C. Microscopic Anatomy

1.  Boundaries and Organization of Neuronal Cell Groups
Using phylogenetic and cytoarchitectonic criteria (46), a number of nuclear groups and fiber tracts are recognized in the vertebrate hypothalamus.  These are organized into three major regions including the lateral, medial and periventricular hypothalamus, each having distinct morphological and functional features.  In the human hypothalamus, the anterior column of the fornix that extends rostro-caudally through the substance of the hypothalamus to end in the mammillary bodies, and the mammillo-thalamic tract that projects from the mammilary bodies upward to the thalamus, create an anatomical boundary that divides the hypothalamus into medial and lateral subdivisions (Fig. 16).  Contained within the medial subdivision is the periventricular subdivision, a 5-6 cell layer thick nuclear group surrounding the third ventricle that is easily recognized in rodents using standard vital stains, but has less clear anatomical boundaries in the human brain.

Fig. 16.  Schematic representation of the human hypothalamus in coronal orientation (A-D: rostral to caudal), demonstrating the location of major nuclear groups. Drawings correspond to MRI images in Fig. 26.  Using the fornix (fx) as an anatomic landmark as it passes through the mid-portion of the hypothalamus on each side of the third ventricle, it is convenient to divide the hypothalamus into medial and periventricular zones (that lie largely medial to the fornix) and a lateral zone (that lies lateral to the fornix).  The medial and periventricular zones contain most of the hypothalamic cell groups, and the lateral zone contains relatively fewer neurons.  This is because the lateral zone is largely composed of a massive bidirectional fiber pathway – the medial forebrain bundle – that extends through the hypothalamus and interconnects it with the limbic system and brainstem autonomic centers.

   Both the medial and periventricular subdivisions of the mammalian hypothalamus contain a high density of neuronal cell bodies organized into nuclear groups (Tables 5 and Fig. 16) and in the human brain, has been classified with a number of different synonyms (Table 6) (6).  Both subdivisions are crucial for the regulation of the anterior and posterior pituitary gland.  The medial hypothalamus also contains nuclear groups that serve as relay centers for highly differentiated neural information coming from the limbic system and autonomic sensory centers in the brainstem involved in initiation phases of specific homeostatic behaviors such as thirst, hunger, thermoregulation, the sleep-wake cycle, and reproductive behavior (46).
The lateral hypothalamus occupies the largest portion of the hypothalamus by volume.  However, it has relatively fewer neurons compared to the medial hypothalamus, and only a limited number of nuclear groups intercalated within a massive fiber system, the medial forebrain bundle (MFB). It is through this fiber system that information from the medial forebrain (amygdala, hippocampus, septum, olfactory system) and the brainstem is carried to the medial and periventricular hypothalamic subdivisions, delegating an important role to the lateral hypothalamus to influence homeostatic control systems elaborated by the medial hypothalamus.  Figure 17 schematically depicts major interrelationships between the periventricular, medial and lateral hypothalamic subdivisions and the rest of the brain.

Fig. 17. Schematic representation of the major neural pathways connecting the periventricular, medial and lateral hypothalamic subdivisions with the rest of the brain.  Groups with identical colors are functionally linked.

    Each of the three hypothalamic subdivisions can be further divided along the rostral-caudal axis into the: a) anterior or chiasmatic region, extending between the lamina terminalis and the anterior limit of the infundibular recess; b) median or tuberal region, extending between the infudibular recess and the surface of the anterior column of the fornix; and c) posterior or mammillary region, extending between the anterior column of the fornix and the caudal limit of the mammillary bodies.

1. Circumventricular Organs

a. Median Eminence (ME)
One of the most important regions in the hypothalamus that is essential for regulation of the pituitary gland is the median eminence, a midline structure located in the basal hypothalamus ventral to the third ventricle and adjacent to the arcuate nucleus.  It is here that all hypophysiotropic hormones converge before they are conveyed to the pituitary gland.  The median eminence is one of seven so called circumventricular organs situated as midline structures in the walls of the lateral, third or fourth ventricles (47,48).  Other circumventricular organs include the organum vasculosum of the lamina terminalis, subfornical organ, choroid plexus, pineal gland, subcommissural organ and area postrema (Fig. 18).  Characteristically the circumventricular organs contain a rich capillary plexus and with the exception of the subcommissural organ, have a fenestrated endothelium rendering the structures outside of the blood brain barrier.  This morphologic feature together with the presence of neural elements contacting the fenestrated capillaries allows the secretion of brain-derived products into the peripheral circulation and/or makes circumventricular organs targets for blood-born information which can then be transmitted to the brain (49).

Fig. 18.  Location of circumventricular organs in the rat brain.  AP = area postrema, ME = median eminence, OVLT = organum vasculosum of the lamina terminalis, P = pineal gland, PP = posterior pituitary, SFO = subfornical organ.  (Modified from Saper and Breder, New England Journal of Medicine 330: 1080-1886, 1994.)

