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Hypothalamus
Introductory article
Article Contents
J Patrick Card, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
. Structural Organization of the Hypothalamus
The hypothalamus is a functionally diverse region of the forebrain which exerts profound
regulatory influences over physiological and behavioural processes essential for survival. It
possesses extensive synaptic connections with other regions of the nervous system and is
the region of the brain responsible for controlling the functional activity of the pituitary
gland. An essential feature of its function is the ability to convert synaptic information to
humoral signals that exert regulatory control over peripheral organ systems and to respond
to the functional activity of the peripheral systems that it controls.
. Functional Parcellation of the Hypothalamus
. Summary
Structural Organization of the
Hypothalamus
The hypothalamus is found at the base of the forebrain,
immediately in front of the midbrain flexure (Figure 1a). In
the human brain, three distinct superficial landmarks on
the ventral surface of the brain define its location
(Figure 1b). The most rostral of these is the optic chiasm,
formed by the convergence of the two optic nerves. The
intermediate portion of the hypothalamus is defined by the
pituitary stalk. This structure lies immediately behind the
optic chiasm and is one of the most functionally important
regions of the hypothalamus. It connects the hypothalamus and pituitary gland, and contains the nerve fibres and
blood vessels through which hypothalamic neurons control the secretory activity of this important endocrine
gland. The caudal portion of the hypothalamus is defined
by two prominent spherical protuberances behind the
pituitary stalk, known as the mammillary bodies. These
protuberances define the location of cell groups that are the
site of termination of a distinct fibre tract, the fornix, which
arises from the limbic system and traverses the full
longitudinal extent of the hypothalamus.
The superficial landmarks noted above also define the
location of major subdivisions of the hypothalamus that
were initially defined on the basis of cell morphology and
packing density (Figure 2). These areas, known as the
chiasmatic, tuberal and mammillary subdivisions contain
morphologically distinct groups of neurons that were
initially visualized in tissue sections stained with basophilic
dyes and the Golgi silver impregnation method. The
application of contemporary staining methods has shown
that many of these neurons are also distinguished by their
peptide or neurotransmitter phenotype as well as by their
connections.
Transverse (frontal) sections through the hypothalamus
reveal a central fluid reservoir, the third ventricle, flanked
by grey matter containing a mixture of neurons, glia and
fibre tracts (Figure 2b). Basophilic stains reveal cytoarchitectural differences in neuronal morphology and packing
density that define longitudinal columns extending
Figure 1 The location of the hypothalamus in the ventral forebrain is
illustrated in midsagittal and ventral exposures of the human brain. (a) The
midsagittal exposure reveals the hypothalamus (marked with an asterisk)
immediately in front of the midbrain flexure. This same exposure is shown
at higher magnification in Figure 2a. (b) The ventral exposure reveals the
three major superficial landmarks that mark the position of the
hypothalamus in the ventral diencephalon. The most rostral portion is
marked by the optic chiasm (OC), which is formed by the convergence of
the optic nerves and gives rise to the optic tracts. The pituitary stalk (PS) lies
immediately behind the chiasm in the tuberal hypothalamus. The pituitary
has been removed from this specimen, revealing the central lumen, which
is continuous with the third ventricle. The bilaterally paired mammillary
bodies (MB) mark the caudal-most extent of the hypothalamus.
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Hypothalamus
Functional Parcellation of the
Hypothalamus
Although the classical literature defined hypothalamic
subdivisions solely on the basis of cell morphology and
packing density, it is now known that many of these
structural subdivisions also reflect differences in function.
A large literature has contributed to the definition of
numerous, functionally distinct, cell groups in the
hypothalamus that are beyond the scope of this short
overview. Instead, a subset of those regions that effectively
illustrate the functional diversity of the hypothalamus and
the profound influence that it exerts over central and
peripheral systems are discussed.
The hypothalamus contains a biological clock
Figure 2 (a) The rostrocaudal extent of the hypothalamus, defined by the
optic chiasm (OC) and mammillary body (MB), is shown in midsagittal
exposure. (b) The internal structure of hypothalamus as revealed in a
myelin-stained transverse section passing through the level of the pituitary
stalk. The homogeneous tissue on either side of the third ventricle (V3)
constitutes the bilaterally paired cell masses that form the hypothalamus.
