The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus: The former opens to the portal blood and the latter to the cerebrospinal fluid

Abstract

The blood-brain barrier (BBB) is a single uninterrupted barrier that in the brain capillaries is located at the endothelial cells and in the circumventricular organs, such as the choroid plexuses (CP) and median eminence (ME), is displaced to specialized ependymal cells. How do hypothalamic hormones reach the portal circulation without making the BBB leaky? The ME milieu is open to the portal vessels, while it is closed to the cerebrospinal fluid (CSF) and to the arcuate nucleus. The cell body and most of the axons of neurons projecting to the ME are localized in areas protected by the BBB, while the axon terminals are localized in the BBB-free area of the ME. This design implies a complex organization of the intercellular space of the median basal hypothalamus. The privacy of the ME milieu implies that those neurons projecting to this area would not be under the influence of compounds leaking from the portal capillaries, unless receptors for such compounds are located at the axon terminal. Amazingly, the arcuate nucleus also has its private milieu that is closed to all adjacent neural structures and open to the infundibular recess. The absence of multiciliated cells in this recess should result in a slow CSF flow at this level. This whole arrangement should facilitate the arrival of CSF signal to the arcuate nucleus. This review will show how peripheral hormones can reach hypothalamic targets without making the BBB leaky.

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Review
The design of barriers in the hypothalamus allows the median eminence and the
arcuate nucleus to enjoy private milieus: The former opens to the portal blood and
the latter to the cerebrospinal fluid
Esteban M. Rodrı
´guez
a,
*, Juan L. Bla
´zquez
b
, Montserrat Guerra
a
a
Instituto de Anatomı
´a, Histologı
´a y Patologı
´a, Facultad de Medicina, Universidad Austral de Chile, Valdivia, Chile
b
Departamento de Anatomı
´a e Histologı
´a Humana, Facultad de Medicina, Universidad de Salamanca, Salamanca, Spain
Contents
1. Blood-brain barrier: concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
2. Circumventricular organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
3. The median eminence as a circumventricular organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759
4. Tanycytes: a key component of the blood-brain and CSF-brain barriers of the medial-basal hypothalamus ............................. 759
4.1. Tanycytes: ontogeny and cell lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
4.2. Spatial relationships of tanycytes subtypes with discrete regions of the medial-basal hypothalamus . . . . . . . . . . . . . . . . . . . . . . . . 760
4.3. Tanycyte subtypes: morphological and immunochemical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
4.4. Tanycytes subtypes are joined together by different junction complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
4.5. Functional differences between tanycyte subtypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
5. Microscopic arrangement of the median eminence barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
5.1. In the external region of the median eminence neurosecretory axons and
b
tanycytes end on the fenestrated portal capillaries ..... 762
5.2. The median eminence-AN barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
5.3. The median eminence-CSF barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
5.4. The lack of barrier between CSF and the nuclei of the medial-basal hypothalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
6. Dynamic aspects of the median eminence barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
6.1. The median eminence milieu, the perivascular space of the portal vessels, and the subarachnoid space are in open
communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
6.2.
b
2
tanycytes establish an efficient barrier between the median eminence milieu and the ventricular CSF . . . . . . . . . . . . . . . . . . . . 766
Peptides 31 (2010) 757–776
ARTICLE INFO
Article history:
Received 11 December 2009
Received in revised form 9 January 2010
Accepted 10 January 2010
Available online 20 January 2010
Keywords:
Blood-brain barrier
Tightness
Hypothalamus
Arcuate nucleus
Cerebrospinal fluid
Choroid plexuses
Peripheral hormones
ABSTRACT
The blood-brain barrier (BBB) is a single uninterrupted barrier that in the brain capillaries is located at
the endothelial cells and in the circumventricular organs, such as the choroid plexuses (CP) and median
eminence (ME), is displaced to specialized ependymal cells. How do hypothalamic hormones reach the
portal circulation without making the BBB leaky? The ME milieu is open to the portal vessels, while it is
closed to the cerebrospinal fluid (CSF) and to the arcuate nucleus. The cell body and most of the axons of
neurons projecting to the ME are localized in areas protected by the BBB, while the axon terminals are
localized in the BBB-free area of the ME. This design implies a complex organization of the intercellular
space of the median basal hypothalamus. The privacy of the ME milieu implies that those neurons
projecting to this area would not be under the influence of compounds leaking from the portal capillaries,
unless receptors for such compounds are located at the axon terminal. Amazingly, the arcuate nucleus
also has its private milieu that is closed to all adjacent neural structures and open to the infundibular
recess. The absence of multiciliated cells in this recess should result in a slow CSF flow at this level. This
whole arrangement should facilitate the arrival of CSF signal to the arcuate nucleus. This review will
show how peripheral hormones can reach hypothalamic targets without making the BBB leaky.
ß2010 Published by Elsevier Inc.
* Corresponding author. Tel.: +56 63 29 30 31; fax: +56 63 22 16 04.
E-mail address: erodrigu@uach.cl (E.M. Rodrı
´guez).
Contents lists available at ScienceDirect
Peptides
journal homepage: www.elsevier.com/locate/peptides
0196-9781/$ – see front matter ß2010 Published by Elsevier Inc.
doi:10.1016/j.peptides.2010.01.003

Author's personal copy
6.3.
b
1
tanycytes establish a lateral barrier on either side of the median eminence, separating the intercellular space of the
median eminence from that of the AN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766
7. How do hypothalamic hormones reach the portal circulation without making the BBB leaky? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
8. AN barriers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
8.1. The AN is in open communication with the ventricular CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
8.2. The intercellular space of the AN is segregated from the intercellular space of the median eminence and of the
adjacent hypothalamic nuclei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768
9. How do peripheral hormones may reach hypothalamic targets without making the BBB leaky? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
9.1. Specific transport systems at the choroid plexuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
9.2. The CSF as a pathway for neuroendocrine integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
9.3. Insulin growth factor-I: transport through choroid plexuses, presence in the CSF and targeting to tanycytes . . . . . . . . . . . . . . . . . . 771
9.4. Thyroid hormones: transport of T4 at choroid plexuses (role of transthyretin), and bioconversion into T3 at tanycytes. . . . . . . . . . 771
9.5. Prolactin: transport through the choroid plexuses, presence in the CSF and probable effect on dopaminergic neurons of the AN . . 772
9.6. Leptin: transport through choroid plexuses, presence in the CSF and targeting to periventricular receptive neurons . . . . . . . . . . . . 772
10. Trying to bridge apparently contradictory findings and views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772
11. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 774
1. Blood-brain barrier: concept
The blood-brain barrier (BBB) is an efficient system to prevent
direct exposure of nervous tissue to the blood circulation, allowing
the brain milieu to be maintained in a constant state. The stability
of the unique internal environment of the brain is essential for
normal brain development and function.
The BBB is a highly dynamic and complex structure formed by
the specialized endothelial cells of the brain capillaries and the
ependymal cells of the circumventricular organs. These cells
establish an interphase between the blood in the cerebral vessels
on one side and the intercellular fluid of the brain parenchyma and
the cerebrospinal fluid (CSF), on the other. Tight junctions between
cells forming this interphase represents a fundamental physical
barrier preventing free movements of compounds through the
intercellular spaces between endothelial cells in the BBB and
ependymal cells in the choroid plexuses blood–CSF barrier [134].
This design allows ions and other compounds to be transported by
specific transfer mechanisms operating within the cells of the
barrier interfaces. These mechanisms include (i) blood-to-brain
transfer by carrier- and receptor-mediated transport of a series of
selective compounds (e.g., amino acids, glucose, vitamins, pro-
teins); (ii) two-way exchange of ions that provides the ionic
stability necessary for neuronal activity, and (iii) brain-to-blood
transfer mechanisms, such as p-glycoprotein [105,106].
It may be proposed that the BBB functions as a single and
unique barrier that in the brain capillaries is located at the
endothelial cells and in the choroid plexuses and other circum-
ventricular organs (see below) is displaced to the specialized
ependymal cells lining these periventricular structures. It may also
be suggested that for a barrier system to be efficient it must be
tight throughout the whole barrier; otherwise any molecule
transposing the barrier through a weak or leaky region could reach
any target beyond the barrier.
There are discrete areas of the brain, the circumventricular
organs, where neural cells, either neurons or specialized glia, may
establish an open communication with blood capillaries; but this
should not be misinterpreted as indicating open access from blood
to brain.
2. Circumventricular organs
Neurons secreting hormones into the blood stream must be in
open communication with the blood capillaries. In the central
nervous system (CNS), there are discrete areas localized in the
ventricular walls, known as circumventricular organs (CVO)
[63,55], in most of which the BBB displays special characteristics.
These ‘‘brain windows’’ may serve two purposes, namely, to allow
peptides and proteins secreted by the neural tissue to reach the
blood stream, and to allow neural cells to sense the plasma. The
CVO share some characteristics, namely, their location close or
within the ventricles; their vascular supply formed by capillaries
endowed with fenestrations and a perivascular space, and their
ependymal cells highly specialized in transport and/or secretion.
With the exception of the choroid plexuses of the lateral ventricles,
all other CVOs display the unique feature of being single, unpaired
structures located along the midline of the CNS (Fig. 1A and B).
CVOs undergo relevant phylogenetic and ontogenetic changes.
Many of them, such as the subcommissural organ, pineal organ,
choroid plexuses and median eminence are ancient structures
conserved throughout evolution. Others, such as the paraventri-
cular organ and infundibular recess organ are only found in non-
mammalian species. The mammalian CVOs are the neurohypoph-
ysis (neural lobe and median eminence), the vascular organ of the
lamina terminalis, the subfornical organ, the subcommissural
organ, the pineal gland, the collicular recess organ, the area
postrema, and the choroid plexuses [151] (Fig. 1A and B).
