Download INTRODUCTION Corticotrophin-releasing hormone (CRH) is a 41

NOVEL REGULATORS OF THE IN VITRO RELEASE OF HYPOTHALAMIC
CORTICOTROPHIN-RELEASING HORMONE TWO DECADES AFTER ITS DISCOVERY:
A REVIEW
Alfredo Costa, Rossella E. Nappi*, Antonella
Smeraldi, Matteo Bergamaschi, Pierluigi
Navarra**, Ashley Grossman***
IRCCS C. Mondino Institute of Neurology, Laboratory of Neuroendocrinology, “Maugeri - Mondino University of Pavia” Research Center, Pavia, University of Pavia; * Department of Obstetrics and
Gynaecology, IRCCS S. Matteo Hospital, University of Pavia, Italy; **Institute of Pharmacology,
Catholic University Medical School, Rome, Italy;
*** Department of Endocrinology, St
Bartholomew’s Hospital, London, UK.
Reprint requests to: Dr A. Costa, C. Mondino Institute of Neurology, University of Pavia, via Palestro 3, 27100 Pavia, Italy.
E-mail: [email protected]
Many environmental and occupational chemicals are
known to affect the central and/or peripheral nervous
system, causing changes that may result in neurological and psychiatric disorders. Because of the limited
accessibility of the mammalian nervous tissue, new
strategies are being developed to identify biochemical
parameters of neuronal cell function, which can be
measured in easily obtained tissues, such as blood
cells, as potential markers of the chemically-induced
alterations occurring in the nervous system. This review includes a comparative analysis of the effects of
mercurials on calcium signalling in the neuroadrenergic PC12 cells and rat splenic T lymphocytes in an attempt to characterize this second messenger system as
a potential indicator of subclinical toxicity. The suitability of neurotransmitter receptors in blood cells,
such as the sigma binding sites, as biological markers
of psychiatric disorders is also discussed.
KEY WORDS: AVP, CRH, hypothalamus, in vitro,
neuropeptides, stress.
FUNCT NEUROL 2001;16 (SUPPL.): 205-216
INTRODUCTION
Corticotrophin-releasing hormone (CRH)
is a 41 amino-acid peptide which was isolated
and characterised exactly two decades ago (1).
It is distributed heterogeneously throughout the
central nervous system (CNS) (2) and, due
both to its ability to stimulate the release of
adrenocorticotrophic hormone (ACTH) from
the pituitary and to its numerous extra-hypophyseal biological effects, is now considered
one of the major neuropeptides involved in
adaptive processes: CRH indeed coordinates
the neuroendocrine, behavioural, autonomic
and immune responses to stressors (3-5). The
recent cloning of specific CRH receptors (6-8)
has further facilitated research on CRH in
physiology and in the development of neuropsychiatric diseases, including depression,
anxiety and eating disorders. For this reason,
and thanks to the availability of specific antagonists and laboratory animal models (such as
transgenic mice), CRH is still a peptide under
active investigation 20 years after its discovery
(9).
While CRH-containing neurons are widely
distributed within the CNS, the hypothalamic
paraventricular nucleus (PVN) is the principal
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A. Costa et al.
site of origin of the parvocellular neurosecretory neurons which deliver the neuropeptide into
the hypophyseal-portal system, thereby initiating the response of the pituitary-adrenal axis.
The process of CRH release from the hypothalamus, which appears to be increasingly complex (10), is under the control of a considerable
number of mediators, each of which has a different effect. The PVN is, indeed, at the very
centre of several intra- as well as extra-hypothalamic inputs, such that a given amount of
CRH released represents the net result of a
complex interplay of modulatory (inhibitory or
stimulatory) factors.
In order to characterise these factors, various techniques have been used, but it would
appear that the in vitro hypothalamic preparations have, to date, been among the most productive model systems (11,12). Methods used
to study the rat hypothalamus in vitro differ to
a considerable extent according to a number of
factors, such as the age of the hypothalamus
(foetal or adult), the type of incubation system
(acute or chronic), and the specific perifusion
techniques used (incubation or continuous
flow). In this paper, we describe the in vitro
acute incubation system of hypothalamic explants that has been in use for several years in
our laboratories. The advantages and limitations of this system are compared with other in
vitro methods, and some of the data obtained
on the effect of various substances on CRH release are reviewed, paying particular attention
to observations on novel regulators recently
made by our groups.