The relative position of periventricular (P), medial (M) and lateral (L)
columns are defined on the left side of the hypothalamus. OT, optic tract: F,
fornix.
through the full extent of the hypothalamus. Groups of
neurons immediately adjacent to the third ventricle
constitute the periventricular column. Prominent among
the functions subserved by the small and densely packed
neurons of this region are biological timing and neuroendocrine regulation of the pituitary gland. The medial
column consists of well defined groups of neurons
immediately adjacent to the periventricular subdivision
which are involved in the expression of motivated social
behaviours. The lateral column consists of diffusely
distributed neurons that communicate extensively with
other regions of the neuraxis.
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One of the principal functions of the hypothalamus is to
provide temporal organization to the expression of
physiological and behavioural processes. This is accomplished by compact cell groups in the rostral hypothalamus, known as the suprachiasmatic nuclei (SCN). SCN
neurons exhibit a genetically derived circadian rhythm of
neuronal activity in which neuronal activity is high for 12 h
of the light dark cycle and low for the remainder. Available
evidence supports the conclusion that each SCN neuron is
an independent oscillator and that communication between SCN neurons allows them to synchronize their
activity and act as an integrated oscillatory unit.
The oscillatory activity of SCN neurons is synchronized
with the external environment through a ‘retinohypothalamic projection’ which arises from ganglion cells in the
retina and synapses in the SCN. Retinal ganglion cells are
functionally heterogeneous and one subpopulation is
responsible for bringing information on luminance in the
external environment to neurons in the SCN. The
entrained oscillatory output of the SCN is transmitted to
other cells groups through axonal projections of SCN
neurons. The synaptic targets of these projections are
found largely within the hypothalamus, but the temporal
information is distributed to a number of cell groups in the
central and peripheral nervous systems through polysynaptic circuits. The temporal information distributed from
the biological clock provides important integrating influences necessary for homeostatic regulation, reproduction
and behaviour.
Regulation of the secretory activity of the pineal gland
provides a vivid example of how the SCN exerts it temporal
influence upon other areas. Melatonin, the secretory
product of the pineal gland, is synthesized and released
into the circulation in a circadian manner which persists in
constant darkness or in blind individuals with an intact
retinohypothalamic tract. Levels of melatonin and its
synthetic enzymes are highest during the dark phase of the
photoperiod and low during the light phase. However,
ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
Hypothalamus
exposure to light during the dark phase of the photoperiod
leads to an abrupt drop in the levels of melatonin
synthesis and release. The entrained circadian release of
melatonin provides an accurate means of measuring day
length that is essential to the regulation of mammalian
physiology, particularly in seasonal breeders. Melatonin
exerts an influence upon the circadian timing system and
endocrine systems through specific binding sites in the
brain, most notably within the SCN and pars tuberalis of
the pituitary gland. The ability of melatonin to influence
the circadian system has led to its use for treatment of
disturbances of biological timing that result from jet lag
and shift work.
Available evidence supports the conclusion that both the
circadian and light influences upon melatonin secretion are
mediated through a trisynaptic pathway which impinges
upon the autonomic outflow to the pineal gland. The first
synapse in this circuit is the retinohypothalamic projection
to the SCN which, in turn, projects upon neurons in the
paraventricular nucleus of the hypothalamus. The subpopulation of PVN neurons receiving this input project to
neurons in the thoracic spinal cord that give rise to the
dysynaptic autonomic outflow to the pineal gland. Interruption of any component of this circuitry abolishes the
circadian and photic regulation of pineal function. Thus,
the SCN imposes a temporal influence upon diverse
physiological systems through a multisynaptic circuit,
which ultimately is converted to a humoral signal exerting
feedback regulatory influences upon the brain.