According to what is known about the function of CVOs, they
may be grouped into the following: (i) sensing organs (vascular
organ of the lamina terminalis, subfornical organ, area postrema):
The cell body and dendrites of the neurons forming these CVOs are
not protected by a BBB and remain in open communication with
peripheral blood so that they may respond to blood borne signals,
such as the subfornical neurons responding to angiotensin II
plasma levels. The axons of the neurons of these sensory CVOs
project to multiple areas of the CNS; therefore, such axons leave
the BBB-free area of the CVO and enter the brain areas protected by
the BBB. This is diferent from providing direct access of the
substances into the CNS. (ii) Neurosecretory organs (pineal gland,
median eminence and neural lobe of the pituitary): In the medial
hypothalamus the cell body and dendrites of the neurons secreting
neuropeptides and monoamines are protected by the BBB whereas
their axons enter the BBB-free areas of the median eminence and
neural lobe to deliver their secretions into the portal capillaries and
systemic capillaries, respectively. (iii) Ependymal secretory organs
(subcommissural organ, choroid plexuses): These CVOs are formed
by ependymal cells highly specialized to secrete proteins into the
CSF. (iv) Transporting CVOs organs (choroid plexuses, median
eminence). The choroidal cells and the tanycytes of the median
eminence are cells endowed with a transport machinery to transfer
substances from blood to CSF and from CSF to blood [55,74,75,151]
(see below).
E.M. Rodrı
´guez et al. / Peptides 31 (2010) 757–776
758

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It must be emphasized that a complex barrier system surrounds
the BBB-free CVOs preventing blood-borne compounds from
entering the BBB-protected adjacent structures.
3. The median eminence as a circumventricular organ
The median eminence of the hypothalamus, the main issue of
this review, is one of the brain windows in which peptides and
monoamines synthesized in different hypothalamic nuclei reach
the portal circulation [85]. The cell body, dendrites, and a long
segment of the axon of these neurons are localized in areas
protected by the BBB, whereas the terminal segment of the axon
and the terminal proper are localized in an area lacking a BBB
[55,151]. This implies a complex organization of the intercellular
space of this area, involving the existence of discrete compart-
ments of this space, so that the median eminence would behave as
a canal lock, with inlet and outlet gates. In this way, neurohor-
mones can enter the median eminence by axonal transport and,
upon release, they can reach the perivascular space, the portal
blood, and the intercellular space of the median eminence but are
prevented from traveling back into either the CSF or the
intercellular space of the adjacent hypothalamic region, the
arcuate nucleus (AN). Similarly, plasma compounds could escape
from the fenestrated portal capillaries and be confined to the
intercellular space of the median eminence.
4. Tanycytes: a key component of the blood-brain and CSF-
brain barriers of the medial-basal hypothalamus
Tanycytes are a unique cell type of the mature brain, lining the
floor of the third ventricle [88,91,153,154]. Due to their shape,
Horstmann [64] called these cells ‘‘tanycytes’’ (from the Greek
word tanus = ‘‘elongated’’). A distinct structural feature of tany-
cytes is that they possess a single and long basal process that
Fig. 1. (A and B) Line drawing and scanning electron microscopy of the rodent brain showing the circumventricular organs, namely the neural lobe (NL), median eminence
(ME), vascular organ of the lamina terminalis OVLT), subfornical organ (SFO), subcommissural organ (SCO), pineal gland (PIN), collicular recess organ (CRO), area postrema
(AP), thalamus (Th), and choroid plexuses (CHP). (C) Frontal section of the rat medial basal hypothalamus immunostained for vimentin. The four subpopulations of tanycytes
are shown. ME median eminence [132]. Bar 100
m
m. (D) Line drawing depicting the distributions of
a
1,2
,
b
1,2
tanycytes. ME median eminence, 3V third ventricle. (E) Double
immunostaining for caveolin 1 (green) and
b
IV-tubulin (red) showing
a
tanycytes ending on a capillary (arrow). Bar 10
m
m. (F) Line drawing of a
b
1
tanycyte showing the
unidirectional transport from CSF to portal capillaries. (G) Golgi preparation of the rat hypothalamus showing a few
b
1,2
tanycytes impregnated and some axons entering the
median eminence (black arrows).
b
1
tanycytes establish a frontier between the median eminence and the arcuate nucleus (AN), highlighted by a broken red line. The red
arrows point to portal capillaries. [128]. Bar 45
m
m. (H) Double immunostaining tuberalin 2 (green), a secretory protein of the pars tuberalis, and
b
IV-tubulin (red). Bar 70
m
m
(see [57]). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
E.M. Rodrı
´guez et al. / Peptides 31 (2010) 757–776
759

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project to discrete regions of the hypothalamus (Fig. 1C–H). This
led Lo
¨fgren [88–91] to suggest, for the first time, that tanycytes
may link the cerebrospinal fluid (CSF) to neuroendocrine events.
4.1. Tanycytes: ontogeny and cell lineage
A good body of evidence indicates that tanycytes may be
regarded as genealogical descendents of the transient embryonic
radial glia [132]. During the perinatal period most radial glial cells
are transformed into astrocytes, whereas a subpopulation of radial
glial cells is differentiated into tanycytes. Tanycytes, although
sharing some properties with astrocytes and with the radial glia,
display unique and distinct morphological, molecular, and
functional characteristics [132]. Furthermore, tanycytes do not
constitute a homogeneous cell population; instead, they further
differentiate into four subtypes, each one with distinct properties.
4.2. Spatial relationships of tanycytes subtypes with discrete regions
of the medial-basal hypothalamus
In the rat, four types of tanycytes have been described:
a
1
,
a
2
,
b
1
,
b
2
[3–5,128,132] (Fig. 1C and D). They present different
characteristics with respect to their location, spatial relationships,
morphology, cytochemistry, ultrastructure, and certain functions.
a
1
Tanycytes line the ventromedial nucleus and part of the
dorsomedial nucleus, and project their basal processes into these
nuclei (Fig. 1C and D).
a
2
tanycytes line the AN and most of them
project their processes within this nucleus [5,128,132] (Fig. 1C and
D). Whether or not the processes of
a
tanycytes end on neurons is a
matter of controversy. In Golgi preparations, some authors have
found the presence of such an ependyma-neuron contacts
[15,16,128], whereas others have not [23,95,96]. Unfortunately,
ultrastructural studies of this aspect are missing.
b
1
tanycytes line the lateral evaginations in the infundibular
recess [5,6,128,132] and project their processes to the latero-
external region of the median eminence where they form a cuff of
ependymal endings separating the neurosecretory terminals from
the portal capillaries located in this region [128,132] (Fig. 1C–F,
Fig. 2A). None of these processes has been seen to end on a neuron
[128].
b
2
tanycytes line the floor of the infundibular recess and their
basal processes end on the portal capillaries of the medial zone of
the median eminence [14,51,78,110,128,132] (Fig. 1C–H). In brief,
a
tanycytes bridge the lumen of the third ventricle with neurons
and blood vessels of the medial basal hypothalamus and
b
tanycytes establish an anatomical link between the ventricular CSF
and the portal blood [5,73,88,124,125,128,132].
4.3. Tanycyte subtypes: morphological and immunochemical
characteristics
The four subtypes of tanycytes present distinct ultrastructural
features [4,128,132]. The morphological characteristics of tany-
cytes have been confirmed and extended by immunocytochemical
studies. Thus, antibodies against the glial-fibrillary acidic protein
[115], vimentin [86] (Fig. 1C), a protein of 85 kDa apparently
specific of tanycytes [14], and
b
IV-tubulin [58] (Fig. 1H) immu-
nostain tanycytes. Immunocytochemistry is contributing to a
further characterization of the four types of tanycytes. Worth
mentioning for this review is that
a
1,2
and
b
1
tanycytes express the
glucose transporter 1, whereas
b
2
tanycytes do not [110] (Fig. 2A)
and that
b
1
but not
b
2
tanycytes express insulin-like growth factor
binding protein [24]. Tanycyte subtypes can also be distinguished
by the differential expression of other proteins such as glutamate
transporters GLT-1 and GLAST [12], somatostatin sst
2(a)
receptor
[60], and various proteins of the endocytotic and transcytotic
pathways [111].
4.4. Tanycytes subtypes are joined together by different junction
complexes
Immunocytochemistry and electron microscopy display rele-
vant differences between the junction complexes of tanycytes
subtypes. The cell body and basal process of
a
1
tanycytes are
moderately reactive for
a
-catenin whereas
a
2
tanycytes display a
weak reaction at the ventricular cell pole and along the basal
process. At variance, the cell body of
b
1,2
tanycytes as well their
processes and endings are strongly reactive for
a
-catenin (Fig. 4A
and Fig. 5A). Immunocytochemistry for N-cadherin reveals that
only the processes and endings of
b
2
tanycytes are reactive [132].
A recent study of tight junction proteins in the tanycytes of the
mouse medial basal hypothalamus has revealed that tanycytes
lining the floor of the infundibular recess, most likely correspond-
ing to
b
2
tanycytes, express occludin, ZO-1, and claudins 1 and 5
(Fig. 3C); such proteins appear arranged as a continuous belt
around the cell bodies of these tanycytes [97]. At variance with the
endothelial cells of the brain capillaries,
b
2
tanycytes do not
express claudin 3 [97]. On the other hand, tanycytes lining the AN,
most likely corresponding to
a
2
and
b
1
tanycytes, do not express
claudin 1; instead they exhibit a disorganized expression pattern of
occludin (Fig. 3G), ZO-1 and claudin 5 [97].
