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Corticosterone modulation of stress-induced neuropeptide gene expression in the paraventricular nucleus

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Corticosterone modulation of stress-induced neuropeptide gene expression in the paraventricular nucleus

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CORTICOSTERONE MODULATION OF STRESS-INDUCED
NEUROPEPTIDE GENE EXPRESSION IN THE
PARAVENTRICULAR NUCLEUS
by
Susan Mariko Tanimura
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)
August, 1999
© 1999 Susan Mariko Tanimura
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This dissertation, written by
Susan^Mariko^Tanim ....
under the direction of h.ss. Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School in partial fulfillment of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
DISSERTATION COMMITTEE
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of the copyright owner. Further reproduction prohibited without permission.
Acknowledgements:
I would like to take this opportunity to thank my thesis advisor, Alan G. Watts, for
his guidance, enthusiasm, and patience. I would also like to thank the members of my
committee for their guidance and insight: Drs. Michel Baudry, William McClure, Michael
Stallcup, and Larry Swanson. I am sincerely grateful for the expert technical training
provided by Graciela Sanchez-Watts, and her contributions to die research presented here.
I am especially thankful for the love and support from my parents, Mitsuru and
Michi Tanimura, Thomas Majchrowski, and all my friends during my graduate career.
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Table of Contents
Chapter 1. Introduction
1
Chapter 2. Materials and Methods
14
Chapter 3. Peptide Gene Expression, Secretion, and Steroid Feedback during
Stimulation of Rat Neuroendocrine CRH Neurons
21
Chapter 4. Corticosterone Can Facilitate as Well as Inhibit CRH Gene
Expression in the Rat PVH
39
Chapter 5. Adrenalectomy Dramatically Modifies the Dynamics of the
Neuropeptide and c-fos Gene Responses to Stress in the PVH
56
Chapter 6. Discussion
72
Bibliography 89
Appendix
108
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List of Tables and Figures
Figure 2.1
Figure 3.1
Figure 3.2
Figure 3.3
Figure 3.4
Figure 3.5
Table 4.1
Figure 4.1
Figure 4.2
Figure 4.3
Comparison in percentage differences in mean (+SEM) MGL of
CRH mRNA hybridization in the PVHmpd at 5h, as a function of
exposure time in animals injected sc with 5mi 40% PEG.
A, Mean (± SEM) hematocrits and equivalent plasma volume deficit;
plasma ACTH (B) and corticosterone (C) concentrations in rats
injected sc with either 5 ml vehicle or 40% PEG. D, Individual
plasma ACTH concentrations of all animals as a function of plasma
volume deficit.
Brightfield photomicrographs of hybridization for CRH mRNA (A)
and CRH hnRNA (B) on sections counterstained with thionin
to show cell nuclei.
A, Mean (±SEM) of the number of CRH hnRNA-labeled cells in
rats injected sc with either 5ml vehicle or 40% PEG, and (B)
individual CRH hnRNA cell counts plotted as a function of plasma
volume deficit. C, Mean (±SEM) MGL of CRH mRNA hybridization
in the PVHmpd expressed in arbitrary units of animals injected sc
with either vehicle or 40% PEG.
A, Mean (+SEM) MGL of c-fos, and pENK mRNA (B)
hybridizations in the PVHmpd after sc injection of either
vehicle or 40% PEG.
Schematic summary of the temporal response observed along the
HPA axis as sustained hypovolemia progresses.
Mean (±SEM) hematocrits, plasma volume deficit, and plasma
corticosterone concentrations in intact and each corticosterone
treatment group injected sc with either 0.9% saline or 40% PEG.
A, Mean (±SEM) plasma corticosterone concentrations (ng/ml) in
each steroid replacement group and thymus weights (mg) in
intact and steroid treatment groups (B).
A, Mean (+SEM) MGL expressed in arbitrary units of CRH mRNA
hybridization in the PVHmpd of saline-injected groups and each
corticosterone treatment group. B, Mean (+SEM) changes in
the MGL of the CRH mRNA response to sc injection of 40% PEG
in intact and replacement groups.
Mean (+SEM) MGL of CRH mRNA (A), CRH hnRNA (B),
and pENK mRNA (C) hybridization signal of the PVHmpd seen
after sc injection of either vehicle or 40% PEG.
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Figure 4.4
Figure 5.1
Figure 5.2
Figure 5.3
Figure 5.4
Figure 5.5
Figure 6.1
Images from Cronex microvision x-ray film of three serial sections
hybridized for CRH mRNA, CRH hnRNA, or pENK mRNA in
the PVHmpd of representative intact and ADX animals 5h after sc
injection of either vehicle or 40% PEG.
A, Mean (±SEM) hematocrits of rats injected sc with either 5ml
vehicle or 40% PEG. B, Mean (±SEM) relative plasma volume
deficit as determined by the hematocrit in intact and ADX rats as
a function of time after sc PEG injection.
A, Mean (±SEM) number of CRH hnRNA-labeled cells in rats
injected sc with either 5ml of vehicle or 40% PEG. B, Mean
ttSEM ) MGL of CRH mRNA hybridization in the PVHmpd
expressed in arbitrary units of rats injected sc with 5ml of either
vehicle or 40% PEG.
A, Mean (±SEM) number of AVP hnRNA-labeled parvicellular
cells in the PVHmpd in rats injected sc with either vehicle or
40% PEG. B, Mean (±SEM) MGL of AVH hnRNA hybridization
in the supraoptic nucleus expressed in arbitrary units of rats
injected sc with either vehicle or 40% PEG.
A, Mean (±SEM) MGL of c-fos, or pENK mRNA (B) hybridization
in the PVHmpd expressed in arbitrary units of rats injected sc with
either vehicle or 40% PEG.
Mean (±SEM) plasma ACTH concentrations in rats injected sc
with either vehicle or 40% PEG.