    The median eminence is a highly organized structure containing three zones: the ependymal zone, the internal zone (or zona interna) and the external zone (or zona externa) (50,51) (Fig. 19A).  The ependymal zone forms the floor of the third ventricle and has some very specialized features including densely formed tight junctions between adjacent cells and highly specialized cells, tanycytes, that extend bleb-like protrusions and microvilli into the cerebrospinal fluid (CSF) at their ventricular surface and long cytoplasmic processes ventrally into the substance of the median eminence (51,52).  Since the portal capillaries in the median eminence lie outside of the blood brain barrier, one of the functions of the ependymal zone is to create a barrier to the brain, preventing substances released into the periportal capillary spaces from entering the cerebrospinal fluid (52,53).  Tight junctions also can be found at the dorso-lateral margins of the median eminence adjacent to the neuropil of the arcuate nucleus created by perivascular tanycyte processes (41), thereby compartmentalizing all substances entering the periportal capillary spaces within the confines of the median eminence, itself.  Norsted et al (42), however, have demonstrated the absence of endothelial barrier antigen in blood vessels at the far ventro-medial aspect of the arcuate nucleus close to the border between the walls and floor of the third ventricle, and propose the lack of a blood brain barrier in this region.  Allowing blood-born substances including gut peptides, glucose, amino acids and fatty acids to enter the arcuate nucleus in this region may be an important homeostatic mechanism that contributes to the regulation of appetite and satiety (see later).

    Tanycytes are presumed to have other important neuroendocrine functions that may supersede their function as barrier cells.  The close association of tanycyte foot processes with the basal lamina of the portal capillaries and with individual axon terminals (Fig. 20) could create a retractable barrier to regulate the diffusion of secretory products entering or exiting specific regions of the portal capillary plexus or from axon terminals (55,56).  Similar dynamic interactions between glial cells and secretory nerve endings in the posterior pituitary have been described by Beagley and Hatton (57).  In addition, the absorption of substances from the CSF at its apical surface for transport to the portal capillaries 52,58,59) could result in a mechanism whereby secretory products released into the CSF have access to the anterior pituitary.  Tanycytes may also serve as a scaffolding for axons entering the median eminence during embryologic development, guiding them to their ultimate destination in the external zone (60).  Finally, tanycytes express one of the highest concentrations in the brain of type 2 deiodinase (D2), the enzyme responsible for the conversion of thyroxine (T4) into its more biologically potent product, triiodothyronine (T3), suggesting a role in control of the hypothalamic-pituitary-thyroid axis (61).

    The internal zone of the median eminence lies directly below the ependymal zone and is primarily composed of unmyelinated axons of passage of the hypothalamic-neurohypophysial system en route to the posterior pituitary (Fig. 19B).  Characteristic of these axons are dilatations or Herring bodies, in which collect large numbers of neurosecretory granules measuring 200 to 350 nm in diameter (Inset, Fig. 19B).  The internal zone also contains cytoplasmic processes of tanycytes and axons of passage of the hypothalamic tuberoinfundibular system as they descend into the external zone.
The external zone underlies the internal zone and in addition to the portal capillaries and cytoplasmic extensions of the tanycytes described above, it contains numerous fine caliber, unmyelinated axons and axon terminals of the hypothalamic tuberoinfundibular system (Fig. 19C).  Characteristic of these axon terminals are dense-core vesicles ranging from 50 to 130 nm in diameter (Fig. 20).  The close proximity of many of the axon terminals to the portal system suggests that these axons are capable of secreting the material stored in their vesicles into the pericapillary spaces and by percolating through the fenestrated endothelium of the portal capillaries, reach the anterior pituitary by way of the long portal vessels.  These substances, commonly referred to as hypothalamic releasing and inhibitory hormones on the basis of their ability to stimulate or inhibit anterior pituitary hormone secretion respectively, have been chemically identified and are listed in Table 7
Many axon terminals, however, do not abut directly on portal capillaries or terminate at some distance from the portal capillary plexus, may be to serve a modulatory role on other axon terminals rather than secrete into the portal plexus and explain the large numbers of peptides in the median eminence that either have no certain, direct action on anterior pituitary cells or can not be measured in the portal blood (62).  Axon terminals containing dopamine, for example, are located in close proximity to axon terminals containing GnRH (63) at the lateral margins of the external zone of the median eminence and can modulate the secretion of GnRH by presynaptic inhibition (64,65).  Galanin containing axon terminals have also been observed to overlap with GnRH terminals in the lateral portion of the median eminence (66) but stimulate GnRH release from median eminence fragments (67).  Although axo-axonal synapses are uncommon in the median eminence of most animal species studied using morphologic criteria (50), receptors for several different peptide hormones have been identified on axon terminals in the external zone suggesting that axo-axonal interactions can take place.  Given the slow circulation time of blood perusing the median eminence (68), synaptic specialization in the median eminence may be unnecessary.