Anti-
b
IV-tubulin is a good marker of tanycytes and ciliated
ependyma; it strongly labels cilia [58]. Connexin 43, a gap junction
protein, is expressed by multiciliated cells. The combined use of
antibodies against
b
IV-tubulin and connexin 43 has confirmed
that the four subtypes of tanycytes are not multiciliated and reveal
that the multiciliated ependyma lining the walls of the third
ventricle and
a
1
tanycytes express connexin 43 whereas
a
2
and
b
1,2
tanycytes do not (Fig. 4D and E). Expression of connexin 43 in
multiciliated ependyma is expected to occur since coupling
through gap junctions is required for synchronized cilia beating.
However the expression of connexin 43 by
a
1
tanycytes, devoid of
cilia, suggests that this cell population is coupled to perform an
unknown function different from cilia beating. This unexpected
finding places
a
1
tanycytes as a distinct cell population. On the
other hand, the lack of connexin 43 and, consequently, the
probable absence of gap junctions in
a
2
and
b
1,2
tanycytes
indicates that these cells are not coupled, highlighting the view
that they are involved in transport and secretory functions.
The complete absence of multiciliated cells in the walls of the
infundibular recess implies that CSF flow at this level is different
from that occurring in all other regions of the ventricular system.
Since cilia beating of the multiciliated ependyma is responsible for
the laminar flow of CSF, such a flow would be missing in the
infundibular recess. Either a slow or turbulent CSF flow in the
infundibular recess could be envisaged.
Transmission electron microscopy of the walls of the infundib-
ular recess confirms and extends the immunocytochemical
findings concerning junction complexes of tanycytes.
a
tanycytes:
Laterally they are joined by zonula adherens; tight junctions are
missing ([4] unpublished observations by the authors). The
terminal region of neighboring basal processes are joined together
by tight junctions [79].
b
1
tanycytes: At the ventricular cell pole,
b
1
tanycytes are joined by zonula adherens but tight junctions are
missing (Fig. 3D and F) [128]. An amazing feature of
b
1
tanycytes is
that they are joined together by zonula and macula adherens
localized throughout the lateral surface of the cell body, basal
processes and terminals, as revealed by transmission electron
microscopy and immunocytochemistry for
a
-catenin (Figs. 4A and
C, 5A and C). This unique feature shared by
b
1
and
b
2
tanycytes,
may explain the formation of bundles of tanycyte processes
(Fig. 5A and C). However, puzzling is the exceptional high degree of
N-cadherin and
a
-catenin expression in the median eminence
(Fig. 4A) and the relatively low frequency of maculae adherens
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seen under the electron microscope, including that between axon
terminals (Fig. 4C). Although the functional significance of such an
arrangement is unknown, the possibility that these junction
proteins distributed along the basal processes of
b
tanycytes play
an additional role to that of forming maculae and zonulae adherens
should be considered.
b
2
tanycytes: Two distinct ultrastructural characteristics of
b
2
tanycytes are: (i) apically (ventricular cell pole), they are joined
together by zonula adherent and tight junctions (Fig. 3A and B)
[19,20,122,132], (ii) the basal process receives numerous synap-
toid contacts throughout its length [4,76,128,132,154].
4.5. Functional differences between tanycyte subtypes
The function of tanycytes is poorly understood. However, some
functional differences between the four subtypes of tanycytes have
been found.
a
1,2
tanycytes do not have barrier properties, whereas
it is important to note that
b
1
tanycytes establish a barrier between
the AN and the median eminence [120,132] and
b
2
tanycytes
establish a CSF-median eminence barrier [20,129,132,152].
a
1,2
and
b
1
tanycytes express the glucose transporter 1 whereas
b
2
tanycytes do not (Fig. 2A) [110].
b
1
but not
b
2
tanycytes express
insulin-like growth factor binding protein) [24]. Different types of
tanycytes use different mechanisms to internalize and transport
cargo molecules.
b
1
,
2
tanycytes express caveolin-1 at the
ventricular cell pole and at their terminals contacting the portal
capillaries, but
a
1
,
2
tanycytes do not contain this protein (Fig. 7A),
suggesting that caveolae-dependant endocytosis only occurs in
b
tanycytes) [111]. Compounds internalized via a clathrin-depen-
dant endocytosis enter tanycytes from the CSF but not through
their terminals at the perivascular space of the portal capillaries
(Fig. 1F). All tanycyte subtypes internalize wheat germ agglutinin
Fig. 2. (A) Rat medial basal hypothalamus [110]. Immunocytochemistry for Glut 1. The cell body and the basal processes of
b
1
tanycytes are reactive but those of
b
2
tanycytes
are not. Blood vessels of the hypothalamus are reactive (black arrows) but the capillaries of the portal system are not (red arrows). Bar 45
m
m. Inset: Detailed magnification
showing that in
b
1
tanycytes Glut 1 is localized in the plasma membrane (arrow). IR infundibular recess, N arcuate neurons. Bar 10
m
m. (B) Lateral zone of the median
eminence (ME)-pars tuberalis (PT) region. Light- and electron-microscopy immunocytochemistry using anti-Glut 1. Some of the capillaries localized within the pars tuberalis
and in the external region of the median eminence display Glut 1 immunoreactivity in the non-fenestrated endothelium (large arrows). Bar 700 nm; insert: 60
m
m. (C) Medial
region of the median eminence. Ultrastructural immunocytochemistry of an area similar to that framed by circle in inset. A fenestrated (arrows) portal capillary and
ependymal endings of
b
2
tanycytes (T) are devoid of anti-Glut 1 immunoreactive sites. PVS Perivascular space. Bar 700
m
m; insert: 60
m
m.
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injected into the CSF;
a
1
and
b
1,2
tanycytes transport the tracer
along their basal processes but
a
2
tanycytes do not [111].
a
1
tanycytes but not the other subtypes would be functionally
coupled through gap junctions (see Section 4d). A subpopulation of
tanycytes of adult normal rats, most likely corresponding to
a
2
tanycytes, behaves as neural progenitor cells that differentiate into
hypothalamic neurons [132,156].
5. Microscopic arrangement of the median eminence barriers
The median eminence is a discrete hypothalamic compartment
with its dorsal aspect bathed by the ventricular CSF, its ventral
aspect exposed to the perivascular space of the portal capillaries
and to the subarachnoidal CSF, and its two lateral aspects
establishing a border with the hypothalamus.
5.1. In the external region of the median eminence neurosecretory
axons and
b
tanycytes end on the fenestrated portal capillaries
The primary plexuses of the hypophyseal portal system are
formed by a superficial capillary network and deep capillary loops.
The former is formed by a mesh-like network of anastomosed
capillariesoccupying most of the space between the external surface
of the median eminence and the pars tuberalis (Figs. 2 and 5). The
deep capillaryloops display (i) an afferentbranch that penetrates the
median eminence neuropile, (ii) a segment running horizontally in
the subependymal region and (iii) and an efferent branch draining
into the superficial plexuses [45,46,124].The long loops may arrange
as a large subependymal network (Fig. 6A). The long capillary loops
are mainly surrounded by terminals of
b
1,2
tanycytes whereas the
superficial capillary network is surrounded by both neurosecretory
endings and tanycyte terminals [124]. The functional significance of
such an arrangement is unknown. Both types of portal vessels are
endowed with a wide perivascular space and fenestrated endothe-
lium that do not express Glut 1 (Fig. 2A and C).
According to Duvernoy [45], vessels of the AN communicate
with the portal capillaries. If this were the case, the communicating
vessel should, at a given point, have a sealed perivascular space
protecting the AN from the median eminence milieu [79] (Fig. 9G).
Glut 1 immunoreactive capillaries may be seen to extend between
the AN and the lateral subependymal layer of the median eminence
corresponding to the territory of
b
1
tanycytes, but they were never
seen to communicate with portal vessels (Fig. 2A) [110].
Consequently, there would not be a communication between
Fig. 3. (A) Transmission electron microscopy of
b
2
tanycytes displaying tight (TJ) and adherent (ZA) junctions. Arrowheads and thin arrows point to components of the
endocytic pathway [111]. Bar 300 nm. Insert: Junction complex (ZA, TJ) between
b
2
tanycytes. Bar 300 nm. (B) Drawing depicting the tight junctions (TJ) between
b
2
tanycytes. (C) Confocal microscopy of the median eminence (ME) using antibodies for occludin (green) and vimentin (red). The honeycomb pattern of immunoreactive
occludin in
b
2
tanycytes is distinct. [97].Bar20
m
m. (D) Transmission electron microscopy of the cell body of
b
1
tanycytes displaying adherent (ZA) junctions and lacking
tight junctions. Bar 1.5
m
m. (E) Detailed magnifications of a junction complex (ZA) between
b
1
tanycytes. (F) Drawing depicting the adherent junctions (ZA) between
b
1
tanycytes. (G) Confocal microscopy using antibodies for occludin (green) and vimentin (red) showing the unorganized distribution of occludin in tanycytes lining the arcuate
nucleus (AN). [97]. Bar 20
m
m. (C and G): courtesy of Mullier et al. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.) [97].
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the wide perivascular space of the portal capillaries and the
intercellular space around the AN vessels.
At the interphase between the lateral region of the median
eminence and the pars tuberalis are Glut 1 immunoreactive
capillaries endowed with a perivascular space, a non-fenestrated
endothelium that expresses Glut 1 (Fig. 2A and B). The functional
significance of these vessels remains to be elucidated.