ASSESSING HYPOTHALAMIC CRH RELEASE IN VITRO
Cellular processes underlying hormone secretion from the hypothalamus are relatively
difficult to study in the whole animal. On the
other hand, knowledge of the nature of the
mechanisms that regulate hypothalamic func-
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FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001
tion is indispensable for a complete understanding of endocrine homeostasis. Consequently, over the past few years, several investigators have begun to use isolated hypothalamic preparations to elucidate the physiological,
pharmacological, and biochemical characteristics of the hypophysiotropic neurons. However,
different techniques have been used by various
research groups to maintain the hypothalamus
in vitro. These techniques can be roughly divided into three major groups: synaptosomal
and other subcellular preparations, neuronal
cell cultures, and tissue cultures (including
short- and long-term maintenance of hypothalamic preparations).
Hypothalamic explants, coupled with indirect measurements (bioassays) and subsequently with direct measurements [radioimmunoassay (RIA) and immunoradiometric assay (IRMA)] of peptides, have been used over the past
30 years by several authors, and have included
both incubated and perifused hypothalami
(minced or whole) and median eminence fragments (13-16). Prior to the development of
these techniques, relatively little work was undertaken with a view to improving understanding, through the use of direct measurements, of
the regulation of hypothalamic hormone release. The system used in our laboratory was
developed by Lengyel et al. (17), based on a
technique originally devised by Jones et al.
(18), in order to: a) establish the characteristics
of peptide secretion in vitro, b) identify the nature of the immunoreactive forms released in
vitro (e.g., by means of chromatographic techniques), c) investigate the direct/indirect effects
of various classic neurotransmitters, putative
neurotransmitters and neuromodulators on hypothalamic secretion, and d) characterise the
particular receptors involved, by using specific
agonist and antagonist drugs.
The characteristics of the system have been
previously described in full detail (11). Briefly,
the freshly dissected rat hypothalamic halves are
placed in sterile polyethylene vials containing
Release of CRH in vitro
Earle's balanced salt solution (EBSS), supplemented with 0.2% human serum albumin, 60 ug
of ascorbic acid/ml to prevent oxidation, and
100 kallikrein-inactivating units (kIU) of aprotinin/ml to prevent peptide degradation by hypothalamic peptidases. Two hypothalami (four
halves) are incubated in one vial, in 500 ul of
EBSS, in order for the basal release of CRH to
be measurable by RIA. Vials are kept in a water
bath at 37°C and gassed in an atmosphere of
O2/CO2 (95%/5%), at a 1 litre/min flow rate. An
80-min preincubation period is allowed for peptide stabilisation, during which time the medium
is aspirated every 20 min and replaced with
fresh medium. After preincubation, the tissue is
usually incubated in fresh medium for a 20-min
control period, followed by another 20-min period in either medium alone (control group) or in
medium containing test substances (test group).
Viability of the tissue is always tested by incubation in the presence of 56 mM KCl at the end
of each experiment. Media collected throughout
the studies are stored at -20°C until assay.
L I M I TATIONS AND ADVA N TAGES OF
THE SYSTEM
A system that provides information regarding neuroendocrine regulation in the rat
hypothalamus should simulate the physiological situation as closely as possible. Indeed, the
biggest criticism of is that investigations are
performed on the isolated hypothalamus, which
is a structure removed from its natural environment; thus, its function in vitro does not necessarily mirror what actually occurs in vivo. Although the incubation devices (temperature,
oxygen and nutrient supplies) recreate, as far as
possible, the physiological conditions in a
CSF-like milieu, inputs from the supra- and extrahypothalamic brain areas are inevitably
lacking. Moreover, by their very nature, the
preparations are no longer subject to the diverse and complex neuronal feedback controls
that are known to operate in vivo. Although
such characteristics may be advantageous
when assessing putative modulatory substances, the true physiological relevance of the
in vitro set-up should be considered when investigating the control of neuropeptide release.