The hypothalamus participates in the
regulation of sleep
It has long been known that the hypothalamus participates
in the regulation of sleep. Early studies identified ‘sleep
centres’ in different portions of the hypothalamus that were
thought to regulate opposite components of the sleep–
wake cycle. These studies were based upon the loss of
function that resulted from lesions of different portions of
the hypothalamus in animals, and suggested that the
rostral hypothalamus contained a centre involved in the
induction of sleep while the caudal hypothalamus contained a centre responsible for arousal. Recent studies
support these basic observations and have begun to reveal
the specific neurons and underlying circuitry that provide
the basis for this regulation. For example, specific
populations of neurons in the rostral preoptic area of the
hypothalamus are now known to become functionally
active shortly after animals fall asleep, and these same cells
inhibit the activity of histaminergic neurons in the caudal
hypothalamus thought to be involved in arousal. These
compelling observations suggest that the hypothalamus
subserves an important role in the larger set of systems
involved in the regulation of this complex behaviour.
The preoptic hypothalamus influences
thermoregulation
Numerous observations suggest that multiple systems in
the rostral hypothalamus participate in the regulation of
temperature. For example, thermosensitive neurons are
present in the rostral hypothalamus that are activated by
temperature probes. Warming the preoptic area during
waking in animals suppresses the activity of arousalassociated neuronal discharge in the caudal hypothalamus,
as well as in basal forebrain neurons with diffuse cortical
projections. Similarly, this manipulation reduces motor
activity, metabolic function and respiratory rate, suggesting that the compensatory responses to thermal stress may
be expressed through the same circuitry involved in the
initiation of sleep. The well documented circadian rhythm
in body temperature also implicates the SCN in thermoregulatory mechanisms, and there is a large literature
indicating that fever is mediated by the anterior hypothalamus. Collectively, these observations implicate the
rostral hypothalamus as an important site of body
temperature regulation.
Neuroendocrine regulation is an important
hypothalamic function
One of the most important and well documented functions
of the hypothalamus is to regulate the hormonal outflow
from the pituitary gland. This is achieved through an
important structural modification of the mediobasal
hypothalamus through which synaptic information is
converted to bloodborne humoral signals. The pituitary
gland contains two distinct lobes, which are distinguished
by their developmental origin. The anterior lobe derives
from an outpocketing of the ectoderm known as the
Rathke pouch. The posterior lobe is an evagination of the
portion of the neural tube that gives rise to the
hypothalamus. Thus, the posterior lobe is continuous with
the floor of the hypothalamus through this stalk-like
evagination, whereas a dedicated series of long portal
vessels on the surface of the pituitary stalk provides the
only connection between the hypothalamus and the
anterior lobe of the pituitary.
In spite of the structural and developmental differences,
the secretory output of both lobes of the pituitary is under
the direct control of the hypothalamus. Two general
systems of neurons contribute to this regulation. Large
‘magnocellular’ neurons in the hypothalamus give rise to
axons that traverse the pituitary stalk to terminate within
the posterior lobe. These neurons receive synaptic inputs
from other regions of the brain, but release their peptide
contents into the peripheral vasculature in the posterior
pituitary. In this manner, a synaptic signal is converted to a
bloodborne humoral signal that influences peripheral
targets. Small ‘parvocellular’ neurons produce releasing
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Hypothalamus
hormones and inhibiting hormones that control the
secretory activity of cells in the anterior pituitary.
However, the parvocellular neurons release their secretory
products into fenestrated capillaries that drain into the
long portal vessels that drain into the anterior lobe.
The magnocellular neurons secrete either vasopressin
or oxytocin, and are largely concentrated in the
supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. Although both the PVN and SON contain
magnocellular neurons expressing those peptides, the
neurons are sequestered within different subdivisions of
these two hypothalamic nuclei and participate in different
regulatory processes. Oxytocin is best know for its
effect on milk letdown in lactating females. Vasopressin,
also known as antidiuretic hormone, is involved in the
regulation of blood pressure and provides an excellent
example of feedback regulation of peripheral systems
upon the functional activity of the hypothalamus. In
response to low blood pressure, the kidney releases renin
into the vasculature which is subsequently metabolized to
angiotensin II. Angiotensin II has access to neurons in a
region of the nervous system known as the subfornical
organ (SFO) that lacks a blood–brain barrier. SFO
neurons express angiotensin II receptors and also
project to the magnocellular vasopressinergic neurons in
the PVN. Interestingly, the SFO neurons involved in this
projection also appear to use angiotensin II as a
neurotransmitter. The SFO neurons activated by peripherally circulating angiotensin II elicit the release of
vasopressin from the magnocellular endings in the posterior pituitary, which acts at the level of the kidney to reduce
fluid excretion and thereby increase blood fluid volume.