Schematic diagram depicting a potential model for corticosterone
modulation of CRH gene expression in response to a sustained
stress event.
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Chapter 1
Corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular
nucleus define the central component of the hypothalamo-pituitary-adrenal (HPA) axis that
regulates the neuroendocrine stress response. At the simplest level of control, activation of
the HPA axis rapidly increases circulating levels of corticosterone which in turn negatively
regulate hypothalamo-pituitary function. Under stressed conditions, the operation of this
regulatory mechanism becomes more complex. It is likely that corticosterone interacts with
stressor-specific neural and other humoral factors to bring about the appropriate regulatory
control, of which negative feedback is but one aspect. This thesis will focus on ACTH
secretagogue gene regulation in neuroendocrine neurons located in the dorsal aspect of the
medial parvicellular subdivision of the hypothalamic paraventricular nucleus (PVHmpd) in
response to a viscerosensory stressor: sustained hypovolemia generated in response to a
subcutaneously (sc) colloid injection. The primary purpose is to investigate how
corticosterone modulates ACTH secretagogue gene expression during a sustained stress
event.
A series of three experiments were designed to investigate the overall organization of the
ACTH secretagogue gene responses to sustained hypovolemia and their modulation by
corticosterone.
1 1 What is the temporal organization of ACTH secretagogue gene expression in the
PVHmpd during sustained hypovolemia?
Data was generated every hour for the first 6h after colloid-injection given during the early
part of the light period to investigate the temporal profile of secretory and synthetic
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responses to sustained hypovolemia. Plasma concentrations of ACTH, corticosterone, and
prolactin were measured to address the secretory response. In these same animals levels of
CRH, c-fos, pENK mRNAs, and the primary transcripts of CRH and arginine vasopressin
(AVP) in the PVHmpd were measured using in situ hybridization to determine the synthetic
responses. Three questions were asked: First, do neuropeptide secretion and gene
activation share the same stimulus threshold? Second, does corticosterone modulate
mechanisms regulating CRH gene expression during sustained stress? Third, how are
neuropeptides commonly colocalized with CRH affected?
21 How do basal levels of circulating corticosterone modulate CRH gene transcription in
the PVHmpd?
To address this question, animals were left intact, or adrenalectomized and implanted with
replacement steroid pellets designed to maintain consistent levels of corticosterone. Three
doses of corticosterone were given to different groups of ADX rats: first, a low dose that
was not adequate to normalize thymus weights or CRH mRNA levels in the medial
parvicellular subdivision of the PVH (PVHmp); second, a dose that resulted in thymus
weights and CRH mRNA levels similar to those in intact animals; and lastly, a higher dose
that reduced thymus weights and CRH mRNA levels below those seen in intact animals.
Sustained hypovolemia was induced and 5h later (peak response time in the intact) rats
were killed. Two questions were asked: First, does pre-stressed CRH hnRNA expression
parallel the dose dependent suppression observed in CRH mRNA levels? Second, is a
dose-dependent CRH gene response observed in response to sustained hypovolemia?
31 In the absence of corticosterone, what is the temporal organization of ACTH
secretagogue gene expression in the PVHmpd during sustained hypovolemia?
The same indices were employed to measure synthetic and secretory response (except
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prolactin) to investigate the temporal dynamics of the hypothalamo-pituitary limb of the
HPA axis in response to sustained hypovolemia in the adrenalectomized rat. The design of
the experiment was similar to the intact time course experiment. Three specific questions
were asked: First, do CRH or AVP gene transcription occur in the neuroendocrine neurons
of the PVHmpd; if so, how is the temporal response organized? Second, what is the
expression pattern of other genes known to be colocalized in the PVHmpd? Third, if
ACTH secretion occurs, what is its temporal profile?
INTRODUCTION
The viability of an animal depends largely on its ability to maintain the dynamic internal
environment. In the late 1800s, Claude Bernard defined this principle of physiological
balance of the “ fi-xite “ of the “milieu interieur ”. Walter Cannon (1932) coined the term
homeostasis and later extended this principle to include emotional parameters (Chrousos &
Gold, 1992). If homeostasis is threatened by internal or external factors either real or
perceived, physiological and behavioral adaptive responses are evoked to counteract the
effects of the perturbation and reestablish internal parameters within preset limits. Hans
Selye (1936) adopted the word “stress” from physics to describe these types of
perturbations that act on the body. Thus, the state where homeostasis is threatened can be
defined as stress. He recognized that under stressful conditions, an animal develops
stressor-specific and general, defensive responses. The latter consisting predominantly of
the activation of the sympathetic nervous system and hypothalamo-pituitary-adrenal (HPA)
axis to bring about overall state of readiness. In fact, he postulated that brief and
controllable exposure to stressful conditions could be conceived as positive stimuli and
beneficial to health and emotion (i.e. eustress). However, it was the more severe,
uncontrollable situations where physiological and psychological distress predisposed an
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individual to disease and depression (Selye, 1946). Seyle’s hypothesis complemented
Cannon’s concept of homeostasis and played a pivotal role in directing studies regarding
the mechanisms related to stress and adaptation toward the control of the hypothalamo-
pituitary-adrenal axis.
HPA AXIS
Although there are several subdivisions within the PVH. the neuroendocrine CRH neurons
in the PVHmpd define the central component of the HPA axis. CRH is considered the
predominant secretogogue responsible for stimulating release of ACTH from corticotropes
in the anterior pituitary. Axons from these neurons terminate in the external zone of the
median eminence, where CRH is released into the hypophysial portal vasculature to induce
the release of ACTH and stimulate transcription of the POMC gene, the ACTH precursor.
ACTH released into the systemic circulation stimulates secretion and synthesis of
corticosterone from cells in the zona fasciculata of the adrenal cortex. In the unstimulated
rat, corticosterone operates as a negative feedback signal to inhibit the synthesis and
secretion of forward components of the system.