In other words, results obtained from this technique can define intrinsic biological mechanisms in a reliable manner, but only to a limited extent, and without taking into account their
systemic consequences. Combination of these
data with those from complementary in vivo
studies is clearly indispensable, and may make
it possible to relate in vivo expression to in vit ro changes. Another major point regarding this
kind of tissue incubation is the actual survival
time of the tissue in the medium. Histological
studies of oxygen consumption and maintainance of cellular integrity have shown that
incubation (or perifusion) in CSF-like media
allows the whole hypothalamus to survive for
at least 3 hours (19). The main limiting factor
in the adequate maintainance of the hypothalamus is the size, and possibly the shape, of the
tissue examined. There appears to be a gradient
of diffusion of nutrients into, and of toxic
metabolites out of, the central part of the explant. Although some investigators may continue to use the isolated hypothalamus whole,
many others have tried to circumvent this major problem by employing fragmented materials, such as quartered hypothalami, or slices of
various widths. These preparations are advantageous in terms of nutrient diffusion, but the
procedures inevitably lead to a loss of interneuronal connections. In this respect, we have
found the use of halved hypothalami more satisfactory, since the volume of tissue is actually
decreased but the neuronal projections remain
largely unaffected (17). It should be added,
however, that there is still controversy regarding data obtained from whole tissue as opposed
to those obtained from hypothalamic halves.
The bisection of the tissue, in our experience,
allows a larger surface to be exposed to the
FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001
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A. Costa et al.
medium, thus prolonging cell survival and facilitating the access of test substances to the
neuronal .nuclei close to the midline. This is
supported by the observation that the average
survival of hypothalamic explants in our system exceeds 4 hours, as indicated by the good
responses to incubation with 56 mM KCl seen
to date (11).
One of the advantages of this system is the
fact that minimal trauma to the tissue can be
ensured through the taking of extreme care in
brain removal and dissection procedures, as
well as thanks to the CSF-like fluid surrounding the preparations in the vials. Then, during
the experiments themselves, damage to the hypothalami can be avoided providing the experimenter collects and renews the incubation
medium with care. Moreover, the system guarantees the retention of a high degree of cellular
integrity and the preservation of the constituents of the tissue, such as the intrinsic neuronal networks, the supporting glia, and other
neuronal cells. Therefore, compared to other
systems, such as synaptosomes or incubated
slices, this method seems to mirror more closely the previous physiological state. Another advantage is that the technique allows precise
control of the composition of the medium, and
thus the study of the effects of various neuroactive substances. Finally, the day-to-day running
of the system is relatively inexpensive.
As mentioned above, the use of this incubation system has provided a considerable
amount of data on the mechanisms of the hypothalamic release of CRH. The effects of classical neurotransmitters (such as acetylcholine,
catecholamines, serotonin, GABA), excitatory
amino-acids, endogenous opioids, various neuropeptides, cytokines, bacterial lipopolysaccharide and gaseous transmitters – nitric oxide
(NO) and carbon monoxide (CO) – have been
extensively reviewed by ourselves and other
groups (11,12,20-24). This review will focus
on recent data on the in vitro regulation of
CRH release by two different agents: the prod-
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uct of the obese gene, leptin, and the human
immunodeficiency virus-1 (HIV-1)-related protein, gp120.
R E G U L ATION OF CRH RELEASE BY
LEPTIN
Increasing evidence suggests that energy
balance is regulated at central level by brain
circuitry controlling both energy production
(food intake) and energy expenditure (thermogenesis) (25). Peripheral factors, produced by
changes in adipose tissue mass, can signal to
the brain, informing it of the level of accumulated fat depots in order to regulate energy input and output. The obese (Ob) gene product,
leptin, is a 16kDA peptide synthesised and secreted by adipose tissue. While it appears to
have various neuroendocrine functions, being
responsible, for example, for biological effects
in reproduction, adaptive responses of the newborn, and control of GH, TRH-TSH and PRL
secretion (26), leptin is currently considered as
the main afferent signal in the regulatory mechanisms of energy homeostasis. Its effect, which
is probably generated at the level of the hypothalamus, an area particularly rich in leptin
(Ob) receptors (see 26 for review), appears to
be, predominantly, the suppression of appetite
(27), although its activation of the sympathetic
nervous system may also be important (28).