Thus, the hypothalamus is not only an effector system for
modulating peripheral functions, but is also subject to
feedback regulation by the physiological processes that it
controls.
As noted above, hypothalamic control of anterior
pituitary function also involves conversion of synaptic
information to humoral signals. This system draws upon a
heterogeneous population of neurochemically distinct,
parvocellular neurons in the hypothalamus and a dedicated vascular plexus between the hypothalamus and the
pituitary. The long portal vessels that constitute this
neurohaemal link arise from fenestrated capillaries in the
floor of the hypothalamus and traverse the pituitary stalk
to gain access to sinuses in the anterior pituitary gland.
Parvocellular neurons project to the circumscribed region
of the pituitary stalk, known as the median eminence,
where fenestrated vessels drain into the long portal vessels
supplying the anterior pituitary. Parvocellular neurons
that contribute to anterior pituitary regulation are
concentrated in a number of cell groups in the anterior
two-thirds of the hypothalamus. The largest concentration
of cells is found in the arcuate and paraventricular
hypothalamic nuclei. However, substantial numbers of
cells are distributed throughout other regions of the
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hypothalamus, such as the periventricular nucleus and
preoptic area.
The neurochemical phenotype of parvocellular neurons
plays an extremely important role in determining the
secretory responses of the anterior pituitary gland.
Often, two neurochemically distinct populations of
parvocellular neurons will have opposing effects upon the
same group of cells in the anterior pituitary. For
example, release of growth hormone (GH) from the
anterior pituitary is determined by the differential release
of two parvocellular populations of hypothalamic neurons. Arcuate neurons that synthesize growth hormonereleasing hormone stimulate the release of GH from the
anterior pituitary, and the opposite is achieved when
parvocellular neurons in the periventricular nucleus
release somatostatin (growth hormone-inhibiting hormone) into the portal plexus. Thus, appropriate regulation
of GH release from the pituitary is dependent upon the
regulated release of opposing peptides at the level of the
median eminence, a complex process that is dependent
upon both the hormonal output of the pituitary and
feedback from the metabolic products that are driven by
GH secretion.
Reproductive function is controlled by the
hypothalamus
The essential role of the hypothalamus in the production
of the gametes necessary for reproduction and survival of
the species has been documented thoroughly. This is
achieved through the aforementioned neuroendocrine
regulatory mechanisms within which hypothalamic nuclei
control anterior pituitary function. However, it is equally
clear that the hypothalamus plays an essential role in the
expression of the complex reproductive behaviours necessary for fertilization of ova. Experimental studies in a
number of species have demonstrated that different
populations of neurons mediate sexual behaviour in males
and females, with male sexual behaviour being mediated by
the neurons in the preoptic area and female sexual
behaviour by neurons in the tuberal hypothalamus.
Dimorphism in hypothalamic structure and connectivity
in males and females has also been reported in animals and
humans. The significance of this dimorphism remains to be
determined, but there can be little doubt that the
differences in structure are determined by the hormonal
milieu present during different stages of brain development.
Autonomic function is influenced by the
hypothalamus
The hypothalamus is also an important integrative
centre that modulates the output of the autonomic
nervous system (ANS). Dense descending projections of
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Hypothalamus
hypothalamic neurons terminate upon preganglionic
neurons of both the parasympathetic and sympathetic
subdivisions of the ANS in the brainstem and spinal cord.
Several hypothalamic cell groups contribute to this
projection, but particularly prominent monosynaptic
projections from neurons in the PVN and lateral hypothalamic area (LHA) have been demonstrated.
Insight into the identity of neurons that modulate the
functional activity of sympathetic preganglionic neurons
in the intermediolateral cell column of thoracic
spinal cord has recently been provided by studies employing neurotropic alpha herpesviruses for transneuronal
labelling of synaptically linked circuits. These viruses
replicate within permissive neurons and pass transynaptically to infect other neurons in a polysynaptic circuit. Thus,
they can be used as self-amplifying transynaptic tracers to
identify populations of neurons with a common function.