Arginine vasopressin (AVP) coexists with CRH in approximately 50% of parvicellular
CRH-positive axons and terminals in normal resting animals (Whitnall et al., 1985, 1987).
Although AVP alone is a poor ACTH secretogogue in most animals, it acts synergistically
with CRH to augment the release of ACTH in vitro (Gillies, 1982; Turkelson et al., 1982;
Vale et al., 1983) and in vivo (Rivier & Vale, 1983).
Under stressed conditions, the operation of this regulatory mechanism becomes more
complex. For example, some abrupt transient stressors increase CRH mRNA levels
(Harbuz & Lightman, 1988; Imaki et al., 1991; Darlington et al., 1992) even though
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coincidentally increasing plasma corticosterone concentrations attain levels that would
reduce its accumulation if persistently maintained in the unstimulated animal (Swanson &
Simmons, 1989; Watts & Sanchez-Watts, 1995a). This suggests that during abrupt stress
the suppression of CRH gene expression in the PVHmp seen with chronically elevated
corticosterone levels either does not occur because of the short duration of the
corticosterone surge or is inhibited by other processes.
CIRCADIAN RHYTHM
Under basal conditions, the HPA axis of virtually all mammals exhibit a diurnal rhythm that
is synchronized to the sleep/wake cycle. In the rat, a nocturnal animal, the onset of dark
corresponds with the beginning of the activity period. An anticipatory corticosterone surge
precedes lights out and peaks shortly after the dark period has begun (Krieger, 1975).
Corticosterone facilitates energy mobilization when activity is most likely to occur and may
also function to dampen the defense reactions during the activity period (Ingle, 1954,
Munck et al., 1984, Munck & Naray-Fejes, 1992). During the act of foraging, the rat is
most susceptible to predation. However, it must remain alert and capable of responding to a
possible threat, yet not overreact and become inefficient in the amount of energy and time
consumed with defense responses that are not warranted.
The diurnal increases in both ACTH and CORT concentrations are preceded by an increase
in CRH gene expression ( Watts & Swanson, 1989; Kwak et al., 1992). CRH mRNA
levels display a rhythm that is roughly the inverse of corticosterone plasma levels with
maximal CRH gene expression observed around the beginning of the light period, and
trough levels measured in the middle of the dark period. The suprachiasmatic nucleus of the
hypothalamus (SCH) provides the primary timing signal that determines the overall shape
of the circadian pattern. This influence most likely occurs transsynaptically, since a direct
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innervation to the neuroendocrine part of the PVH appears sparse (Watts et al., 1987) and
is mediated by an as yet unknown mechanism. The importance of the circadian drive is
supported by studies showing that lesions of the SCH diminish (Szafarczyk et al, 1979) or
abolish (Moore & Eichler, 1972; Stephan & Zucker, 1972; Krieger et al., 1977) the rhythm
of the HPA axis (Buijs et al., 1993). However, other factors have been shown to influence
the circadian rhythm (Dallman et al., 1987) particularly the temporal dependency on
restricted feeding schedules (Krieger, 1977; Moberg et al., 1975) and splanchnic nerve
integrity (Dijkstra et al., 1996). Additionally, comparing patterns in HPA activity between
intact and ADX rats have provided insight about the existence of adrenal dependent and
independent factors. Kwak and coworkers (1993) showed that ADX in itself, does not
produce differences in CRH mRNA levels in the evening but produced augmented levels
during the light period suggesting that CORT may act to suppress CRH gene transcription
during this time frame, but does not affect the pattern observed just prior to lights-out.
CORTICOSTERONE RECEPTORS
Corticosterone is a steroid hormone that, because of its lipophilic nature, readily crosses the
blood brain barrier and enters the neuropil. There it can easily enter the cytoplasm of all
cells in the brain. Once in the cytoplasm corticosterone can bind to its receptor and be
translocated to the nucleus where, it can either act as a ligand-activated transcription factor
(McEwen et al. 1986) to regulate gene expression, or it can interact with other transcription
factors and mediate gene transcription indirectly. There are two receptor subtypes that bind
corticosterone: a high affinity mineralocorticoid (MR or type I) and a lower affinity
glucocorticoid (GR or type II) receptor. MRs have a dissociation constant (Kd) for
corticosterone of 0.5-1 nM and exhibit binding preference for corticosterone > aldosterone
» dexamethasone (Funder et al., 1973; Reul & De Kloet, 1985). GRs have a lower
affinity for corticosterone (Kd= 5-10 nM), with binding affinity for dexamethasone >
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corticosterone » aldosterone (Funder et al., 1973; Reul & De Kloet, 1985). Because of
these different binding affinities, corticosterone receptor subtypes are differentially
occupied throughout the circadian day. Thus, at the circadian trough, when plasma
corticosterone levels are below 25nM and aldosterone levels are between 0. l-0.5nM or
lower, more than 70% of the MRs and only about 10% of the GRs in the hippocampus are
occupied. Through the course of the circadian day, between trough and peak and MR
occupancy varies between 70-90%. while GR occupancy varies between 10-90% (Reul &
de Kloet, 1985). The dissociation rate of corticosterone from receptors appears to be
biphasic with a half-life of 2.2h from GRs and 34h from MRs, in rat hippocampal cytosol
at 0°C (Sutanto et al., 1989). Furthermore, the availability of free corticosterone to bind to
either receptor subtype is largely determined by the level of its binding protein,
corticosterone binding globulin (CBG) that circulates in blood. CBG is also present in the
pituitary where it may compete for intracellular corticosterone (de Kloet et al., 1977;
Hammond, 1990). Additionally, aldosterone specificity can be conferred by the enzyme 11
B-hydroxysteroid dehydrogenase (11B-OHSD; Funder et al., 1988) which catabolizes
corticosterone into its 11-oxo metabolite, 11-dehyrocorticosterone, and binds with much
lower affinity to MRs or GRs. This enzyme is present in the rat hippocampus, cortex,
cerebellum, hypothalamus and anterior pituitary (Lakshmi et al., 1991; Moisan et al., 1992)
and is thought to confer aldosterone preference.
