN and the LHN (Abraham et al., 2002; Abraham et al., 2003) remains to be established in future studies.

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4. Whether the avian SCN receives direct retinal input or not is still elusive, although it has been investigated in a rather large number of bird species. In contrast to that, retinal projections to the LHN were found in all species studied so far. However, neither tracing retinal projections, nor the investigation of immediate early response gene expression allowed nal conclusions on whether the SCN or the LHN might represent the equivalent to the mammalian SCN. In the house sparrow, photic stimulation during night induces c-fos expression in the rostral part of the SCN, the ventral and dorsal portion of the LHN, the preoptic nucleus, and the ventro-lateral geniculate nucleus, indicating that the light signal may be rapidly distributed over the suprachiasmatic and preoptic hypothalamus of birds independent of whether direct retinal input is present or not (Fig. 6). 5. The avian hypothalamus has been studied regarding the distribution of various neurotransmitters and neuropeptides in a large number of species, but the results were too inconclusive to assign circadian oscillator function to any particular cell group. Neither VT nor VIP were found in the SCN of birds. Instead, VT was found in the preoptic nucleus, close to the SCN in the periventricular preoptic nucleus, as well as in the LHN of most species investigated; whereas VIP-immunoreactive cells were only found close to or within the LHN. In conclusion, a combination of anatomical, morphological, and functional observations is essential for a better understanding and an unambiguous identication of functional cell groups in the avian hypothalamus. Investigations at selected section planes may be misleading when neglecting the full rostro-caudal extension of a cell group, since certain functional compartmentalizations may be present and particular properties may only occur in a certain portion of a cell group, as exemplied by retinal input to the ventral portion of the LHN, immediate early response gene expression that is exclusively present in the most rostral portion of the SCN following a light stimulus (Fig. 6), and a complex spatio-temporal pattern of per gene expression in the SCN (Brandstatter et al., 2001; Abraham et al., 2002; Abraham et al., 2003). Morphological demarcation of cell groups based on Nissl staining or even more sophisticated techniques, such as neuron-specic immunocytochemistry, allow insight that is limited to structural and cytoarchitectonic properties. However, functionally distinct cell types do not always respect the boundaries of traditional cell groups that have been delineated in Nissl stainings, as exemplied by neurons of the neurosecretory hypothalamo-hypophyseal system (Oksche et al., 1974). Thus, an integrative approach utilizing different techniques is needed to obtain unambiguous insight into the organization of complex neural systems. In the avian suprachiasmatic hypothalamus, neither light responsiveness nor rhythmic clock gene expression is conned to a single cell group, as is the case in mammals, but can be found in the SCN as well as in the LHN. Functional studies are needed now to detect possible interaction mechanisms among these cell groups and their function in circadian pacemaking, and to clarify possible integration mechanisms of the melatonin signal to better understand how the complex circadian system of birds works.

ACKNOWLEDGMENTS Thanks are due to T. Roenneberg for coordinating the circadian Schwerpunktprogramm of the German Research Council and his never ending commitment to

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promote chronobiology. Financial support by the German Research Council (DFG grants no. Br-1899=1-1 and Br-1899=1-2) is gratefully acknowledged.

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