Using this approach. Loewy and colleagues have postulated that neurons in the PVN, LHA and perifornical
hypothalamic nucleus are part of a group of ‘central
command neurons’ in the diencephalon and brainstem that
function under circumstances where it is necessary to
coordinate responses in functionally separate components
of the sympathetic outflow. Although this interesting
hypothesis requires further examination, the findings of
this and other studies emphasize the importance of the
hypothalamus in the control of autonomic function and
identify output pathways through which this regulation is
achieved.
The hypothalamus influences immune
function
A large body of evidence now supports the conclusion that
the central nervous system influences immune function
through both the ANS and neuroendocrine regulation of
the pituitary gland. Felten and colleagues have demonstrated that postganglionic noradrenergic neurons of the
sympathetic nervous system densely innervate compartments of the spleen and other lymphoid organs involved in
immune function. Although the central neurons that
modulate the functional activity of this sympathetic
outflow remain to be defined, it seems probable that the
established descending projections of hypothalamic neurons to the intermediolateral cell columns of thoracic
spinal cord contribute to this innervation. Further
evidence for a hypothalamic influence upon the immune
system can be found in studies of the neuroendocrine
system. Work championed by Besedovsky and colleagues
in the 1970s, which has been confirmed and expanded by
subsequent investigators, revealed that the neuroendocrine
outflow of the hypothalamus influences the activity of the
immune system which, in turn, exerts a feedback regulatory control on the brain.
The hypothalamus influences food and fluid
homeostasis
The hypothalamus functions within a larger set of central
nervous system circuits responsible for homeostatic
mechanisms involved in the regulation of food and fluid
intake. Neuronal markers of activity have been particularly informative in defining the cell groups that respond to
challenges of each of these systems, and have provided
considerable insight into the organization of these systems.
Stimuli that evoke adaptive changes in fluid osmolarity and
blood volume (e.g. dehydration or manipulation of blood
sodium levels) have revealed functional parcellation in the
regions of the brain responsible for regulating fluid
homeostasis. For example, neurons sensitive to fluid
osmolarity have been localized to a circumscribed region
in the rostral hypothalamus, whereas neurons that respond
to changes in blood pressure are found in the SFO and
caudal brainstem. Nevertheless, these areas utilize the
same effector systems (magnocellular–posterior pituitary
system) to elicit compensatory changes in fluid volume.
Alterations in food intake are also the product of a
balanced interaction between effector pathways that elicit
food intake and feedback regulation that inhibits consumption. Sensory feedback to the nervous system after a
meal takes the form of excitation of the sensory neurons
innervating the viscera as well as the release of neuroactive
compounds from the gastrointestinal tract. These sensory
stimuli act in the caudal brainstem in regions that maintain
dense reciprocal connections with hypothalamic nuclei. In
addition, hypothalamic nuclei are the primary target of
hormones released into the circulation after a meal. Both of
these regulatory pathways have the capacity to alter the
functional activity of hypothalamic neurons involved in
feeding.
The limbic system influences the output of the
hypothalamus
The functional diversity of the hypothalamus is reflected
not only by its many and varied output pathways, but also
by the projections that it receives from other regions of the
nervous system. This extensive connectivity emphasizes the
importance of the hypothalamus as a dynamic integrative
centre with profound influences upon both central and
peripheral nervous systems. A particularly poignant
example of this integrative capacity can be appreciated
by considering the projections of the limbic system upon
the hypothalamus. Areas that contribute to the limbic
system, such as the hippocampus and amygdala, project
heavily upon the hypothalamus and there have been
considerable advances in our understanding of the function of these connections.