MRs are expressed in discrete areas with high levels found in the hippocampal formation,
dorsal septum, medial and central nuclei of the amygdala, and brainstem sensory and motor
nuclei, but are generally not detected in the hypothalamus (Arriza et al. 1988; van Eekelen
et al., 1988). High expression of GRs is found in the lateral septum, hippocampus, PVH
and ascending monoaminergic neurons of the brain stem (Fuxe et al., 1985; Reul & de
Kloet, 1985). Moderate GR levels are also found in the central nucleus of the amygdala.
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Because the hippocampus expresses the highest levels of both receptor subtypes, it has
been proposed as the primary target for corticosterone modulation (de Kloet et al., 1974;
Akana et al., 1986, 1992).
Two groups have been instrumental in identifying MR and GR mechanisms mediating
corticosterone negative feedback upon the HPA axis. Dallman and colleagues have focused
on the role that these two receptors have on plasma ACTH and corticosterone responses
both across the circadian cycle, as well as in response to stress (Akana et al., 1986;
Dallman et al., 1987; Dallman et al., 1989; Bradbury et al., 1991; Akana et al., 1992;
Bradbury et al., 1994). They have shown that corticosterone can act within three time
domains: immediate (second to minutes), intermediate (2-10 hours) or delayed (hours to
days: Keller-Wood & Dallman, 1984). On the other hand, de Kloet and coworkers have
concentrated on characterizing receptor subtype distribution and hormone binding
properties; the functional implications of MR and concurrent GR occupation on neuronal
excitability and the influence they may have on HPA activity during basal and stimulated
states (de Kloet & Reul, 1987; Ratka et al., 1989; De Kloet et al., 1993). Through the
efforts of these two groups, along with many other investigators, a basic model of steroid
receptor mediated effects on HPA axis activity has been proposed: (1) MR-dependent
actions maintain basal activity; (2) higher levels of corticosterone seen during the circadian
peak and at times of stress, act via GRs to restrain HPA activity (Dallman, et al., 1987; de
Kloet & Reul, 1987; de Kloet et al., 1993).
SUSTAINED HYPOVOLEMIA
A seemingly paradoxical relationship exists between corticosterone and CRH gene
expression during periods of stress. That CRH gene expression can increase in the
presence of high levels of corticosterone suggests that its negative feedback action is but
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one aspect of steroid-mediated effects on HPA activity. Because each homeostatic stressor
represents a unique challenge that is carried via humoral and neural afferent signals, data
have the potential to contain greater meaning when the physiological and behavioral
responses are well characterized. For this reason, sustained hypovolemia has been
employed throughout this thesis to investigate corticosterone modulation of ACTH
secretagogue gene expression in neurons of the PVHmpd. It is a homeostatic stressor that
has a physiological onset that can be identified with some precision, its intensity is
quantifiable, and it can be maintained for some hours (Strieker & Jaloweic, 1970).
Furthermore, it is an exclusively viscerosensory stimulus, at least in its early phase, and
reduces extracellular fluid volume to trigger a set of very well defined and coordinated
neuroendocrine (secretion of vasopressin, oxytocin, aldosterone, corticosterone,
angiotensin), autonomic (eg maintenance of blood pressure) and behavioral (fluid
consumption, modified sodium appetite) responses aimed at restoring fluid homeostasis
(Sticker, 1981).
Sustained hypovolemia can be induced experimentally by sc injections of the colloid
polyethylene glycol (PEG). A large molecule that cannot pass through the basilar
membrane of blood vessels, which acts as a semi-permeable membrane and through
osmosis, pulls fluid and small ions from the extracellular compartment and vessel space.
The sequestering of isotonic, protein-free extracellular fluid results in a biologically
inaccessible edema. The decreased vascular volume equilibrates with interstitial fluid so that
a general depletion of extracellular fluid results. Therefore, the higher the colloid
concentration, the higher the oncotic pressure and the larger the edema. PEG injections
increase plasma corticosterone and renin concentrations (Strieker et al., 1979) as well as
PVHmpd levels of CRH, pENK, and neurotensin mRNAs (Watts & Sanchez-Watts,
1995b). Unlike hemorrhage, PEG-induced hypovolemia is gradual and does not result in a
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loss of red blood cells which could cause anemia and other complications. Additionally,
cellular hydration remains unchanged (Strieker 1968; Fitzsimons, 1971).
If water and saline are made available after PEG-injections, animals will begin to drink
water one to two hours after injection, and conserve water and sodium by reducing
urination (Strieker, 1966, 1968; Fitzsimons, 1961) partly due to increased levels of AVP
(Dunn et al., 1973) and aldosterone (Strieker et al.. 1979). The majority of the increased
drinking associated with hypovolemia appears to be stimulated by reduced baroreceptor
output on the venous side of the circulation (Kaufman, 1984). Approximately 6h after
injection, salt appetite First becomes prominent, independent of PEG concentration (Strieker
& Jaloweic 1970; Strieker, 1981). Although the renin-angiotensin system undoubtedly
plays an important role as a vasoconstrictor on vascular smooth muscles and indirectly
stimulates peripheral sympathetic nerves and brain mechanisms that influence vasomotor
functions (Pacak, 1995), it does not appear to mediate the drinking behavior as
nephrectomized rats present the same increased water intake after PEG (Fitzsimons, 1961;
Strieker, 1973).
AFFERENTS
Neural afferents that convey stressor-specific information to the neuroendocrine PVH can
be segregated into three general classes: First, viscerosensory information is relayed to the
CRH neuron in the PVHmp by groups of neurons, which are generally catecholaminergic
(McKeller & Loewy, 1981; Sawchenko & Swanson, 1982; Cunningham & Sawchenko,
1988), and by afferents from the neurons of the vascular organ of the lamina terminalis
(OVLT) and subfornical organ (SFO) that are sensitive to the humoral status of the animal.