Several neuropeptides are known to be involved in eating behavior: CRH is a potent appetite-suppressant agent, acting at least in part
by inhibiting neuropeptide Y (NPY) release
(29,30), and it also increases energy expenditure (30,31). Due to its presence in several
brain areas, including the adrenergic nuclei,
CRH forms a network of neuronal pathways,
and can function in concert with another peptide, urocortin, to coordinate anorectic and
thermogenic actions and to optimise energy
losses (25).
In the light of previous studies demonstrat-
Release of CRH in vitro
ing that during the response to fasting leptin
may signal to the rat hypothalamus and thus alter the expression of genes encoding for both
NPY and CRH (32), we speculated that the primary effect of leptin in the hypothalamus is via
stimulation of CRH secretion. To evaluate this
hypothesis, we tested the effect of murine leptin on CRH release from hypothalamic explants in our in vitro system. As expected, biologically-active leptin (but not the heat-denatured peptide) was found to stimulate CRH release in a dose-dependent manner, and via a
non-adrenergic mechanism (33). These findings were thus consistent with the view that
leptin, through the reported presence of specific leptin receptors in the brain, participates in
the feedback regulation of body adiposity by
acting directly in the brain to reduce food intake. CRH appears to be one key mediator of
the central effects of leptin, a fact supported by
several other later studies. Indeed, in the mouse
leptin was found to elicit CRH release from superfused brain slice preparations containing
hypothalamus or amygdala (34), although it
was apparently unable to alter NPY or CRH secretion in another in vitro study (35). The intracerebroventricular administration of leptin in
mice was found to stimulate CRH and to reduce NPY synthesis in the PVN, and this effect
is restrained by the presence of endogenous
corticosterone (34). In the same experimental
conditions, leptin markedly increased hypothalamic CRH mRNA, but did not reduce NPY
mRNA (36), while in another study it increased
hypothalamic CRH content and inhibited food
intake at 2h in food-deprived rats (37). Consistently, central leptin administration in the rat
increased the expression of CRH mRNA and
AVP mRNA in the PVN, and plasma AV P,
ACTH and corticosterone levels (38). Furthermore, using primary cultures of rat hypothalamic cells, other authors also observed a stimulatory role of leptin on CRH secretion (39). Finally, the systemic (i.p.) administration of leptin in the rat was found to acutely increase the
expression of CRH mRNA in the PVN as well
as CRH receptor-2 (CRHR-2) mRNA in the
ventromedial hypothalamus (40).
There is still uncertainty over the site of
leptin action in the brain. A highly-specific saturable transport system by means of which the
peptide crosses the blood-brain barrier (BBB)
has been described (41), but the CNS regions
where it primarily acts are unclear. Leptin receptors are present in several hypothalamic nuclei, but apparently not in the PVN (25). This
may imply that leptin acts indirectly, other factors mediating its effect on CRH. Classical
neurotransmitters may be involved, although
we were unable to show any modulatory role
of adrenergic pathways in vitro (33). Interleukins, which are known to stimulate CRH release directly (see 11, for review), are other
possible candidates. A recent in vitro study reported that NO-synthase (NOS)- or cyclooxygenase (COX)-mediated mechanisms were not
involved, whereas leptin was able to enhance
the production of prostaglandin E 2 and F 2α,
which can themselves stimulate CRH release
(39). However, it is extremely likely that other
peptides are also involved in the central regulation of appetite, such as NPY, urocortin, orexin,
glucagon-like peptide-1, galanin, ghrelin and
melanin concentrating hormone, which may
mediate some of the effects of leptin at hypothalamic level.
It would therefore appear that leptin suppresses appetite and stimulates the hypothalamo-pituitary-adrenal (HPA) axis via CRH release, which is consistent with the idea that
high circulating leptin levels may be involved
in the HPA axis activity changes generally
characteristic of human obesity and of most
animal models of obesity (42). However, the
interactions between leptin, CRH and the
HPA axis are still poorly understood and are
likely to be complex. Since leptin and cortisol
show an inverse circadian rhythm, it has been
suggested that a regulatory feedback is present (43). Several studies have reported that
FUNCTIONAL NEUROLOGY (16) SUPPL. 4 2001
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A. Costa et al.
high-dose exogenous glucocorticoids increase
circulating leptin concentrations in humans;
conversely, leptin has been shown to exert inhibitory effects on the HPA axis responses in
some studies, both at hypothalamic and at
adrenal levels (44,45). However, there is now
general agreement on the existence of a
closed, bi-directional, circuit linking HPA axis
function and adipose tissue metabolism, with
different types of stress possibly being associated with many phenotypes of obesity (43).