Alternatively, axons terminating at a distance from the portal capillaries may be held in reserve and only secrete to the anterior pituitary under certain physiological conditions.  This phenomenon has been described for several peptides such as neuropeptide Y, whose concentration increases in portal capillary blood during an ovulatory surge to potentiate the action of GnRH on gonadotropin secretion (69,70).  Similarly, VIP/PHI, which shows a minimal immunocytochemical staining pattern in the median eminence in the basal state, increases during suckling to stimulate prolactin release (71) and vasopressin markedly accumulates in the external zone following adrenalectomy (72).  The anatomical correlate of these physiologic observations may be suggested by the work by King and Letourneau (73) on gonadotropin regulation in which GnRH-containing axon terminals in the median eminence can be found at different distances from the portal capillaries in intact animals vs gonadectomized animals.  This indicates the potential for a dynamic association between axon terminals of the tuberoinfundibular system and the portal capillaries under specific physiologic conditions.  Marked reorganization in the median eminence of several different peptide-containing axon terminals in the median eminence has also been observed following hypophysectomy (74).
A further complexity to the physiology of axon secretion in the external zone of the median eminence is the common occurrence of more than one peptide or transmitter coexisting in the same axon terminal.  For example, TRH and preproTRH 160-169 coexist in the same axon terminals in the median eminence (75) and together have important potentiating effects on anterior pituitary TSH secretion (76).  Galanin coexists with GHRH in the majority of GHRH-tuberoinfundibular neurons (77) and although does not stimulate growth hormone secretion by itself in dispersed anterior pituitary cells (78), when administered together with GHRH, it increases GH secretion over what can be achieved by GHRH alone (79). Rather than arise as a biosynthetic product of the same precursor molecule as preproTRH 160-169 and TRH, galanin and GHRH are derived from two separate gene products, expanding the possible sources for peptides that potentiate anterior pituitary secretion (80).  The coexistence of substances in axon terminals may also help to coordinate the secretion of separate anterior pituitary hormones as has been proposed for VIP/PHI, neurotensin, and enkephalin in CRH-producing neurons (81) to coordinate the secretion of ACTH, GH and prolactin during stress (82).
In addition to axon terminals in the external zone of the median eminence, densely packed fibers that contain VIP and the nitric oxide-synthesizing enzyme, nitric oxide synthase (83), have been described on the ventral surface of the median eminence separated from the external layer (84).  These fibers surround portal vessels and innervate smooth muscle of precapillary arterioles that supply the portal capillary plexus of the median eminence.  Since both VIP and NO are potent vasodilators (85,86), these substances may play an important role in regulating the rate of blood flow to the median eminence and hence to the anterior pituitary, thereby exerting a separate level of control over anterior pituitary secretion.  As opposed to axon terminals in the external zone of the median eminence that derive from the hypothalamus (see below), axons involved in regulation of portal blood flow appear to arise from other regions such as the sphenopalatine ganglion (83,84).
Consistent with the concept that the median eminence lies outside of the blood-brain barrier, claudin-5 and ZO-1, markers for tight junctions, are absent from vessels in the external layer (34a).

    The OVLT is located in the midline of the lamina terminalis as part of the anterior wall of the third ventricle (Fig. 18).  Its dorsal surface protrudes into the third ventricle cavity and its ventral surface is in direct contact with the prechiasmatic cistern.  Thus, OVLT cells are in a position to be bathed by soluble factors in the CSF in both ventricular and cisternal spaces.  In rodents, ultrastructural studies by Weindl et al (77) and Mitro and Palkovits (88) have described a variety of cell types in the OVLT, including specialized neurons, tanycytes, ciliated ependyma, and glial cells (89).  Some of these cells send long processes to the periventricular space, whereas others establish specialized junctions and synaptic contacts or project outside the OVLT (89-91).
As in the median eminence, the OVLT contains fenestrated capillaries.  They are derived from small branches of the preoptic artery that break up into a dense network of small vessels in the pia matter lining the external surface of the lamina terminals, and loop up towards the ventricular lumen (92).  These vessels circumscribe interstitial spaces filled with cellular processes and secretory nerve endings that contain a number of neurotransmitter substances including atrial naturetic peptide, vasopressin, somatostatin, and GnRH (93), suggesting that like the median eminence, the OVLT subserves a neuroendocrine function.  In contrast to the median eminence, however, blood from the OVLT does not drain into a portal plexus, but rather primarily to the medial preoptic region (94), suggesting a close functional interrelationship between the OVLT and this region of the hypothalamus.  In addition, neurons in the OVLT project to the preoptic nucleus, subfornical organ, arcuate nucleus, supraoptic nucleus and parts of the limbic system.  This anatomical organization, therefore, strategically places the OVLT in an ideal location to receive blood-born information and then transmit this information to specific regions of the brain.  Accordingly, the OVLT has been implicated in mediating the febrile effects of circulating cytokines (see Thermoregulation).  In addition, the OVLT is involved in osmoregulation and fluid balance through osmoreceptor cells that express the transient receptor potential vanilloid (TRPV) 1 gene (95), and respond to circulating levels of angiotensin II and relaxin (96,97).  The OVLT is also densely innervated by axon terminals containing GnRH, originating from perikarya in the septum and areas surrounding the OVLT that presumably contribute to the regulation of pituitary gonadotropin secretion (98), perhaps through connections between the OVLT and the median eminence (99).  In female rodents, these axons cross the OVLT en route to the median eminence to trigger pulsatile proestral release of pituitary gonadotropins (100), whereas in males, their gonadotropin-releasing hormone content is regulated by levels of circulating thyroid hormone (101,102).   Direct connections between the OVLT and the median eminence have also been described (99). 

