Download Hypothalamic Circadian Organization in Birds. I. Anatomy

CHRONOBIOLOGY INTERNATIONAL
Vol. 20, No. 4, pp. 637–655, 2003
Hypothalamic Circadian Organization in Birds. I.
Anatomy, Functional Morphology, and Terminology
of the Suprachiasmatic Region
Roland Brandstätter* 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 Brandstätter, Department of Biological Rhythms and Behaviour, MaxPlanck-Research Centre for Ornithology, Von-der-Tann-Strasse 7, D-82346 Andechs, Germany;
E-mail: [email protected].
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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
(Björklund 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 specific 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 identified in the brain of
any nonmammalian vertebrate, and little is known about the exact localization and specific
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).
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; Brandstätter 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 Brandstätter, 2002; Gwinner
and Brandstätter, 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
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 ¼ 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 Brandstätter, 2001) and (Brandstätter, 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 specific 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 defining 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 fiber 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-specific 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 exemplified 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-specific 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 SCN’’—as 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 ¼ 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. flt,
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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sufficient to identify the site of circadian oscillations but to characterize a defined 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 specific
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; Brandstätter 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-specific nuclear protein revealed that neuron-specific 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 first 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 finally 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-specific nuclear protein (B; for details
see Abraham et al., 2002) demonstrate that neuron-specific immunocytochemistry allows a better
recognition of cellular aggregations that can hardly be demarcated in Nissl stainings. Scale
bar ¼ 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-specific 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 zebrafinch (H). Scale bar ¼ 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 flanked
(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 ¼ 200 mm. The rostro-caudal axis within the sections is indicated in (A). DSO ¼ supraoptic
decussation; LHN ¼ 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 identified 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
exemplifies 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 fibers 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 fibers
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 field 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 significance 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
finch (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 (Björklund 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
Blähser (1994) found VIPergic fibers 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 fibers 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 fibers in the
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 ¼ 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 findings 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-specific staining in coronal and horizontal sections (Figs. 3–5). This nucleus can
also be found in various other species, including zebrafinch, 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 (Björklund 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 justification 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 flanked 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 final 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 identification 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 exemplified 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 (Brandstätter 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-specific
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 exemplified 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 confined
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
Hypothalamic Circadian Organization in Birds. I
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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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