As these mechanisms are still far from clear,
studies are in progress to further characterise
the biological mediators of leptin effects in
vitro.
REGULATION OF CRH RELEASE BY gp120
Another substance that we have recently
studied as a possible mediator of CRH release
is the HIV coat protein gp120, a viral component which has been associated with neuropathological changes (46). A broad spectrum
of in vitro studies has documented that gp120
causes excitotoxic, glutamate-mediated death
of neuronal cells (47). In addition, many of the
effects of gp120 appear to involve the production of NO and cytokines (48-50), as well as
activation of the inducible COX pathway (51).
These observations have led to speculation that
gp120 may be the primary pathogenic agent in
the loss of brain cortical neurons (52), accounting for the typical neurological deficits occurring in AIDS (53).
Our interest in gp120 was prompted by the
observation that patients with HIV-1 infection
exhibit deranged activity of the HPA axis (54),
which may have important pathogenetic and
therapeutic implications from the point of view
of both CNS damage and of the clinical progression of disease (55). Several studies in HIV
patients indeed showed increased plasma cortisol and ACTH levels, blunted ACTH and cortisol responses to CRH administration, and
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blunted cortisol responses to ACTH administration (56-59). Interestingly, CNS expression
of gp120 in transgenic mice has been reported
to activate the HPA axis, as indicated by increased plasma ACTH and corticosterone levels and pituitary ACTH content (60). It has also been observed that infusion of gp120 into
the rat brain increases pituitary-adrenal activity
via interleukin-1 (IL-1), and decreases peripheral cellular immune responses (48). In addition, in vitro studies on non-transgenic mouse
hypothalamic preparations have shown that
gp120 directly stimulates AVP release, suggesting that HIV-induced activation of HPA
function may be due to an increased hypothalamic drive (60).
We therefore speculated that the primary
effects of gp120 in the hypothalamus may occur via stimulation of both CRH and AVP secretion, and tested the direct effect of gp120 on
the release of CRH and AVP in our in vitro system. As previous studies had shown that NO is
involved in some of the effects of gp120, we
also considered the possible mediation by NO
pathways of any observed effect of gp120. The
addition of gp120 to hypothalamic explants indeed resulted in a clear stimulation of both
CRH and AVP release, while in the presence of
the NOS inhibitor N G-methyl-L-arginine (LNMMA) the stimulatory effect of gp120 on the
release of both peptides was completely lost
(61). These observations were found to be entirely consistent with the effects seen in the hypothalamic tissue from rats administered gp120
systemically (i.p.). Indeed, gp120 induced the
expression of Fos protein (the product of the
early gene c-Fos, an index of neuronal activation) in both the parvo- and the magnocellular
PVN, while the phenomenon was significantly
attenuated by the co-administration of L-NMMA. In addition, double immunocytochemistry
showed increased co-staining for Fos protein
and CRH or AVP in the PVN following gp120,
the number of double-labelled CRH and AVP
cells for Fos protein being markedly reduced
Release of CRH in vitro
by co-administration of L-NMMA (61). These
data, which also appear to support those of another recent study (62), lend further support to
the hypothesis that HIV-related hyperactivity
of the HPA axis is secondary to increased hypothalamic drive, and that gp120 activates the
HPA axis via both the principal ACTH secretagogues.
Studies are currently under way to elucidate the precise site of gp120 effects in the
brain, as well as the relevant peptide-receptor
interactions. Although gp120 is a large molecule, it has been found to cross the BBB by inducing adsorptive endocytosis (63). However,
alternative/additive mechanisms to explain the
effects of circulating gp120 within the hypothalamus may be that gp120 enters the CNS via
the circumventricular regions which lack the
BBB (such as the organum vasculosum of the
lamina terminalis, OVLT). In this case, the effect of gp120 in the PVN would imply the involvement of other mediators. NO pathways
are certainly involved, and this is in agreement
with previous studies reporting that gp120
stimulates NO production in glial cultures
(64,65). With regard to NO modulation of
CRH and AVP release, experimental evidence
indicates that NO plays in fact a predominant
inhibitory role in CRH and AVP secretion in
conditions of increased inflammatory-induced
secretory drive in vitro (66,67). However, recent in vivo evidence suggests that, in some
circumstances (68), NO stimulates ACTH secretion and the transcription of the genes encoding for CRH and AVP in the rat hypothalamus. Thus, NO may have a dual excitatory/inhibitory role in the control of the pituitaryadrenal axis, depending on the stressor (24).