    The name of this circumventricular organ derives from its midline, anatomical location under the fornix (Fig. 18), at the point where the lamina terminalis joins the tela choroidea of the third ventricle (103).  Embryologically, the SFO arises from the same part of the neural tube as the OVLT, and accordingly, have a similar microarchitecture and share common functions (104).
The SFO can be divided into two regions: a peripheral shell or "perimeter” that is rich in nerve endings arising from neurons intrinsic to the SFO but poor in blood capillaries, and a more densely packed center or "core” crowded with neuronal and glial perikarya and containing a dense vascular network of fenestrated and unfenestrated capillaries.  In caudal portions of the SFO, capillaries are continuous with those of the choroid plexus
(105).  It is presumed that the "core” of the SFO is the locus for major hormonal receptor fields and fiber terminals of its afferent neuronal innervation, particularly the median preoptic nucleus, whereas the "perimeter” is the site of exit for SFO axons projecting to specific target regions in the hypothalamus including the preoptic nucleus, OVLT, supraoptic nucleus, paraventricular nucleus and lateral hypothalamus (106).
The SFO has an important role in coordination of fluid balance with blood pressure and drinking behavior, especially during hemorrhage and hypovolemia (107).  The rich vasculature of the SFO allows circulating angiotensin II to stimulate intrinsic neurons (108) via angiotensin type 1 receptors (109).  Through direct projections to the paraventricular nucleus, supraoptic nucleus and accessory magnocellular cell groups of the hypothalamus (110), SFO neurons induce release of vasopressin from the posterior pituitary (111), activate paraventricular nucleus neurons that descend to sympathetic centers of the spinal cord that regulate vasoconstriction (1127), and possibly favor the release of vasoactive peptides like VIP from the anterior pituitary and a number of other neural sites related to fluid and blood pressure balance (113).  Evidence for intrinsic production of angiotensin II in the SFO (114a) and the antagonistic effects of galanin released from axon terminals that synapse on SFO neurons on angiotensin II-induced drinking behavior and vasopressin release (115), may also contribute to the regulation of fluid homeostasis.  The presence of other peptides in the SFO and/or their receptors including obestatin, somatostatin and thyrotropin-releasing hormone, has suggested that the SFO might play a role in coordinating the ingestive behavior of liquids with solid food and the sleep cycle (116-118).  Indeed, due to its connections with the preoptic nucleus and OVLT, the SFO is also involved in the regulation of thirst and locomotor behavior for drinking (119). 

   The fiber systems that link the hypothalamus to the rest of the brain are numerous and intricate, reflecting the importance of the hypothalamus as an integrating center for the rest of the brain.  Due to the complexity of the fiber systems, however, it is impractical to individually describe each fiber pathway linking each nuclear group, particularly for the human hypothalamus in which nuclear boundaries and relative projections are less clear than in other mammals.  Readers are referred to extensive reviews on this topic (120,121).  We will describe only the major hypothalamic fiber systems with respect to their afferent and efferent connections to the periventricular, medial and lateral hypothalamic nuclear subdivisions.

a. Afferent Connections
Inputs to the mammalian hypothalamus arise primarily from the limbic system, brainstem reticular formation, thalamus, subthalamus, basal ganglia, retina and possibly the neocortex (Fig. 21).  Afferents from the limbic system include 3 main fiber groups, the medial forebrain bundle, the stria terminalis, and the fornix.  The medial forebrain bundle is located in the most lateral part of the hypothalamus and contains fibers originating from more than 50 nuclear groups in different regions of the brain including descending fibers from the olfactory and septal areas, and ascending fibers from the amygdaloid complex and substantia innominata, the latter forming the ventral amygdalofugal component of the ansa peduncularis.  The stria terminalis originates in the amygdaloid complex, and the fornix in the hippocampus, both entering the rostral-medial hypothalamus close to the ventricular surface, and then arching into the substance of the hypothalamus to terminate along the entire extent of the hypothalamus.