5.2. The median eminence-AN barrier
For the whole BBB system to operate efficiently, a barrier
between the median eminence milieu and the AN must exist
[79,125,126] (see below). What is the nature of the median
eminence-AN barrier? Those tanycytes lining the lateral exten-
sions of the infundibular recess (
b
1
tanycytes) have certain distinct
characteristics [6,128] and their basal processes extend along the
border between the median eminence and the AN, thus becoming a
good candidate to be involved in the barrier mechanism operating
in this area. In the median eminence, Glut 1 is missing from the
portal vessels and
b
2
tanycytes, but is present in
b
1
tanycytes
(Fig. 2A). Glut 1 distributes throughout the whole plasma
membrane of
b
1
tanycytes, outlining the cell body, the basal
processes, and their endings at the lateral region of the median
eminence (Fig. 2A) [110]. Thus, in the median eminence, GLUT 1
Fig. 4. (A) Rat medial basal hypothalamus. Immunocytochemistry for
a
-catenin. The cell body and the basal processes of
b
1,2
tanycytes and the BBB-free median eminence are
strongly reactive. IR infundibular recess [132]. Bar 100
m
m. (B and C) Transmission electron microscopy of the rat median eminence B. Adherent junctions (arrow) between
tanycyte terminals (EE) [132]. Bar 400 nm. (C) Adherent junctions (arrow) between terminals of neurosecretory axons (NE). Bar 100
m
m. (D) Double immunostaining for
connexin 43 (green) and
b
IV-tubulin (red). The ciliated ependyma (CE) and
a
1
tanycytes express connexin 43, while other tanycyte types do not. Bar 75
m
m. Left insert:
Detailed view of area framed in figure E. In
a
1
tanycytes express connexin 43 is mostly localized in the apical cell pole (arrow). Bar 20
m
m. Right insert: Detailed magnification
of area framed below, showing that
a
2
tanycytes do not express connexin 43. Bar 28
m
m. (E) High magnification of an area shown in figure D. The ciliated ependyma (CE) and
a
1
tanycytes express connexin 43, but
a
2
tanycytes (below broken line) do not. Bar 35
m
m. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
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has been shifted from the vascular side to the median eminence-
AN barrier, but not to the median eminence-ventricular barrier.
b
1
tanycytes have a distinct ultrastructure. Apically (ventricular cell
pole), they are joined together by adherent junctions but tight
junctions are missing (Fig. 3D–G). The processes of the
b
1
tanycytes establish a border between the AN and median
eminence. As will be shown below, this border actually corre-
sponds to a barrier separating the intercellular space of the
hypothalamus from that of the median eminence. An amazing
feature of
b
1
tanycytes is that they are joined together by zonulae
and maculae adherens localized throughout the lateral surface of
the cell body, basal processes, and terminals (Fig. 5A–C). This
unique feature of
b
tanycytes may explain the formation of
bundles of tanycyte processes. The electron microscope reveals the
frequent existence of adherent junctions and occasional tight
junctions between the
b
1
tanycyte processes as well as between
tanycyte and neurosecretory axons (Fig. 5C). Tight junctions
between tanycyte processes located in the lateral region of the
median eminence (
b
1
tanycytes) have been reported by freeze-
etching studies [79]. Axons entering the median eminence are
tightly surrounded by the processes of
b
1
tanycytes. Lateral finger-
like projections of the processes of
b
1
tanycytes embrace isolated
or small bundles of axons penetrating into the BBB-free area of the
median eminence (Fig. 5C).
The Glut 1 reactive processes of
b
1
tanycytes bordering both
lateral sides of the median eminence are arranged into several
bundles. Located among these bundles are the most ventral
neurons of the AN (Fig. 2A). Blood capillaries of these areas are tight
and express Glut 1. Occasionally, some secretory neurons are found
in the lateral subependymal region of the median eminence, in the
Fig. 5. (A) Bundles of processes of
b
1
tanycytes are immunoreactive for
a
-catenin. Bar 50
m
m. (B) Line drawing of barriers in the medial basal hypothalamus: CSF-median
eminence barrier (red line) formed by
b
2
tanycytes; arcuate nucleus-median eminence barrier formed by the processes of
b
1
tanycytes joined into bundles by adherent and
tight junctions. The median eminence (ME) milieu (pink dots) is opened to the fenestrated portal capillaries; while the arcuate nucleus (AN) milieu is opened to the ventricle
(green dots). The median eminence milieu (pink background) is segregated from the arcuate nucleus milieu (green background). (C) Transmission electron microscopy of a
bundle of processes of
b
1
tanycytes displaying adherent junctions (ZA). Lateral branches of these processes envelope isolated (circle) or bundles (arrows) of axons. Bar
200 nm. Left insert: Bundles of axons (arrows) packed by processes of
b
1
tanycytes. Bar 200 nm. Right insert: Tight junctions (framed area) between a neurosecretory axon
and tanycyte processes (T). Bar 300 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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vicinity of a long loop of the primary plexuses of the portal system.
These neurons are thus located within the BBB-free territory of the
median eminence (see Section 10).
5.3. The median eminence-CSF barrier
The dorsal wallof the median eminence bordering the floor of the
infundibular recess is lined by
b
2
tanycytes. The apical (ventricular
cell pole) cytoplasm of these cells contains the machinery for active
endocytosis, namely, microvilli, coated pits, coated vesicles, early
endosomes, multivesicular bodies (late endosomes), and formations
most likely corresponding to recycling endosomes (Fig. 3A) [111].
Apically (ventricular cell pole),
b
2
tanycytes are joined together by
tight junctionsand zonulae and macculae adherens extending along
the lateral plasma membrane (Fig. 3A and B). The use of an antibody
against a tight junction-associated protein has shown that the
median eminence tanycytes are joined by continuous, unbroken
immunoreactive junctions [112]. In a recent investigation Mullier
et al. [97] have shown that tight junctions of
b
2
tanycytes are a
continuous belt around the cell bodies made up of claudins 1 and 5
and ZO-1 (Fig. 3C). These tight junctions seal the intercellular space
between the cell bodies of
b
2
tanycytes and prevent the free
movement of molecules between the third ventricle and the
intercellular space of the median eminence (see below).
5.4. The lack of barrier between CSF and the nuclei of the medial-basal
hypothalamus
At the ventricular cell pole,
a
1,2
and
b
1
tanycytes are joined
together by adherent junctions but tight junctions are missing
Fig. 6. Distribution of horseradish peroxidase (HRP) 15 min after injection into a lateral ventricle. (A) HRP injected into the lateral ventricle penetrates the walls of the third
ventricle but not penetrate into the median eminence; it also reaches the brain tissue from the subarachnoid space [129,132]. Bar 800
m
m. From Rodrı
´guez et al. (1982). (B)
Detailed magnification of previous figure. HRP penetrates through the intercellular space of
a
1,2
and
b
1
tanycytes (green arrow) but not between
b
2
tanycytes (black arrow).
Bar 45
m
m. (C) HRP localized in the intercellular space of tanycytes is seen. Bar 15
m
m. (D) Electron micrograph of an area similar to that shown by green rectangle in figure B.
Peroxidase reaction product labels the intercellular space between
b
1
tanycytes (black arrows), around nerve profiles and neurons of the arcuate nucleus (red arrows). Bar
1
m
m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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(Fig. 3D and F). The intercellular space of this ependymal zone is in
open communication with the ventricular lumen.
a
2
tanycytes
express much less
a
-catenin than all other tanycyte subtypes
(Fig. 4A) suggesting that at this level adherent junctions are
different from those joining the other tanycyte subtypes. Further-
more, these tanycytes do not express claudin 1 and display a
disorganized expression pattern of occludin, ZO-1 and claudin 5
(Fig. 3G) [97]. Worth mentioning is that
a
2
tanycytes line and their
basal processes project to the middle and dorsal regions of the AN.
Does this arrangement imply a special interphase between CSF and
AN? Evidence for a positive answer is discussed below.
6. Dynamic aspects of the median eminence barriers
6.1. The median eminence milieu, the perivascular space of the portal
vessels, and the subarachnoid space are in open communication
The open communication of the median eminence milieu with
the portal blood is a well-established fact. Indeed, decades of
investigation have demonstrated that peptides and amines
secreted by hypothalamic neurons are delivered into the portal
circulation. On the other hand, a series of tracers (trypan blue,
Evans blue, ferritin, HRP), when administered intravenously escape
from the portal capillaries into the perivascular space and readily
reach the intercellular space of the median eminence and, most
important for the scope of the present review, remain confined to
this region [20,21,97,132] (Fig. 7). This indicates that plasma
compounds can escape from the fenestrated portal capillaries,
reach the intercellular space of the median eminence and be
confined here due to the CSF-median eminence and AN -median
eminence barriers (see below).
Horseradish peroxidase (HRP) injected into the subarachnoidal
CSF (cisterna magna) reaches the perivascular space of the portal
capillaries and the intercellular space of the median eminence,
indicating that the median eminence milieu is in open communi-
cation with the subarachnoidal CSF also [110,132] (Fig. 8). Several
minutes after the administration of HRP into the ventricular or
cisternal CSF the tracer is seen in the median eminence as a
ventrodorsal gradient of peroxidase reaction product occupying
the intercellular space of the median eminence [132] (Fig. 8A). The
gradient, which was patent even 15 min after the HRP injection,
could be an indication that there is a dorsoventral bulk flow of
intercellular fluid toward the perivascular space of the portal
vessels. If this were the case, blood-borne and subarachnoidal CSF-
borne molecules gaining access to the median eminence milieu
would be pushed back to the perivascular space. Krisch et al. [80]
have proposed that a subarachnoidal-median eminence commu-
nication does not occur. This discrepancy and the evidence
presented by several authors supporting the existence of such a
communication have been discussed extensively by Peruzzo et al.