Second, hypothalamic nuclei most likely provide a substrate for local integration to facilitate
appropriate neuroendocrine and autonomic responses (Swanson, 1987). Third, afferents
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arising from the telencephalon that are concerned with cognitive or emotional influence
reach neuroendocrine CRH neurons generally indirectly by way of the BST. Each of these
afferent sets will be described in more detail.
As it pertains to sustained hypovolemia, at least three sets of afferent inputs can convey
relevant sensory information to both CRH neuronal cell bodies and to their terminals in the
median eminence during sustained hypovolemia. First, baroreceptors located in the right
atrium and adjacent walls of the venous return decrease output in response to reductions in
blood volume. Changes in firing rate are conveyed by way of sensory pathways in the
vagus and glossopharyngeal nerves that terminate in the nucleus of the solitary tract (NTS).
The NTS, along with other brainstem catecholaminergic afferents, relays this information
to the PVH (Sawchenko & Swanson, 1981; Cunningham & Sawchenko, 1988).
Additionally, increases in plasma concentrations of angiotensin-II (AH) access the brain
through All-receptors on neurons in the subfornical organ (SFO) which in turn projects to
the PVH. The SFO is a midline circumventricular structure that lacks a blood-brain barrier
and thus provides an entry point through which circulating peptides can gain access to the
CNS (Grossman, 1990). In particular, All increases blood pressure through direct actions
on the SFO, which are apparently independent of the peptide’s peripheral effects, as they
are abolished by SFO lesions (Mangiapane & Simpson, 1980). Anatomical tracing studies
have demonstrated that SFO neurons are retrogradely labeled by tracers injected into the
PVH. These afferents are immunoreactive for AD (Lind et al., 1985a), indicating that AH
may be utilized by these connections (Lind et al., 1985a, 1985b). This contention is
supported by demonstrations of both immunoreactive fibers and AD receptors in the PVH
(Castren & Saavedra, 1989; Gehler et al., 1986; Mendelsohn et al., l984).Recent findings
have shown that the majority of AH receptors in the parvicellular neurons of the PVH
(PVHp) to be of the ATI subtype (Gehlert et al., 1990). These anatomical findings are
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supported by electrophysiological studies demonstrating that application of exogenous All
can specifically excite neurons in the PVH (Akaiski et al., 1981; Harding & Felix, 1987;
Jhamandas et al., 1989). Taken together, these findings implicate AH as a functional
chemical messenger through which the SFO may coordinate the release of CRH and AVP.
Second, most areas in the hypothalamus, with the exception of the supraoptic nucleus
(SON), medial and lateral mammillary nuclei, and the magnocellular preoptic nucleus
(Sawchenko & Swanson, 1983) have been reported to contain at least a few cells that send
projections to the CRH region of the PVH. These projections are predominantly
GABAergic and may provide divergent information, via the medial forebrain bundle and
periventricular system. As such, hypothalamic projections to PVH may be modulated by
signals originating from the limbic regions, reticular formation and central gray.
Lastly, afferents arising from the telencephalon (Sawchenko & Swanson, 1983) along with
limbic structures are thought to subserve the integration of sensory input with cognitive and
emotional information, and reach the PVH generally by way of the bed nucleus of the stria
terminalis (BST: Swanson 1987, 1991). Specifically, the BST receives topographically
organized input from the ventral regions of the hippocampal formation (Swanson &
Cowen, 1977; Cullinan et al, 1993; Hong-Wei Dong- personal communication), the medial
parts of the amygdala (Krettek & Price, 1978) and the medial parts of the prefrontal cortex
(Swanson, 1991). In turn, these three regions each receive input from the neocortex which
suggests that the BST is in a position to integrate and relay cognitive influences to the PVH
(Swanson 1987, 1991). Interestingly, the projection from the BST appears to be
predominantly GABAergic (Cullinan et al., 1993) and thus, has been postulated as a route
through which hippocampal activity exerts inhibitory influence on the PVH in the resting
and stressed states (Herman & Cullinan, 1997).
12
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THESIS OBJECTIVE
The data from this thesis provide insight into the modulatory role that corticosterone exerts
on ACTH secretagogue gene expression in the PVHmpd during a sustained stress event. In
brief, the results demonstrate that sustained hypovolemia is accompanied by a specific,
sequential and coordinate response in the intact rat, with ACTH secretion possessing a
lower activation threshold than CRH gene activation. Additionally, elevated levels of
corticosterone do not appear to act rapidly to inhibit CRH gene transcription. When the
antecedent corticosterone environment is manipulated, low levels of corticosterone appear
to facilitate CRH gene transcription in response to sustained hypovolemia. In the absence
of corticosterone, CRH gene transcription and expression are compromised. These data
suggest that low levels of corticosterone facilitate and direct the CRH gene response to
sustained hypovolemia. In conclusion, a model is proposed whereby CRH gene
transcription may be sustained in the presence of elevated corticosterone levels.
13
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Chapter 2
MATERIALS AND METHODS
The following methods were used in the experiments described in this thesis. Specific
adaptations to experiments are included in each experimental chapter, where pertinent.
Recipes and protocols are described in Appendix 2
Animals: All experiments were performed on adult male Sprague-Dawley rats (225-250g
body weight at the beginning of the experiments), housed 3 per cage and maintained on a
12 h light: 12 h dark photoperiod (lights on at 06:00 h). Animals were supplied with
unlimited water and rat chow and allowed at least 5 days to acclimatize to animal quarters.