   Afferents from the brainstem reticular formation include the dorsal longitudinal fasciculus (fasciculus of Schutz), the periventricular fiber system and the medial forebrain bundle.  The dorsolongitudinal fasciculus receives primarily autonomic inputs from centers in the mesencephalic tegmentum (limbic midbrain area), reticular raphe nuclei in the pons and viscero-sensitive nuclei (e.g. the nucleus tractus solitarius) in the medulla oblongata.  The periventricular system carries fibers ascending from both the central grey (including the raphe nuclei) and medial nuclei of the reticular formation in the mesencephalon or dorsal nucleus of the mesencephalic tegmentum (limbic midbrain area). Collectively, these fibers enter the hypothalamus close to the ventricular wall.  The medial forebrain bundle also receives a well-defined fiber tract, the mammillary peduncle, originating from the medial nuclei of the mesencephalic reticular formation (limbic midbrain area).  It courses ventrally in the cerebral peduncle (within the ventral tegmental area of Tsai), and then laterally to the mammillary bodies.
Afferents from the thalamus originate in nuclei of the median and medial thalamus, and course in the periventricular system and inferior thalamic peduncle of the ansa pednucularis (an extension of the medial forebrain bundle).
Afferents from the subthalamus are believed to originate in the nucleus subthalamicus and zona incerta, and directly enter the hypothalamus along the lateral aspect of the hypothalamic wall (122).
Afferents from basal ganglia (corpus striatum) arise from the nucleus accumbens, located in the ventral portion of the caudate nucleus, and via the substantia innominata, directly and indirectly reach the lateral portions of the hypothalamus.
Afferents from the retina reach the hypothalamus via the retino-hypothalamic tract, and travel through the optic chiasm to terminate in the suprachiasmatic nucleus.   
Finally, direct projections arise from the frontal cortex and course in the medial forebrain bundle to the most lateral part of the ventricular wall.

b.  Efferent Connections
Outputs from the mammalian hypothalamus include fiber pathways to the anterior and posterior pituitary gland, limbic system, brainstem reticular formation, thalamus, subthalamus, basal ganglia, superior colliculi, substantia nigra, cerebellum, and neocortex (Fig. 22).  With exception of projections to the pituitary gland, discussed in detail below (see Hypothalamic Tuberoinfundibular System and Hypothalamic Neurohypophysial Tract) and those directed to locomotor centers such as the optic tectum, susbstantia nigra, and cerebellum, in general, efferent fibers from the hypothalamus reciprocate its afferent fibers in a sort of feedback loop.

Efferents to the limbic system include 2 major fiber groups.  The first course in the medial forebrain bundle (lateral hypothalamus) and carries projections that ascend to the septal areas and descending to the amygdalo-piriform cortex complex.  These latter fibers form the ventral amygdalopetal component of the ansa pedunclaris.  The second, the stria terminalis system with its bed nucleus, is positioned medially, very close to the inner surface of the third ventricle, and projects to the amygdala.
Efferents to the brainstem reticular formation include 2 main fiber groups.  The first is a dorsal and medial group formed by the dorsal longitudinal fasciculus and periventricular system.  These axons descend to the brainstem to innervate visceral motor, sensory and somatic nuclei (occulomotor, trigeminal, facial, glossopharyngeal, vagus and accessory spinal nerves), and to autonomic sympathetic and parasympathetic preganglionic neurons in the spinal cord.  The second courses in the medial forebrain bundle and gives off fibers that project to the medial nuclei of the mesencephalic reticular formation (limbic midbrain area) through the mammillary-tegmental tract and the mammillary peduncle. 
Efferents to the thalamus include 3 main systems.  The first is the mammillo-thalamic tract that links the posterior hypothalamus to the cingulate gyrus of the limbic cortex (a component of the "Papez circuit”).  The second is the periventricular fiber system that projects to the medial (dorsomedian nucleus) and median (midline, intralaminar and reticular nuclei) thalamus and habenula through the stria medullaris.  The third is part of the medial forebrain bundle that courses in the inferior thalamic peduncle of the ansa peduncularis to reach the medial thalamic nuclei.
Efferents to the subthalamic and basal ganglia region course in the lateral aspect of the ventricular wall in the mammillo-thalamic tract (mammillo-subthalamic tract) terminating in the field H1 of Forel, and in the substantia innominata (122,123).  Efferents to superior colliculi, substantia nigra and cerebellum travel in either the stria terminalis  and periventricular fiber system, or in the medial forebrain bundle as a part of the mammillary peduncle.  Finally, efferents to the neocortex are carried laterally within the medial forebrain bundle. 