[110]. This is an important issue, since the existence of such a
communication has some relevant implications: (1) neurotrans-
mitters and neuropeptides released at the perivascular space of the
portal vessels by hypothalamic neurons (and tanycytes?) could
reach the local subarachnoidal CSF; (2) compounds present in the
subarachnoidal CSF could enter the portal circulation; (3) blood-
borne substances would not only reach the median eminence
milieu but also reach the local subarachnoidal CSF.
6.2.
b
2
tanycytes establish an efficient barrier between the median
eminence milieu and the ventricular CSF
HRP injected into the ventricular CSF enters the hypothalamus,
but it does not enter the median eminence due to the tightness of
the
b
2
tanycyte layer [20,110,117,129,132,152] (Fig. 6A–C). A
similar finding is obtained after intraventricular administration of
cationic ferritin [132] and Evans blue (Fig. 9D) [97]. Intraventricu-
larly injected HRP, regardless of the post injection interval, does
not traverse the tight junctions of
b
2
tanycytes, but it may be
visualized in the median eminence as a ventrodorsal gradient of
peroxidase reaction product, similar to that found after intracis-
ternal injection of the tracer [110,132]. This indicates that HRP
injected intraventricularly can readily reach the median eminence
milieu through the ventricular-subarachnoidal flow of CSF.
Alternatively, compounds present in the ventricular CSF can reach
the intercellular space of the median eminence by transependymal
transport occurring in
b
2
tanycytes [132]. The possibility that the
tight junctions between
b
2
tanycytes may be leaky has been
considered [21,114].
6.3.
b
1
tanycytes establish a lateral barrier on either side of the
median eminence, separating the intercellular space of the median
eminence from that of the AN
A good body of evidence supports the existence of an efficient
barrier between the median eminence and AN. Indeed, tracer
molecules injected into the ventricular CSF reach the AN but do not
enter the median eminence [20,132,152]; whereas, when these
molecules are delivered to the systemic blood, they reach the
median eminence milieu but do not pass into the neighboring
neuropile of the AN [20,21,152]. When used at certain doses and
post injection intervals, HRP injected into the ventricle readily
gains access to the intercellular space of AN without reaching the
median eminence (Fig. 6A and B) [20,129,132,152]. At variance,
HRP injected intravenously, although it strongly labels the median
eminence, does not pass into the AN [20,21,80]. Similarly, vital
stains, such trypan blue and Evans blue, when injected intravas-
cularly readily enter the median eminence but not the hypothala-
mus (Fig. 9A, B, C and E), whereas when administered into the
ventricle they reach the AN but not the median eminence (Fig. 9D).
These findings strongly indicate that a barrier between the AN and
the median eminence must operate. This evidence is further
substantiated by the finding that when HRP [120] and trypan blue
(Fig. 9F) are injected directly into the AN they do not enter the
median eminence. In all the experimental protocols discussed, the
labeling of the intercellular space suddenly stopped at both lateral
borders of the median eminence, exactly at the site of location of
b
1
tanycytes and their basal processes. Thus,
b
1
tanycytes become a
good candidate to be involved in the barrier mechanism between
the median eminence and AN (see Section 5b).
As discussed in Section 5b, Glut 1 is missing from the portal
capillaries and
b
2
tanycytes but is present throughout the whole
plasma membrane of
b
1
tanycytes suggesting that these cells
contribute to the lateral barrier of the median eminence [52,110].
This possibility is supported by the following experimental
findings. It is well established that the subcutaneous administra-
tion of monosodium glutamate (MSG) to newborn rats leads to a
severe and selective destruction of the neurons of the AN
[103,104]. Little is known about the mechanisms responsible for
this temporal and spatial selective neuronal damage. The
possibility that the median eminence tanycytes are somehow
involved in this mechanism has been suggested [66]. Interestingly,
the postnatal development of Glut 1 immunoreactivity of
tanycytes parallels that of the susceptibility of arcuate neurons
to MSG treatment [110]. Indeed, the maximal damage of the
arcuate neurons occurs when MSG is administered during the first
postnatal week [17,103,104,108,110] a period when Glut 1
immunoreactivity of
b
1
tanycytes is barely detectable. The
possibility may be suggested that, at this time, the median
eminence-AN barrier is not yet developed, thus allowing MSG from
portal vessels to gain access to the AN. Once the median eminence-
AN barrier is fully developed, around the fourth postnatal week,
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the systemic administration of MSG does not affect the AN [110].
This evidence suggests that, in rats older than 1 month, a median
eminence-AN barrier is operating.
7. How do hypothalamic hormones reach the portal circulation
without making the BBB leaky?
Neurons secreting hormones into the blood stream must be in
open communication with the blood capillaries. Peptides and
monoamines synthesized in different hypothalamic nuclei enter
the median eminence and reach the portal circulation. The cell
body and a long segment of the axon of these neurons are localized
in areas protected by the BBB, whereas the preterminal segment of
the axon and the terminal proper are localized in the median
eminence, an area lacking a BBB. This unique design implies a
complex organization of the intercellular space of this area, which
should involve the existence of discrete intercellular compart-
ments, so that the median eminence would behave as a canal lock,
with inlet and outlet gates (Fig. 5B). Thus, neurohormones can
enter this area by axonal transport and, upon release, they can
reach the perivascular space, the portal blood, and the intercellular
space of the median eminence but are prevented from traveling
back into the cerebrospinal fluid of the third ventricle or into the
intercellular space of the adjacent hypothalamic region. Such an
arrangement also implies that those neurons projecting to the
median eminence are not under the influence of blood borne
Fig. 7. (A–D) Rat medial basal hypothalamus. Double immunostaining for caveolin 1 (green) and
b
IV-tubulin (red). (A) The ciliated ependyma (CE), neurons of the dorsal
medial nucleus (DMN) and smooth muscle cells of an arteriole (ar) express caveolin 1 but
a
1
tanycytes do not. Bar 40
m
m. (B) Detailed magnification of the multiciliated
ependyma (E) shown in the previous figure. These cells strongly express caveolin 1 (green) and the cilia occupying the ventricle lumen are strongly reactive for
b
IV-tubulin
(red). Bar 11
m
m. (C) All
a
2
tanycytes express
b
IV-tubulin (red, full arrow) and some of them also express caveolin 1 (orange, broken arrow). AN arcuate nucleus, IR
infundibular recess. The area framed is shown in figure D. Bar 30
m
m. (D) Detailed view of area framed in C. Arcuate neurons (N) express caveolin 1 strongly. Tanycyte
processes expressing caveolin 1 and
b
IV-tubulin appear orange (broken arrow). Bar 10
m
m. (E) Transmission electron microscopy of an arcuate neuron projecting a single
cilium to the intercellular space (frame area). Bar 1
m
m. (F) Detailed magnification of area framed in E. C cilium, G Golgi apparatus. Bar 250 nm. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
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compounds leaking through the portal capillaries, unless receptors
for such compounds are located at the axonal plasma membrane.
The whole spatial arrangement at the median eminence, as
described in Sections 5 and 6, allows neurons to secrete
neurohormones into the blood stream without making the BBB
leaky (see [70,105]).
8. AN barriers
8.1. The AN is in open communication with the ventricular CSF
A few minutes after its intraventricular administration, HRP is
found in the intercellular space ofthe tanycyteslining the AN and in
the intercellular spaceof the AN (Fig. 6). Thespace around the arcuate
neurons is filled with the tracerand HRP may be seen internalized by
the arcuate neurons. Arcuate neurons express caveolin 1 as
numerous dots distributed throughout the cell body (Fig. 7Cand
D). When Evans blue is administered into the ventricle it almost
exclusively enters the AN (Fig. 9D) [97], suggesting that the AN-
ventricular interphase is even more permeable to CSF compounds
than the ventromedial/dorsomedial nuclei-ventricular interphase.
Conversely, when trypan blue is microinjected into the left AN it
labels this nucleus fully, the wall of the infundibular recess, and
partially the right AN (Fig. 9F), indicating that the tracer has moved
from the AN intercellular space to the lumen of the ventricle.
8.2. The intercellular space of the AN is segregated from the
intercellular space of the median eminence and of the adjacent
hypothalamic nuclei
Peroxidase injected directly into the AN does not enter the
median eminence [120]. The microinjection of trypan blue into the
Fig. 8. Distribution of horseradish peroxidase (HRP) 15 min after injection into the cisterna magna. (A) HRP reaches the subarachnoid space but not the ventricles; it
penetrates the brain tissue from the subarachnoid space. The arcuate nucleus-median eminence barrier prevents HRP to reach the arcuate nucleus. An area similar to that
framed by square is shown in figure B. Bar 50
m
m. Insert: Some HRP present in the subarachnoid space reaches the neuropile of the median eminence where a ventro-dorsal
gradient can be seen (asterisk) but it does not reach the layer of
b
1,2
tanycytes (arrows). Bar 15
m
m. (B) Peroxidase reaction product strongly labels the perivascular space
(PVS), the external limiting membrane of the brain and its extensions into the median eminence and the intercellular spaces around nerve profiles and processes of
b
2
tanycytes [110]. Bar 700 nm.
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left AN results in the following findings: (i) the tracer is
circumscribed to the injected AN, outlining this nucleus distinc-
tively; (ii) it enters neither the median eminence nor the adjacent
ventromedial nucleus; (iii) it reaches the lumen of the infundibular
recess and labels its wall (Fig. 9F). When Evans blue is injected into
the ventricle it enters the AN fully, but it does not progress beyond
the boundary of this nucleus, namely the median eminence, the
ventromedial nucleus, and the adjacent neuropile (Fig. 9D) [97].