The hippocampal projection to the hypothalamus
through the fornix is one of the most well described and
ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net
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Hypothalamus
long acknowledged monosynaptic projections of the limbic
system into the diencephalon. In 1937, James Papez
proposed that the projection of hippocampal neurons to
the mammillary nuclei through the fornix was part of a
multisynaptic circuit involved in the expression of emotion. This hypothesis has not been confirmed, and it has
subsequently become apparent from contemporary studies
that the cells that give rise to this projection are found in the
subiculum rather than the hippocampus. Nevertheless,
recent work of Risold and Swanson has demonstrated that
the hippocampus does exert a profound influence upon
hypothalamic function via disynaptic projections through
the lateral septal nucleus. This projection is topographically organized such that subpopulations of hippocampal
neurons project into subfields of the lateral septum which,
in turn, project upon longitudinal columns extending
through the rostrocaudal extent of the hypothalamus. The
columns that receive these projections are multifunctional,
but contain cell groups that can be grouped into general
functional categories. Thus, neurons in the periventricular
zone are associated with neuroendocrine and autonomic
function as well as ingestive behaviours, while those in the
rostral medial zone influence motivated reproductive and
agonistic behaviours, and those in the mammillary region
feed processed information back to the hippocampus.
These disynaptic projections into hypothalamus are far
more extensive and target many more functionally distinct
hypothalamic systems than the monosynaptic projections
to the mammillary nuclei that pass through the fornix.
The amygdala also gives rise to extensive projections to
the hypothalamus that pass through the stria terminalis.
There is now strong evidence that the amygdala plays an
integral role in the expression of fear and anxiety, and that
effector pathways responsible for the expression of
behaviours include both the hypothalamus and ANS.
Activation of the sympathetic component of the ANS in
response to fearful or stressful stimuli is thought to occur
through projections of the amygdala and locus coeruleus
upon hypothalamic nuclei. These projections also activate
neurons in the PVN, which ultimately lead to the increased
levels of cortisol in the circulation by virtue of their
regulation of anterior pituitary function. This dual
activation of hypothalamic systems is a prominent
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contributor to the stereotypic responses to stress and fear,
and vividly illustrates how integration of information by
the hypothalamus can activate multiple effector systems
that contribute to complex behavioural responses.
Summary
In conclusion, the hypothalamus is an important integrative centre in the forebrain that subserves a variety of
essential functions. Its ability to fulfil effectively the diverse
functional demands placed upon it is integrally related to
its ability to convert synaptic information to humoral
signals and to respond to feedback regulation by the
peripheral systems that it controls.
Further Reading
Alder R (1996) Historical perspectives on psychoneuroimmunology. In:
Friedman H, Klein TW and Friedman AL (eds). Psychoneuroimmunology, Stress, and Infection, pp. 1–24. Boca Raton, FL: CRC Press.
Charney DS, Grillon C and Bremmer JD (1998) The neurobiological
basis of anxiety and fear: circuits, mechanisms, and neurochemical
interactions (Part I). Neuroscientist 4: 35–44.
Loewy AD (1990) Central autonomic pathways. In: Loewy AD and
Spyer KM (eds) Central Regulation of Autonomic Function, pp. 88–
103. New York: Oxford University Press.
Martin JB and Reichlin S (1987) Fundamental aspects of neuroendocrinology. In: Clinical Neuroendocrinology, chap. 1, pp. 1–62.
Philadelphia: FA Davis.
Moore RY (1992) The organisation of the human circadian timing
system. Progress in Brain Research 93: 101–117.
Ramsay DJ and Thrasher TN (1990) Thirst and water balance. In:
Stricker EM (ed.) Handbook of Behavioral Neurobiology, pp. 353–386.
New York: Plenum Press.
Risold PY and Swanson LW (1997) Connections of the lateral septal
complex. Brain Research Reviews 24: 115–195.
Saper CB (1990) Hypothalamus. In: Paxinos G (ed.) The Human Nervous
System, pp. 389–414. San Diego: Academic Press.
Sherin JE, Shiromani PJ, McCarley RW and Saper CB (1996) Activation
of ventrolateral preoptic neurons during sleep. Science 271: 216–219.
Stellar E (1990) Brain and behaviour. In: Stricker EM (ed.) Handbook of
Behavioral Neurobiology, pp. 3–22. New York: Plenum Press.
Swaab DF and Fliers E (1985) A sexually dimorphic nucleus in the
human brain. Science 228: 1112–1115.
ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net