c.   Hypothalamic Tuberoinfundibular System
The hypothalamic tuberoinfundibular system comprises all neurons in the brain that send axonal projections to the external zone of the median eminence.  Although the arcuate nucleus and inferior portion of the periventricular nucleus were thought primarily responsible for this pathway on the basis of silver stains (124), the relative paucity of myelin in these neurons and high density of perikarya in the medial and periventricular zones of the hypothalamus made it impossible to elucidate the full extent of the tuberoinfundibular system using this technique.  In addition, the inability of pharmacologic ablation of the arcuate nucleus to significantly reduce the concentration of TRH and GnRH in the median eminence (125), made it likely that the origin of neurons contributing to the tuberoinfundibular system is considerably broader than just the medial basal hypothalamus.  Using retrogradely transported marker substances that are taken up in axon terminals in the external zone of the median eminence and transported back to the cell body of origin, a detailed analysis of the tuberoinfundibular system has been possible (126-129).
Retrogradely labeled cells of the tuberoinfundibular system concentrate in four major hypothalamic regions: the arcuate nucleus, periventricular nucleus, paraventricular nucleus, and medial preoptic-septal region (Fig. 23).  Within the arcuate nucleus the labeled cells accumulate in two distinct clusters (Fig. 23 G-I), a dorsomedial group of small to medium size neurons in the distribution of the dopamine-containing A12 group of Dahlstrom & Fuxe (130) and a basolateral group of medium sized cells that contain dopamine, GHRH, galanin, and neurotensin (131-134).  Occasional enkephalin-producing neurons in the arcuate nucleus are retrogradely labeled from the median eminence (135) but the majority of these cells, as well as ACTH-producing neurons (personal observations), do not contain the retrogradely transported marker substance.  These observations emphasize that only a small subset of chemically coded neurons in the arcuate nucleus project to the median eminence.
In the periventricular nucleus (Fig. 23 A-F), a thin layer of retrogradely marked neurons in the subependymal neuropil contains somatostatin and dopamine (132,136).  These retrogradely labeled cells can be identified even in the most rostral portions of the periventricular nucleus but the majority concentrate between the middle of the optic chiasm and anterior portion of the median eminence (Fig. 23 B).  Often cells interdigitate between the ependymal wall and even extend into the third ventricular space suggesting possible secretion into the CSF.  Axonal projections from these cells to the median eminence is through a circuitous pathway that extends laterally into the lateral hypothalamus toward the ventral surface of the hypothalamus (retrochiasmatic area) and then medially to enter the median eminence, although some fibers also descend directly in the periventricular neuropil.

    The most remarkable finding of studies using retrogradely transported marker substances from the median eminence is the massive accumulation of the marker substance in neurons of the paraventricular nucleus (Fig. 23 A-F).  This winged-shaped nucleus at the dorsal margin of the third ventricle can be divided into two major portions based on the size of the neuronal perikarya, including a magnocellular division of large neurons and a parvocellular division of small to medium sized neurons (137).  The parvocellular portion is located in the most medial portion of the nucleus adjacent to the ependymal wall of the third ventricle and can be broken down into several, smaller subdivisions shown in detail in Fig. 24.  Retrogradely labeled cells of the tuberoinfundibular system are located primarily in the anterior, medial and periventricular subdivisions of the paraventricular nucleus with relatively few or no neurons in the dorsal, ventral and lateral parvocellular subdivisions.  Many of these retrogradely labeled cells contain TRH, corticotropin-releasing hormone (CRH), enkephalin, somatostatin, and VIP (135,136,138-142).  Not all neurons in the anterior, medial and periventricular parvocellular subdivisions project to the median eminence, however.  This is particularly apparent for TRH neurons in the anterior parvocellular subdivision that cannot be retrogradely labeled by marker substances introduced into the median eminence (143).  These neurons are also immunocytochemically distinct from hypophysiotropic TRH neurons in the medial and periventricular parvocellular subdivisions in that they do not co-express the peptide, cocaine and amphetamine-regulated transcript (CART) (144).  The true, physiologic function of TRH neurons in the anterior parvocellular subdivision is not known.