Furthermore, the existence of a barrier between the ventromedial
nucleus milieu and the AN milieu is further supported by the
finding that HRP injected into the ventromedial nucleus diffuses
into adjacent areas with the exception of the AN, which remains
free of label [120]. All these findings support the amazing
possibility that the AN milieu is closed to all adjacent neural
structures while is open to the CSF present in the infundibular
recess. The nature of the barrier surrounding the AN is not known
and certainly it is a most interesting task for future investigations.
The AN is a complex structure including various subpopulations
of neurons that secrete different neurotransmitters and neuropep-
tides, and that project to a BBB-free area such as the median
eminence and to BBB-protected areas such as the paraventricular
nucleus. What is the subcellular arrangement around the AN
Fig. 9. (A–C) Trypan blue was injected into a carotid artery of a mouse and five minutes later the brain was fixed by vascular perfusion with Bouin, and cut frontally to visualize
this vital stain. (A) Scanning electron microscopy of the mouse brain. The arrow indicates the plane of section of figure B. (B) Frontal section of hypothalamus. Trypan blue is
exclusively localized in the median eminence (arrow). Bar 250 nm. (C) Detailed magnification of previous figure showing the localization of trypan blue in the wall of the
portal capillaries and the neuropile of the median eminence, with a clear ventro-dorsal gradient (arrows). The arcuate nucleus-median eminence barrier (broken line) and the
CSF-median eminence barrier (blue line) prevent the dye to escape from the median eminence milieu. Bar 70 nm. (D) Evans blue injected into the cerebrospinal fluid
penetrates into the arcuate nucleus but not into other hypothalamic nuclei (VMN, DMN) or the median eminence [97]. Bar 100
m
m. (E) Evans blue injected into the blood is
confined to the median eminence milieu. Broken line, arcuate nucleus-median eminence barrier. [97]. Bar 100
m
m. (F) Trypan blue was microinjected into the left arcuate
nucleus of a medial basal hypothalamus explant using a glass cannula, 5
m
m external diameter. The dye was confined to the arcuate nucleus (arrows) and the wall of the third
ventricle. Bar 500 nm. Inset: The large arrow indicates site of injection of trypan blue. The dye fills the left arcuate nucleus and partially labels the right arcuate nucleus (small
arrow). (G) Line drawing depicting the barriers of the medial basal hypothalamus. The layer of
b
1
tanycytes is open and that of
b
2
tanycytes is tight. The capillaries of the
median eminence are fenestrated (f) and display a perivascular space (asterisks) while those of the arcuate nucleus are tight (t) and lack a perivascular space. Orange line,
arcuate nucleus-median eminence barrier. (D and E): courtesy of Mullier et al. [97]. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
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allowing both, the existence of an AN private barrier and the
projection of the AN efferent axons through such a barrier? The
cellular organization of the AN-median eminence barrier is
partially known (see Sections 5.3 and 6.3) whereas that of the
ventromedial nucleus-AN is completely unknown.
What is the functional significance of the AN having its own
private milieu, which in turn is open to the ventricular CSF? The
fact that the infundibular recess, the most ventral part of the third
ventricle, is devoid of multiciliated ependymal cells should result
in the slowing of the CSF flow at this level. It may be speculated that
this whole arrangement facilitates the arrival of CSF borne signal to
the AN (Fig. 10).
9. How do peripheral hormones may reach hypothalamic
targets without making the BBB leaky?
9.1. Specific transport systems at the choroid plexuses
Choroid plexuses are outpouchings of the vascular system into
the ventricle, covered with ependyma that has become morpho-
logically and functionally differentiated from its neighboring
regions, and is called the choroidal epithelium. Through active
transport of salts associated with movement of water, the CSF is
secreted by the choroid plexuses [38,39]. The ependymal cells
forming the choroid plexuses project numerous microvilli to the
ventricle and display extensive infoldings of the basolateral plasma
membrane, thus providing a large surface area at the blood-
choroidal cell and the choroidal cell-CSF interphases. The total area
for transport at the four choroid plexuses is the same order of
magnitude as the whole BBB [67,71,72,138].
The capillaries of the choroid plexuses are fenestrated and
endowed with a wide perivascular space. At the choroid plexuses
the BBB has been shifted from the vascular side to the ependymal
side, as is occurs in other circumventricular organs (see Section 2).
Indeed, choroid cells are joined together by tight junctions that
completely seal the intercellular space of the choroidal epithelium.
This cellular arrangement results in a barrier preventing sub-
stances from freely moving from the perivascular space to the CSF
[67]. Therefore, for blood-borne compounds to reach the CSF they
have to use specific transport system. Indeed, the choroidal cells do
contain a wide variety of transport systems that mediate both the
entry of essential nutrients (glucose, amino acids) and regulatory
substances into the brain [138]. Entry of peripheral peptide/
proteins into CSF is mediated by specific mechanisms for receptor-
and adsorptive-mediated transcytosis [10,32,138,139,150].
Several peptides of peripheral origin are thought to be actively
transported by the choroidal cells to the CSF and exert an effect on
central nervous structures, the hypothalamus in particular.
Receptors for several hormones, such as growth hormone,
prolactin, corticotrophin-releasing factor, insulin, insulin-like
growth factor-I and II, leptin as well as the low-density lipoprotein
receptor have been identified in the choroid plexuses [138]. It has
been suggested that these receptors transport the bound hormone
from blood to CSF via a receptor-mediated transcytosis. A good
body of evidence indicates, indeed, that at the choroidal cells there
are specific transport systems for leptin, insulin, insulin growth
factor I and prolactin (see Sections 9.3–9.6).
Choroid plexuses not only provide central nervous structures
with a selective group of peripheral compounds but they also
synthesize and release into the CSF several polypeptides that can
be regarded as ‘‘CSF-specific’’ proteins, namely, transferrin [146],
nerve growth factor [142], transforming growth factor-
b
[147],
vascular endothelial growth factor [100], transthyretin [41], and
vasopressin [31].
All the findings discussed above substantiate an early view by
Cserr [37] that a fascinating aspect of the choroid plexuses
concerns their possible role in the overall activity of the CNS.
9.2. The CSF as a pathway for neuroendocrine integration
It has early been proposed that CSF should be considered as a
pathway for neuroendocrine integration [126]. This proposal was
based on the presence of peripheral hormones in the CSF and the
existence in the ventricular walls, especially those lining the
hypothalamus, of structures specialized in transport and/or
secretion. New evidence further supports such an integrative
view. Two main categories of periventricular structures may be
recognized, namely, ependymal specializations and neuronal
formations.
The ependymal lining of the ventricular cavities is heteroge-
neous, with zones of conventional multiciliated ependymal cells
alternating with specialized ependymal areas. There are ependy-
mal structures that are clearly specialized as secretory. The
subcommissural organ, the ependyma of the aqueduct of Sylvius,
and the choroid plexuses belong to this type of ependyma. On the
other hand, there are ependymal areas that are specialized for
Fig. 10. Integrative pathways involving the CSF. Upper panel. By receptor mediated
transport at the choroid plexus (CP), leptin (Ob-Ra), insulin growth factor I
(megalin), thyroid hormones (MCT8/OATP14) and prolactin (PRLr) are transported
from blood to CSF. Transthyretin (TTR) is secreted by choroid plexus into the CSF.
Most CSF T4 is bound to TTR. Lower panel: TTR-T4 complexes are taken up by
tanycytes that express deiodinase 2. Here, T4 is converted to T3 and then released
into the intercellular space of the arcuate nucleus or into the CSF to reach the TRH-
parvocellular neurons of the paraventricular nucleus. Main panel: The milieu of the
arcuate nucleus (green background) is especially exposed to molecules present in
the CSF and closed to the median eminence and ventromedial nucleus. Leptin
present in the CSF may readily reach the neurons expressing the Ob-Rh receptor of
the arcuate, ventromedial and dorsomedial nuclei of the hypothalamus. CSF
prolactin may reach the dopamine-secreting neurons (DA) of the arcuate nucleus
that project to the portal capillaries of the median eminence (light pink
background). CSF insulin growth factor I is internalized by
b
tanycytes and
transported along their processes. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
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transport, such as the choroid plexuses, the tanycytes of the
median eminence, and the ependyma of the subfornical organ and
of the organum vasculosum of the lamina terminalis.
Three main types of the periventricular neuronal system may
be recognized. In one of them, the neurons are not in direct contact
with the ventricular fluid, but belong to circumventricular organs,
such as the subfornical organ and the organum vasculosum of the
lamina terminalis. In these organs there are neurons responsive to
CSF and plasma levels of angiotensin II and trigger a neuroendo-
crine response stimulating the release of vasopressin and a
behavioral response leading to thirst and water intake [55]. Thus,
the CSF plays a key role in the mechanism responsible of the water
and volume balance.
In the second periventricular neuronal system the neurons are
in direct contact with the CSF forming the CSF-contacting neuronal
system [148,149]. Most of these neurons are bipolar with the
dendritic process reaching the CSF. Only occasionally has it been
possible to trace the axons of these neurons. CSF-contacting
neurons may be cholinergic, aminergic, or peptidergic [126,148].
The dendritic ventricular processes of this neuronal system,
endowed with a single cilium, most likely play receptive functions
sensing CSF composition [126,148,149]. Neurons of the AN should
also be regarded within this group, since they are fully exposed to
the CSF and many of them display a single 9 + 0 cilium (Fig. 7E and
F) [125].