Subcutaneous in jection: Under brief halothane anesthesia (2-bromo-2-chloro 1,1,1,-
trifluoroethane, Halocarbon Laboratories, N. Augusta, SC), 5 ml subcutaneous injections
of either 40% (w/v) PEG (MW 8000; Sigma, St. Louis MO) dissolved in 0.9% saline or
0.9% saline at room temperature were performed between 07:00 - 08:00. Each injection
took less than 3 minutes from the onset of anesthesia to recovery of consciousness. At this
time, all water bottles and food were removed from the cages and animals were left
undisturbed until sacrifice.
Perfusion and tissue handling: Perfused rats were deeply anesthetized by intraperitoneal
injection of tribromoethanol (Aldrick, Milwaukee, WI) and a single 1 -1.5 ml blood
sample taken from the external jugular vein into a heparinized syringe for hematocrit
14
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measurement and determination of plasma corticosterone concentrations. Animals were
then perfused through the ascending aorta with a brief saline rinse, at which time the
thymus was removed, dissected free of adjoining tissue and fluid, and weighed. The saline
rinse was followed by 500 ml of ice-cold 4% paraformaldehyde solution in 0.1 M borate
buffer pH 9.5. After perfusion the brain from each animal was removed and post-fixed
overnight in the fixative containing 12% sucrose (w/v).
Decapitation: Rats were rapidly decapitated with trunk blood collected in two cooled vials,
coated with either EDTA-saline for ACTH assay, or heparin-saline for corticosterone and
prolactin assay. Hematocrits were measured and plasma separated and stored at -20°C until
sectioning at a later date. Plasma volume deficit was derived from hematocrit
((hemactocritpEG -hernatocritm ean control) /(hematocritpEG)) X 100. Brains were rapidly
removed and fixed by immersion in ice-cold 4% paraformaldehyde in 0.1M borate buffer,
pH 9.5, overnight. Sucrose was added to the 4% paraformaldehyde solution to attain a
12% sucrose concentration and then fixed for two additional days.
Tissue Handling: Brains were frozen in powdered dry ice and immediately stored at -70°C
until sectioning at a later date. Eight series of one-in-eight fifteen (im coronal sections were
cut through the rostral hypothalamus and saved in ice-cold KPBS buffered saline
containing 0.25% paraformaldehyde. The sections were mounted the same day on poly-L-
lysine coated gelatin-subbed slides, vacuum desiccated overnight, post-fixed in KPBS-4%
paraformaldehyde for 1 h at room temperature, rinsed for 5 X 5 min in clean KPBS, air
dried, and then stored at -70°C in air-tight containers containing silica-gel desiccant for
hybridization at a later date. Serial sections were saved for thionin staining.
*
15
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In Situ Hvbridi?ation: Serial sections through the rostral hypothalamus were each
hybridized with 35S-UTP-labelled complementary RNA (cRNA) probes transcribed using
the Promega Gemini kit (Promega Inc., Madison WI), with appropriate RNA polymerases,
from either a 700bp cDNA sequence coding for part of the mRNA encoding prepro-CRH,
a 935bp cDNA sequence for the entire coding sequence of pre-proenkephalin, a 536 bp
PvuH fragment complementary to the sequence within the single CRH intron, a
complement to a 2.1 kb region of the cDNA sequence coding for c-fos, or a complementary
sequence to the 700 bp Pvu II fragment of intron 1 of the AVP gene. Control hybridization
experiments for each probe have been previously described (Watts & Sanchez-Watts,
1995b; Herman et al., 1992). In situ hybridization with the 35S-labeled cRNA probes was
performed as described elsewhere (Watts & Sanchez-Watts, 1995b) with post
hybridization modifications for CRH hnRNA as follows. After the RNase incubation and
room temperature washes from 4X to 0. IX SSC, slides were incubated at 70°C for 30 min
with slight agitation every 10 min. Controls for all 3 in situ hybridization probes consisted
of incubating sections with cRNAs synthesized from cDNA sense strands, or the
incubating sections pre-treated with RNAse and then hybridizing with antisense generated
probes. In all cases no hybridization signal was seen. Sections were exposed to Cronex
Microvision X-ray film (Dupont, Wilmington, DE) for appropriate exposure periods (4-21
days), then dipped in nuclear track emulsion (Kodak NTB-2, diluted 1:1 with distilled
water), exposed for 5-25 days, developed and counterstained with thionin.
Thionin Staining: Slides with mounted sections were dried overnight and processed
through increasing ethanol concentrations (50%, 70%, 95%X2, 100%X3) for a minimum
of I minute each to dehydrate tissue. Slides were then soaked in xylene for at least 30
minutes to defat tissue. Slides were then rehydrated using the same ethanol concentrations,
16
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placed in distilled water and dipped in thionin for approximately 20-30 seconds. Slides
were rinsed in distilled water, briefly exposed to distilled water containing two drops of
concentrated HC1, rinsed briefly in distilled water and then placed through dehydration
process again. Slides were then placed in xylene for a minimum of 30 minutes then
coverslipped using DPX (Fluka). Slides that were dipped in emulsion were stained using
the same method, without exposure to acidic water.
Radioimmunoassay: Plasma corticosterone, prolactin, and ACTH were measured in
duplicate unextracted samples by double-antibody radioimmunoassays. Plasma ACTH
concentrations for chapter 2 and 3 were determined using an 1 2 5 I-ACTH double antibody
RLA as previously described (Fink, et al. 1988). The lower sensitivity was 20 pg/ml and
the intra-assay coefficient of variation was 1.8%. Plasma ACTH was determined using
l25I-ACTH double antibody RIA supplied in kit from (ICN Biochemicals Inc. Costa Mesa,
CA) with a lower sensitivity limit of 15 pg/ml and intra-assay coefficient <4.1%. Plasma
corticosterone concentrations were determined as described elsewhere (Watts & Sanchez-
Watts, 1995a) using an I25I-corticosterone double-antibody RIA supplied in kit form (ICN
Biochemicals Inc. Costa Mesa, CA). The lower sensitivity limit was 15 ng/ml and the intra
assay coefficient of variation was <8%. Plasma prolactin concentrations were determined
by double-antibody RIA as previously described (Watts et al., 1989). PRL RP-3
(NIAMDD) was used as the reference standard. The lower sensitivity limit was 1.0 ng/ml
and the intra-assay coefficient of variation was 6.1%. All samples were measured in single
assays.