    Tuberoinfundibular neurons in the paraventricular nucleus project to the median eminence either by arching laterally and inferiorly through the lateral hypothalamus through the retrochiasmatic area before turning medially to terminate or by descending along the wall of the third ventricle to directly enter the median eminence.  As the ependymal wall underlies both the paraventricular and periventricular nuclei and is likely permeable to the diffusion of CSF (55), these cells of the hypothalamic tuberoinfundibular system could be influenced by substances carried in the CSF or secrete directly into the CSF as an alternative way to reach the median eminence.
Finally, small, bipolar and multipolar, retrogradely labeled cells that can be immunostained with GnRH (142,145), are found in the rostral hypothalamus in the ventral wings of the diagonal band of Broca, lamina terminalis, medial septum, and medial preoptic nucleus, while few cells extend more caudally in the basolateral hypothalamus.  In primates, however, retrogradely labeled GnRH cells are located more caudally in the basal hypothalamus (146).  Axonal projections to the external zone of the median eminence occur either by joining the medial forebrain bundle in the lateral hypothalamus or along the wall of the third ventricle.  The tendency for GnRH neurons of the tuberoinfundibular pathway to be more deeply embedded into the substance of the hypothalamus than is typical for the periventricular distribution of the majority of the hypothalamic tuberoinfundibular system relates to the embryologic origin of GnRH neurons from the nasal epithelium (147), as opposed to primordial cells in the walls of the third ventricle.
Although the bulk of tuberoinfundibular neurons arise from periventricular and medial portions of the hypothalamus, some brain stem neurons also have direct projections to the median eminence, explaining the presence of catecholamines in addition to dopamine in this structure.  Retrogradely neurons can be identified in C1-C2 adrenergic neurons and A2 noradrenergic neurons (148), but since the tracer was injected into the bloodstream in this study, uptake could have occurred from other circumventricular organs in addition to the median eminence.  Lesions of the brainstem, however, do result in degeneration of axon terminals in the median eminence (149).

d.  Hypothalamic Neurohypophysial Tract
The hypothalamic neurohypophysial tract defines the neuronal system terminating in the posterior pituitary and is best known for its secretion of vasopressin and oxytocin into the peripheral circulation to regulate water balance (antidiuresis), milk ejection and uterine contraction (143).  Neurons of this tract arise primarily from the magnocellular division of the paraventricular nucleus and the supraoptic nucleus (151), the latter situated as a cluster of cells dorsal and lateral to the optic chiasm (Fig. 25A-C).  The axon trajectory from magnocellular neurons to the posterior pituitary is by way of arching fibers extending laterally and inferiorly from the paraventricular nucleus above and below the fornix toward the supraoptic nucleus, where it gathers fibers from the supraoptic nucleus and continues medially along the base of the hypothalamus into the internal zone of the median eminence.  Vasopressin-containing axon terminals have also been demonstrated in the external zone of the median eminence, particularly following adrenalectomy (152), but largely arise from a separate population of parvocellular neurons in the paraventricular nucleus that contain CRH (153).  Vasopressin is a weak corticotropic factor but potentiates the secretion of ACTH in the presence of CRH (154) and is responsible for the ACTH rise following hypoglycemia (155).

    Magnocellular neurons of the paraventricular and supraoptic nucleus possess large perikarya and prominent dendrites that interdigitate with adjacent perikarya and dendrites, respectively, of other magnocellular neurons.  These dendrites contain numerous hormone-laden neurosecretory granules that can be released by exocytosis (156) and may be important to coordinate the secretion of vasopressin or oxytocin from individual neurons in unison through somato-somatic and/or dendro-dendritic interactions or alter the sensitivity of these neurons in response to a biologic stimulus such as suckling.  Regulation of magnocellular neurons may also depend upon dynamic glial-neuronal interactions in response to specific stimuli, reducing or enlarging the cell to cell contact area between magnocellular neurons by retraction or extension of astrocytic processes that separate perikarya and dendrites (157) or to permit the formation of new synaptic contacts on magnocellular neurons (synaptic plasticity) (158).
In addition to vasopressin and oxytocin, magnocellular neurons of the hypothalamic neurohypophysial tract also produce and transport numerous other peptides to the posterior pituitary.  These peptides include dynorphin, enkephalin, galanin, cholecystokinin, dopamine, TRH, VIP, neuropeptide Y, substance P, CRH, and endothelin (159-162).  A number of different neuropeptides are also carried into the posterior pituitary by axons from parvocellular neurons including GnRH, TRH, somatostatin, enkephalin, neurotensin CRH and dopamine (163).  Furthermore, there is evidence that messenger RNA for vasopressin, oxytocin (164) and tyrosine hydroxylase (165) can be transported in axons of the hypothalamic neurohypophysial tract, particularly during osmotic stress.
The functional significance of numerous biologically active substances in axons from both magnocellular and parvocellular neurons other than vasopressin and oxytocin, is of great interest.  Since their concentration in the posterior pituitary is low, release into the peripheral circulation for action at a distant locus seems remote.  Endothelin-1 may be an exception, however, where co-release with vasopressin into the periphery may assist the effect of vasopressin on water conservation by decreasing glomerular filtration rate (140).  Other substances are likely involved in the regulation of vasopressin and oxytocin secretion by a paracrine or autocrine mechanism or by acting presynaptically on nerve endings in the posterior pituitary.  Dopamine, for example, may be important in stimulating vasopressin release during an osmotic challenge (166).  Neuropeptide Y has also been shown to enhance the secretion of vasopressin (167) and NPY Y2 receptors are present on nerve endings in the posterior pituitary in high density (168).  Some neuropeptides in the posterior pituitary may act as trophic hormones (74), important to promote regeneration of its axon terminals following injury or to increase the proliferation of endothelial cells to promote changes in its vascularization (169).
There is considerable evidence, however, that some of the peptides in the posterior pituitary are destined for transport to the anterior pituitary via the short portal vessels, thereby utilizing the posterior pituitary as an accessory median eminence.  The most convincing data that the posterior pituitary can influence anterior pituitary function comes from animal studies in which the posterior pituitary is surgically removed.  Consequently, the blood flow to the anterior pituitary from the median eminence through the long portal veins is preserved, but the blood flow from the posterior pituitary through the short portal veins is disrupted.  These animals have a number of abnormal neuroendocrine responses including a diminished ACTH response to stress (170), elevation in basal prolactin levels (165), and the loss of either suckling or estrogen induced prolactin release (171,172).  These responses indicate the requirement of magnocellular derived-vasopressin or some other posterior pituitary secretogogue (CRH, oxytocin, dopamine, TRH, other prolactin releasing factor) to achieve normal physiologic responses.