The third periventricular neuronal system includes those
neurons projecting their axons to the ventricles. These neurons
are secretory and release monoamines and neuropeptides into the
CSF, such as the serotoninergic raphe-ventricular neuronal system
releasing serotonin into the CSF in a circadian pattern [2,39,121]
and the neurophysinergic hypothalamic neurons releasing vaso-
pressin and oxytocin into the CSF [127,155].
On the other hand, most of the hormones of the hypophysis and
those of the peripheral glands have been found to be present in the
CSF. One interesting feature is that for each of them there is a
particular plasma-CSF ratio, which is always higher than one,
suggesting that the hormones reach the CSF not by simple diffusion
but by a saturable active transport system [39,155] (see below).
Many neurotransmitters and their metabolites [131] and a series of
neuropeptides [155] have also been detected in the CSF. These
neuroactive compounds present in the CSF may originate: (i) by
bulk flow of the brain intercellular fluid, (ii) by specific blood-CSF
transport at the choroid plexuses, (iii) from neurosecretory CSF-
contacting neurons, or (iv) from secretory ependymal structures,
such the choroid plexuses and the subcommissural organ
[39,87,93,129,130,149,155].
Melatonin may also be regarded as a signaling molecule carried
by the CSF. Melatonin enters the CSF through the pineal recess
[145] and its CSF concentration is 20-fold higher than that of
plasma [137]. Radioactively labeled melatonin administered into
the CSF rapidly penetrates into the diencephalic parenchyma
bordering the third ventricle [84]. Furthermore, CSF melatonin
undergoes circadian variations parallel to those of plasma [68,119].
These findings support the possibility that the regulatory function
of melatonin on the suprachiasmatic nucleus and the pars tuberalis
(bathed by CSF) is exerted via the CSF [58].
The integrative role of CSF becomes evident when the analysis
is circumscribed to specific physiological phenomena, such as the
two following examples. Neurons of the raphe nuclei release
serotonin into the CSF; the cells of the choroid plexuses have
serotonin receptors only at the apical (ventricular cell pole) plasma
membrane and serotonin exerts an inhibitory effect on CSF
secretion [39,109]; such a design underlies the circadian produc-
tion of CSF [101]. Dopaminergic neurons of the AN send their axons
to the portal capillaries and are involved in the control of prolactin
release. This latter hormone reaches the CSF by a specific transport
system at the choroid plexuses (see below) and gains access to the
perikaryon of the dopaminergic arcuate neurons, thus closing a
negative feedback loop.
Furthermore, the choroid plexuses do secrete polypeptides into
the CSF (see Section 9a) that gain access to targets in the brain
parenchyma. The presence in the ventricular walls (including the
choroid plexuses) of numerous structures playing secretory,
receptive, and transport functions and the existence in the CSF
of a series of signal molecules have, to a great extent, been
overlooked and should be kept in mind when interpreting new
findings or designing new experiments.
With the aim to substantiate the view that peripheral hormones
may reach hypothalamic targets without making the BBB leaky, the
transport from blood to CSF of insulin growth factor I, thyroid
hormones, prolactin, and leptin, and their targeting to periven-
tricular neuronal and ependymal structures will be discussed
below.
9.3. Insulin growth factor-I: transport through choroid plexuses,
presence in the CSF and targeting to tanycytes
Receptor-mediated transport of insulin growth factor-I (IGF-I)
has been shown to occur through the BBB [118] and the blood-CSF
barrier [18,69]. Megalin is involved in IGF-I transport across the CP
[28]. The evidence strongly suggests that blood-borne IGF-I is an
important neuro-surveillance factor [143]. In response to certain
physiological stimuli, circulating IGF-I enters the brain [27] and
participates in relevant events, such as hippocampal neurogenesis
[144].
Furthermore, IGF-I has been immunocytochemically detected
in the hypothalamic tanycytes [44,53]. Tanycytes display IGF-I
receptor and insulin growth factor binding protein-2 [24].
Tanycytes absorb IGF from the CSF and transport it along their
basal processes [54] (Fig. 10). The capacity of tanycytes to
incorporate and accumulate IGF-I [50] and the fact this capacity
is under the influence of ovarian hormones has led to the
suggestion that this compound is involved in the cyclic plastic
changes of tanycytes associated with the release of gonadotrophin
hormone-releasing hormone [54] (Fig. 10).
Thus, blood-borne IGF-I may enter the CSF and through this
pathway may reach different targets; in the hypothalamus the
target are tanycytes rather than neurons (Fig. 10).
9.4. Thyroid hormones: transport of T4 at choroid plexuses (role of
transthyretin), and bioconversion into T3 at tanycytes
Thyroid hormones require transport across the BBB to carry out
their biological functions in the CNS. Several transporters have
been identified such as the monocarboxylate transporter (MCT)
family members MCT8 and MCT10, and the organic anion
transporter polypeptide OATP14 [61,77]. In the choroid plexuses,
MCT8 appears concentrated on the apical plasma membrane facing
the CSF, whereas OATP14 is detectable at both the apical and the
basolateral membranes [123]. This distribution suggests that both
transporters may function as a pair, transporting T4 from blood to
CSF.
Transthyretin (TTR) is the main thyroid hormones carrier
protein in the CSF. In human CSF, approximately 80% of these
hormones are bound to TTR [42,67]. TTR accounts for 25% of all CSF
proteins and participates in the delivery of thyroid hormone to the
brain [135]. There is evidence that TTR participates in the transport
of thyroxine (T4) from blood to the brain through the blood-CSF
barrier [116].
T4 is the predominant iodothyronine in plasma. However, T3 is
the major receptor-active form of thyroid hormone. Consequently,
T4 has to be converted by deiodination into T3. This function is
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exerted by deiodinases (Dio 1,2). Dio1 is absent from the brain, Dio2
being the primary T3-producing enzyme in the brain [83].
Tanycytes are the main Dio2-expressing cells in the CNS and the
site of intracellular generation of T3 from T4 [49,56,81,82,83,133].
The virtually exclusive location of Dio2 in tanycytes places these
specialized ependymal cells in a strategic position to incorporate
T4 from the CSF (Fig. 10). Interestingly, TTR significantly increases
the uptake of T4 into the ependymal cells. T4 can be converted to
T3 in the cytoplasm of tanycytes and then be released into the
intercellular space of the AN or into the CSF of the infundibular
recess (Fig. 10)[48,82,83,133]. In the former case T3 would
influence the activity of AN neurons that project to parvocellular
neurons of the paraventricular nucleus secreting thyroid stimu-
lating hormone-releasing hormone (TRH) (Fig. 10)[82].T3
entering the CSF could reach several central targets, including
the hypophysiotropic TRH neurons of the paraventricular nucleus
[48,82,83]. Both pathways involve the CSF in the feedback
mechanism regulating TRH/TSH release.
Recently, it has been shown that melatonin regulates the
expression of Dio2 and TSH-receptors in the tanycytes supporting a
role of these cells in the control of photoperiod (melatonin)-
dependent endocrine activity [157,158].
9.5. Prolactin: transport through the choroid plexuses, presence in the
CSF and probable effect on dopaminergic neurons of the AN
Abundant immunoreactive prolactin is present in the choroidal
ependymal cells [107]. Such an intracellular prolactin would
correspond to the hormone being transported from blood to CSF
and vice versa, rather than being the result of a local synthesis.
Indeed, a soluble isoform of prolactin receptor participates in the
transport of prolactin from blood to CSF or from CSF to blood [113].
Choroidal ependymal cells display high amounts of prolactin
receptor mRNA in pregnancy [7]. Prolactin penetrates the blood-
CSF barrier, but not the BBB [67]. Thus, CSF prolactin appears as a
good candidate to feed back to the dopaminergic neurons of the
AN. The choroid plexuses and the ventricular CSF may be regarded
as an important pathway for conveying prolactin from blood to the
hypothalamus as a signal input that controls pituitary prolactin
release [139].
9.6. Leptin: transport through choroid plexuses, presence in the CSF
and targeting to periventricular receptive neurons
Leptin, a 16 kDa protein released by fat cells into the blood, is
the major regulator of body fat. It crosses the BBB to interact with
its receptors located in the hypothalamus and other CNS structures
to regulate feeding, thermogenesis, and other functions [8,9,99].
The presence of leptin in the CSF and leptin receptors
throughout the brain and the absence of leptin mRNA in the
CNS indicate that peripheral leptin has to cross the blood-brain-
barrier. Leptin CSF levels correlate well with those in plasma,
although the relation is not linear [25,59,92,136]. Thus, as plasma
leptin levels rise, the corresponding increase in the CSF level
becomes smaller, indicating that leptin reaches the CSF through a
saturable transport mechanism [9]. Such a mechanism operates at
the choroid plexuses.
The intravenous administration of radioiodinated leptin
revealed a saturable transport into the brain; autoradiography
revealed uptake of the radiolabeled leptin at the choroid plexuses,
AN and median eminence [8–10]. Leptin binds to at least two
receptors in the CNS encoded by the Ob-R gene. The short isoform
of the leptin receptor, ObRa, is highly expressed in cerebral
microvessels and choroid plexuses, so it is considered the main
transporting receptor mediating leptin transport across the
BBB and blood-CSF barrier [22]. The short isoform of leptin
receptor is highly expressed in the choroid plexuses (Fig. 10)
[10,13,40,62,140] and is considered to act merely as a transporter
to convey leptin from blood to CSF via a receptor-dependent
transcytosis [11,22,67,94]. Megalin expressed by the choroid
plexuses can also act as a receptor to transport leptin from blood
to CSF [43]. The hypothesis has been advanced that, under
physiological conditions, the choroid plexuses play a key role in
regulating leptin entry into the CSF [159]. Regulated leptin
transport at the blood-CSF barrier has been demonstrated by
using the perfused sheep choroid plexuses in vitro model [141].