17
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Semi-Quantitation o f35S-UTP-cRNA Hybridization: Mean gray levels (MGL) of the
hybridization signal in the Nissl-defined PVHmpd were measured from images on Cronex
microvision X-ray film as described by Watts & Sanchez-Watts (1995b). The response
linearity of the image analysis system used for measuring MGL of film images from in situ
hybridization was confirmed using a series of l4 C-microscales (Amersham International;
Watts & Swanson, 1989). The response of the film and camera system over the
signal range used in this experiment was linear (R2 =0.9946, F=464, pcO.OOOl)
Intronic hybridization that required cell counting in the PVHmpd were performed using the
method of Kovacs and Sawchenko (1995). The total number of nuclei with numbers of
silver grains 5 times greater than background was counted in three 1:8 sections centered on
the PVHmpd using the adjacent Nissl-stained and CRH-hybridized sections as reference.
AVP hnRNA positive cells were determined using the methods of Herman (1995) whereby
positively labelled cells were counted in the same sections used for CRH hnRNA analysis
and identified as parvicellular neurons by the characteristically small darkly stained nucleus.
Cells were disqualified if they exhibited a magnocellular nuclear profile of a large, lightly
stained nucleus.
A preliminary experiment was performed to determine the best film exposure time frame for
maximal signal differences between treatment groups (Fig 2.1). Sections from intact and
ADX rats were hybridized for CRH mRNA levels and exposed to Cronex microvision X-
ray film for different time periods (ie longer exposure times produce brighter signal) and
semi-quantitatively analyzed. The data demonstrate two points. First, the percent change
reported in in situ hybridization studies represents the signal measured on the film rather
than the absolute mRNA values, that is, at least in part, dependent upon film exposure.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Second, the shorter the exposure period, results in larger percentage differentces between
treatment groups because the signal detected remains within the linear range of the response
curve, avoiding saturation of the film and therefore, reduced sensitivity.
Statistical analysis: The significance of differences between dependent variables across
treatment groups were determined using multifactorial ANOVA, followed by two-tailed
post-hoc tests with intact values taken as control. P<0.05 was regarded as being
statistically significant for all tests. The significance of differences in dependent variables
between saline-injected and PEG-treated animals within each steroid treatment group were
determined using Student’s t test assuming unequal variances. All statistical analyses were
performed using Excel (Mac version 4.0; Microsoft) and Systat (Mac version 5.2).
19
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c
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FIG 2.1 Comparison of percentage increase in mean (+SEM) MGL of CRH mRNA
hybridization in the PVHmpd of ADX compared to intact animals at S h, as a
function of exposure time. Note that shorter exposure times yield greater percent
differences.
20
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Chapter 3
INTRODUCTION
Stimulus/response coupling in CRH neuroendocrine neurons during stress involves at least
four processes: stimulus onset and activation of signal transduction: initiation of
secretogogue (neuropeptide) release; activation of neuropeptide gene expression; and
feedback regulation. The temporal organization and functional interactions between these
components are currently poorly defined in CRH neuroendocrine neurons. To help clarify
how these elements interact during a sustained stress event, in a single set of animals, the
temporal profiles were measured for a number of secretory and synthetic processes
associated with the CRH neuroendocrine neuron during sustained hypovolemia.
Examining the structure of these profiles allows us to address the following questions.
First, how are peptidergic gene activation and secretory events related in the CRH neuron?
Second, does corticosterone act as an inhibitory signal for CRH gene expression in the
PVHmpd during periods of sustained secretion? And finally, what are the concurrent
effects of this stressor on neuropeptide genes colocalized with CRH?
Indices of secretory and synthetic responses to sustained hypovolemia were generated
every hour for the first six hours after PEG injections given during the early part of the
light period. Plasma concentrations of ACTH, corticosterone, and prolactin were measured
to address the secretory response. In these same animals, levels of CRH, c-fos, pENK
mRNAs, and the primary transcripts of CRH and A VP in the PVHmpd were measured
using in situ hybridization to determine the synthetic responses.
21
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MATERIALS and METHODS
Animals were housed and maintained as stated in the Material and Methods (Chapter 2).
Six animals were assigned for each treatment group at every time point with the exception
of the Oh and 2h-PEG-treated groups where 12 animals were used.
Decapitation and tissue handling: Animals were killed by decapitation at assigned time
points between 08:00-13:00.
Radioimmunoassay and Quantitation o f35S-UTP-cRNA Hybridization. Performed as
stated in the Material and Methods (Chapter 2). Most of the data in this thesis are derived
from in situ hybridization, where radioactively-labeled probes are used to estimate relative
levels of mRNA and hnRNAs. Consequently, it is worth noting the technical limitations. It
is important to consider that in situ hybridization is semi-quantitative and therefore only an
approximation of hnRNA and mRNA expression. Additionally, because the CRH mRNA
pool is relatively large, small changes in CRH mRNA signal may go undetected. Other
factors may also contribute to the changes in signal measured such as post-transcriptional
processing and stability of mRNA (Murphy & Carter, 1990; Herman et al., l991).The use
of intron-based in situ hybridization is advantageous since there is a high level of cellular
resolution that allows for estimating gene transcription in individual cells (Herman et al.,
1992a; Kovacs & Sawchenko, 1996a). Furthermore, because the intron is spliced out of
the nascent mRNA molecule soon after synthesis, has a relatively short half-life, and its
abundance is primarily dependent upon transcription, it can be used as a better indication of
gene transcription. It is worth noting that the timing of hnRNA signal generation correlates
well with what has been reported of the rate of transcription estimated from nuclear run-on
22
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assays (Fitzsimmons et al., 1992). Lastly, in situ hybridization only provides a ‘snap shot’
of a dynamic situation and as such, cannot be used to determine kinetic events. With these
factors in mind, the majority of data presented here were performed within a time course to
offer more insight into the dynamics of the CRH gene response to sustained hypovolemia.