On the other hand, gp120 is also known to
stimulate the production of cytokines (4850,64,65), some of which, particularly IL-1,
are recognized activators of the HPA axis (69)
and are able to potently stimulate CRH and
AVP release in vitro (70,71). Excitatory aminoacids may be other possible mediators of the
central effects of gp120 (60,72,73); in addition,
gp120 has been shown to stimulate the inducible isoform of COX (51), and thus the production of prostanoids, which are themselves
able to elicit CRH release (70). Finally, another
possibility which should be taken into account
is that gp120 may act, at least in part, at the
level of nerve terminals in the median eminence.
The central effects of gp120, along with
those of inflammatory agents (such as cytokines) released into the circulation by activated immune cells, may lead to excessive activation of the HPA axis. This endocrine derangement may critically influence the clinical evolution of HIV patients (55,74), since glucocorticoids can cause brain damage in particular
circumstances (75) and worsen gp120 neurotoxicity in various brain regions (76,77); they
may also potentiate the immunosuppression
characteristic of AIDS. Thus, in HIV infection,
excess of glucocorticoids induced by a central
effect of gp120 may in turn act synergistically
with gp120 to cause the variety of neuropathological and cognitive disturbances seen in the
AIDS-dementia complex (78).
CONCLUDING REMARKS
The in vitro system described above has
proved to be a valid model for assessing the activity of the isolated hypothalamus, providing
insight into the role played in CRH release by
traditional neurotransmitters as well as by
many other regulatory factors. Indeed, a
panoply of neuromodulators appear to influence this process, the main results obtained by
our groups over the last few years being summarised in Table I (see over).
The biological agents leptin and gp120
have recently joined the growing list of mediators that signal in the hypothalamus, but their
effects require further characterisation. Recently, other factors have also been reported by dif-
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A. Costa et al.
Table 1 - The role of different substances in the regulation of CRH secretion from in vitro hypothalamic explants (The receptors involved and/or the mediating factors are reported in parentheses).
Excitatory
Noradrenaline (β)
Serotonin
Acetylcholine (M)
NPY
Interleukin-1 (PGs)
Interleukin-2
Interleukin-6 (PGs)
LPS (PGs)
NO, CO
Leptin (PGs)
gp120 (NO)
Inhibitory
No effect
GABA
opioids (µ,κ)
ANP
substance P
NO, CO
MCH
EGF
NGF
melatonin
EAA
Abbreviations: GABA = gamma-aminobutyric acid; MCH =
melanin concentrating hormone; ANP = atrial natriuretic peptide; NPY = neuropeptide Y; EGF = epidermal growth factor;
NGF = nerve growth factor; LPS = bacterial lipopolysaccharide;
NO = nitric oxide; CO = carbon monoxide; EAA = excitatory
amino acids; M = muscarinic receptor; PGs = prostaglandins.
ferent groups as being involved in CRH release: some of these, such as activators of protein kinases A and C, appear to be stimulatory,
while others, such as glucocorticoids, negatively regulate CRH release from the hypothalamus as part of a feedback control loop (12).
Once novel modulators have been identified, the intracellular transduction mechanisms
and the molecular basis of their effect will have
to be characterised, and other methods, such as
cell cultures, appear to be particularly useful in
this regard. While the use of in vitro methods
may help reveal conditions of neuroendocrine
derangement underlying certain diseases, much
care must be taken in extrapolating the findings
to the in vivo context, and particularly to results
in humans. Validation at in vivo level should always be sought: there is indeed little doubt that
such techniques, when integrated with in vivo
data, can greatly expand our understanding of
the central control of the secretion of CRH and
other neuropeptides from the hypothalamus.
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