The relevant role of CSF in conveying the leptin signal to its target
is further supported by the finding that in sheep under a
short photoperiod leptin delivered intraventricularly inhibits
appetite [1].
The long form of the leptin receptor is involved in the
physiological response of the hormone and is expressed by leptin
responsive neurons. In a recent study, this receptor has been found
to be expressed in arcuate, ventromedial, dorsomedial, median
preoptic, and ventral premammillary nuclei of the hypothalamus
and in the nucleus of the tractus solitarius [26]. This wide
distribution of neurons expressing the long form of the leptin
receptor and the periventricular location of these neurons further
support the view that CSF may be regarded as part of the
physiological mechanism conveying leptin to its targets in the
brain (Fig. 10).
10. Trying to bridge apparently contradictory findings and
views
It has been suggested that leptin and other peptides might leak
from the median eminence to reach the AN [29,30,35,36,98,99].
The fact that a subpopulation of arcuate neurons project their
axons to the median eminence has been taken as evidence of a
direct accessibility of these arcuate neurons to circulating levels of
leptin. As shown in the present review, only the terminal segment
of the axon and the axon terminal of these neurons are within the
BBB-free area of the median eminence. Thus, for these arcuate
neurons to be under the influence of leptin leaking out or the portal
capillaries they should either display the leptin receptor at the
axon terminal or have the capacity to internalize leptin at the axon
terminal and transport it by retrograde axonal flow. The former
mechanism has not yet been demonstrated; however all neurons
projecting to the portal capillaries of the median eminence,
including those of the AN, can become labeled after intravascular
or intracisternal administration of tracers, such as wheat germ
agglutinin [111], fast blue [34,35], and fluoro gold [65] because
they internalize the tracer at the terminal (BBB-free area). A good
body of evidence indicates that WGA is internalized by tanycytes
and the axons terminals of the median eminence through clathrin-
mediated endocytosis [111]. WGA has a high affinity for sialic acid
residues that are abundant in clathrin-coated membranes and pits.
In the median eminence, WGA is internalized by axon terminals of
arcuate neurons and transported retrogradely to the cell body
where it accumulates in lysosomes. Such a transport takes about
24 h [111]. Clathrin-mediated endocytosis has mainly been
regarded as receptor-mediated endocytosis. Thus, for a peptide
to be internalized by an axon terminal of the median eminence it
would require a specific receptor-mediated endocytosis. Numer-
ous neuropeptides are released at the axon terminals of the median
eminence and, although they become part of the median eminence
milieu, none of them has been shown to be re-uptaken by axon
terminals. But, even in the case a peptide is internalized by axon
terminals of the median eminence, they would enter the
endocytic/phagocytic pathway and would reach the cell body
one [111] or more days [35,65] later. It is difficult to envisage a
functional significance for such a mechanism.
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There is a small group of secretory neurons located in the lateral
subependymal region of the median eminence and that some
authors have designated as the ventro-medial region of the AN
(vmAN). The vicinity of these neurons to fenestrated portal
capillaries (see Section 5.3) has also been taken as an indication
that leptin or other peptides leaking from the portal capillaries
could directly reach such neurons [34,35]. The possibility that they
correspond to those neurons described by Faouzi et al. [47] that
express the leptin receptor and project their axons to the median
eminence, should be kept in mind. Nevertheless, recent evidence
indicates that in the AN the long form of the leptin receptor is
mostly expressed in neuropeptide Y and proopiomelanocortin
neurons that do not project to the median eminence but to
hypothalamic and extrahypothalamic areas, and whose cell bodies
are located in the ventro-lateral portion of the AN [26]; that is, an
area protected by the median eminence-AN barrier that would
prevent leptin leaking from the portal capillaries to move beyond
the median eminence milieu (see Sections 5.2 and 6.3). Leakage
would also not account for the presence of the leptin receptor in
other hypothalamic and brain stem nuclei [26], or for the
characteristics of the relation between CSF and serum levels of
leptin [9]. Leakage would neither explain peripheral resistance to
leptin; that is, the phase diet-induced obese animals go through in
which they respond to leptin given into the CNS but not into the
circulation, nor the fact that when delivered into the CSF but not
when injected into the blood, leptin inhibits appetite of sheeps
under short photoperiod [1]. The evidence indicates that the bulk
of leptin enters the AN by a saturable transport mechanism. The
non-saturable leakage from the median eminence to AN cannot
explain this phenomenon [8].
The possibility that blood vessels of the AN have a ‘‘weak’’ BBB
allowing circulating compounds to reach directly a subpopulation
of arcuate neurons has been proposed [102]. Indeed, the use of a
series of markers for BBB has revealed the presence in the AN of
blood vessels displaying a different pattern on immunoreactivities
as compared with other vessels of the AN and the median
eminence [102]. Since such vessels are not reactive with antibodies
against the endothelial barrier antigen and the transferrin
receptor, these authors have suggested an absence of an intact
BBB. An alternative explanation for these findings is that such
vessel of the AN have different barrier properties, rather than
lacking them. This is a relevant aspect worth investigating.
In what may be regarded as the most comprehensive study of
the vascularization of the medial basal hypothalamus, Duvernoy
[46] described two types of vascular connections between the
primary portal plexus and the tuberal vessels. (i) Descending
connections correspond to arterioles that penetrate the basal
hypothalamus through the tuberoinfundibular sulcus; after
passing through the ventral region of AN, these arterioles turn
downwards to reach some elements of the subependymal
network of the median eminence or the top of a long capillary
loop. (ii) Ascending connections correspond to spiral arterioles
that after branching form the upper hypophysial arteries
penetrate the pars tuberalis and the median eminence to reach
the anterior region of the medial basal hypothalamus where they
branch out. During its path through the median eminence these
arterioles have lateral branches supplying capillary loops. Worth
noticing is the fact that the vessels connecting the median
eminence and the tuberal vessels are arterioles. According to
Duvernoy [46] this vascular arrangement does not support a short
feedback mechanism.
In a recent study, Ciofi et al. [33,35] have reported the presence
of fenestrated capillaries in the vmAN. By using an antibody
against the plasmalemmal vesicle-associated protein1 (PV1), a
component of the radial fibrils occupying the capillary fenestra-
tions, they were able to distinctively identify the fenestrated
capillaries of the CVOs. At the median eminence anti-PV1 labeled
the short and long capillary loops of the primary plexus of the
portal system, the subependymal capillary network of the median
eminence and a few capillaries located in vmAN. The arcuate
nucleus is an elongated nucleus extending along the pre- and
post-infundibular regions of the median eminence. The fenes-
trated capillaries were found in the ventromedial region of the
caudalmost portion of the AN [35]. Thus, at rostral and medial
levels the vmAN is poorly or not supplied with fenestrated
capillaries. The main body of the AN is devoid of fenestrated
capillaries [35]. The anatomical distribution of the PV1 immuno-
reactive capillaries in the medial basal hypothalamus does not
seem to correspond to any of the two median eminence-tuberal
vascular connections described by Duvernoy [46]. Interestingly,
NPY and
a
MSH neurons were identified in the vmAN; only those
NPY and
a
MSH neurons located in the caudal (postinfundibular)
region of the vmAN would be irrigated by fenestrated capillaries.
However, those NPY and
a
MSH neurons located in other regions
of the vmAN and the bulk of NPY and
a
MSH neurons distributed in
the AN proper are supplied by tight capillaries [35]. What actually
is the vmAN? Does it correspond to a real subpopulation of arcuate
neurons? Where do the vmAN neurons project? Do they display
leptin receptors? Is the vmAN located on the median eminence
side of the median eminence-AN barrier? If so, the neurons of the
vmAN would correspond to those neurons located in the lateral
regions of the ME, described by early workers. At variance, if the
vmAN, especially its caudal portion irrigated by fenestrated
capillaries, is located on the hypothalamus side of the median
eminence-AN barrier, a second lateral barrier separating the
vmAN from the main body of the AN should operate, otherwise the
whole BBB would become leaky. Indeed, any compound entering
the AN milieu can readily flow into the CSF and then reach
virtually any region of the CNS. The Glut 1 reactive processes of
b
1
tanycytes bordering both lateral sides of the median eminence are
arranged into several bundles. Located among these bundles are
the most ventral neurons of the AN (Fig. 2A). Most blood
capillaries of these areas are tight and express Glut 1. It may be
suggested that some processes of
b
1
tanycytes establish a
separate compartment on either side of the median eminence
with its own milieu supplied by fenestrated capillaries and
containing a small group of neurons. This arrangement would
resemble that of the subfornical organ where the cell body and
dendrites of the neurons are located in a BBB-free zone while their
axons project to BBB-protected areas. This possibility needs to be
thoroughly investigated.
11. Conclusion
The title best summarizes this review: The design of barriers in
the hypothalamus allows the median eminence and the arcuate
nucleus to enjoy private milieus: the former opens to the portal
blood and the latter to the cerebrospinal fluid.
Acknowledgements
We are grateful to Drs. Amandine Mullier and Vincent Prevot,
Inserm, Jean-Pierre Aubert Research Center, Development and
Plasticity of the Postnatal Brain, Lille Cedex, France, for generously
providing Figures 3C and G and 9D and E. We also thank Dr.
Antonio Jime
´nez, Departamento de Biologı
´a Celular, Facutad de
Ciencias, Universidad de Ma
´laga, Spain for providing Figure 9B and
C.
This work was supported by grants from DID and MECESUP of
Universidad Austral to M.G. and grant from Fondecyt No. 1070241,
Chile, to E.M.R.
E.M. Rodrı
´guez et al. / Peptides 31 (2010) 757–776
773

Author's personal copy
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