D.  Radiologic Anatomy

E.  Blood Supply

    All arteries carrying blood to the hypothalamus are terminal branches of the circle of Willis, including the internal carotid, anterior cerebral, anterior communicating, posterior communicating, posterior cerebral and basilar arteries (Fig. 13).  Curiously this arterial circle, named for Thomas Willis’s work published in Cerebri Anatome in 1664 (Fig. 27), had already been described between the end of the 16th century and beginning of the 17th century by the Italian anatomists, Fallopius and Casserio (174).  These anatomists noted the existence of small arterial branches entering the floor of the third ventricle and surrounding the tuber cinereum in a position that is now well described as the anastomotic circuminfundibular plexus and prechiasmal anastomotic arteriolar-capillary plexus.  These two anastomotic plexuses are highly developed in species such as Carnivora, Cetacea, Edentata and Ungulata (175) and likely represent the capillary system that Galen described as the rete mirabilis, given that he worked on animal species and not human brains and the anatomical differences between what he described and what is now recognized as the primary portal plexus (see below).

    The vascular supply to the hypothalamus has been extensively studied in man using three-dimensional casts of the hypothalamic vessels (176).  These studies demonstrate that the arterial supply is compartmentalized with respect to three rostro-caudal regions of the hypothalamus, and thus, can be separated into anterior, intermediate and posterior arterial groups (Table 8).  However, only the anterior hypothalamic region is vascularized by a single arterial group (the anterior arterial group), whereas the remainder of the hypothalamus receives blood from both the intermediate and posterior arterial groups.  The preoptic and anterior hypothalamus are primarily supplied by the anterior cerebral and anterior communicating arteries, the tuberal region by the posterior communicating artery, and the mammillary region by the posterior communicating, posterior cerebral, and basilar arteries.  This organization is consistent with the clinical observations that occlusion of the anterior choroidal artery, which has anastomotic branches with the posterior communicating artery, results in damage to the tuberal and mammillary regions of the hypothalamus, whereas occlusion of the thalamoperforate artery, a branch of the posterior cerebral artery, results in damage to the mammilothalamic tract and related thalamic nuclei (176).

As previously described, the blood supply to the median eminence is complex.  In all mammals, including humans, fine branches of the superior hypophysial artery give rise to a capillary plexus, the primary portal plexus of the infundibulum, which is composed of capillary loops in the external zone of the median eminence (external plexus) and that penetrate as pillars vertically toward the ventricular floor to establish the internal plexus.  At this level, a subependymal capillary network can also be recognized in association with the basal membrane of the ependymal cells.  Blood flows from the external to the internal and then back to the external plexus to end in the long portal vessels that reach the anterior pituitary, or from the subependymal network into hypothalamic capillaries of the anterior or intermediate arterial groups (177).  In some species, bidirectional transport of substances has been described in the portal capillary system, allowing the transport of anterior pituitary substances to the external plexus (178), thus supporting the original hypothesis of Popa and Fielding that in the human brain, blood flow in the portal capillary system can be from the pituitary to the hypothalamus (179).  The external plexus is tangential to the ventral surface of the infundibulum and is composed of vessels organized in geometrical arrays (hexagonal in rodents, much more complex in humans), whose central spaces are filled with neuroendocrine axons constituting functional units, called microvascular domains (177) or medianosomes (180).  Neurohemal contacts are established by these axons and by tanycyte processes at the level of both plexuses, whereas neurohypophysial fibers course between the two vascular plexuses without contacting them, en route to the posterior lobe of the pituitary.  This "double-plexus” system provides amplification of the surface area in contact with tuberoinfundibular axons in a given microvascular domain.
Venous drainage from the rest of the hypothalamus is collected into the anterior cerebral, basal, and the internal cerebral veins, ultimately reaching the great vein of Galen.  In general, the anterior cerebral and basal veins drain the majority of the hypothalamus, whereas the internal cerebral vein collects blood from the dorsal portions of the hypothalamus (Table 9).