A preliminary experiment was performed to verify the sensitivity of the CRH hnRNA
hybridization analysis. Six animals were placed under ether anesthesia for five minutes
which has been reported to increase significantly CRH hnRNA levels in the PVH (Kovacs
& Sawchenko, 1996a). Rats were placed singly in a glass chamber saturated with ether
vapor. Once anesthetized, animals were removed from the chamber and exposed to ether
soaked cotton wool applied to the nose as necessary. Animals were maintained under ether
anesthesia for five minutes and then perfused. Four control rats were injected i.p. with
tribromoethanol and immediately perfused. Sections were processed for CRH hnRNA in
situ hybridization as described above.
Statistical analysis. The significance of differences in dependent variables between
euhydrated and PEG-treated animals across the experiment were determined using single
factor ANOVA, followed by Dunnett’s two-tailed post-hoc test (with Oh values taken as
control) or Tukey post-hoc test. The significance of differences between saline and PEG
injected animals at individual times was determined using Student’s ‘t’ test. P < 0.05
regarded as being statistically significant for all tests. All statistical analyses were
performed using Excel (Macintosh version 4.0; Microsoft) and Systat (Macintosh version
5.2).
23
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RESULTS
Hematocrit
Figure 3.1A shows that a significant increase in hematocrit first occurred one hour after
PEG injection (P < 0.05) when compared to saline-injected animals, and was maintained in
all PEG-injected animals for the duration of the experiment (P < 0.002 - 0.0001). The
hematocrit did not significantly increase at any time in saline-injected controls.
S ecreto ry R esponse
Plasma ACTH
Figure 3. IB shows that a significant increase in plasma ACTH concentration occurred two
hours after PEG injection (P <0.01) compared to saline-injected animals at Oh. Elevated
levels were maintained in all PEG-injected animals for the duration of the experiment
(P < 0.05 - 0.001). Plasma ACTH concentrations did not significantly increase at any time
in saline-injected controls. At 6h plasma ACTH concentrations remained significantly
increased (P < 0.001) in hypovolemic rats. However, they tended to decrease when
compared to values at 5h, although this was not statistically significant.
Plasma Corticosterone and Prolactin
Plasma corticosterone concentrations increased in parallel with ACTH levels in
hypovolemic rats (Fig. 3.1C) and were significantly increased above saline-injected
animals at all time points from 2h onwards (P <0.01 - 0.0001). A stimulus-associated
plasma prolactin response did not occur at any time in hypovolemic rats. Plasma prolactin
concentrations did not significantly increase above saline-injected rats; the mean range for
all groups during the entire experiment was between 1.9 ± 0.2 and 5.6 ± 1.7ng/ml.
24
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FIG 3.1. A, Mean (+ SEM) hematocrits and equivalent plasma volume deficit; plasma ACTH (B) and
corticosterone (C) concentrations in rats injected sc with either 5 ml vehicle (0.9% saline; open circles)
or 40% PEG (closed circles). See text for levels of significance. D, Individual plasma ACTH
concentration of all animals as a function of plasma volume deficit. The vertical dashed line depicts the
plasma volume deficit threshold necessary to elicit an ACTH response
25
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Plasma Volume Deficit and ACTH Response
Figure 3. ID shows that significantly elevated ACTH secretion only occurred when a
plasma deficit threshold of about 12% was exceeded. Figures 3.1A and 3. IB shows that
this threshold was only crossed in PEG-injected animals sometime between lh and 2h
when significant increases in plasma ACTH were first measured (P < 0.01).
Synthetic Response
CRH hnRNA
In the preliminary experiment using 5 minutes of ether exposure, a robust increase in the
number of CRH hnRNA labelled cells ( 138.0±9.0) in the PVHmpd was measured
compared to non-exposed controls. The signal from the CRH mRNA hybridization was
localized over the cytoplasm of neurons (Fig. 3.2A), while that for CRH hnRNA was
localized over the neuronal nucleus (Fig.3.2B). The first significant increase in the number
of CRH hnRNA positive cells was seen 3h after PEG injection (Fig. 3.3A; P < 0.01). The
number of labeled cells in PEG-injected animals then remained significantly elevated above
controls until the end of the experiment (P < 0.05 - 0.0001).
Plasma Volume Deficit and CRH Gene Transcription
CRH gene transcription had a higher response threshold to hypovolemic stress (Fig. 3.3B)
compared to the response of ACTH secretion (Fig. 3.2D); CRH hnRNA levels did not
increase until the plasma volume deficit was above approximately 20%. This occurred
between 2 and 3h after PEG injection (Fig.3.3A).
26
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FIG 3.2 Brightfield photomicrographs of hybridization for CRH mRNA (A) and CRH hnRNA (B) on
sections counterstained with thionin to show cell nuclei. Note the cytoplasmic labeling for the mRNA
and the nuclear labeling for the hnRNA (scale bar = 5 micrometers)
27
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CRH mRNA
The content of CRH mRNA in the PVHmpd of saline-injected animals (Fig. 3.3C)
gradually declined from Oh until 5h so that values at 3h (P <0.01) and 5h (P < 0.001) were
significantly lower than at Oh. This decline was prevented in PEG-injected animals; CRH
mRNA levels were significantly greater at 3h (P < 0.01), 4h (P < 0.001), and 5h
(P < 0.001) after injection than saline-injected animals at 3 and 5h. However, between 5h
and 6h, CRH mRNA content was sharply reduced in PEG-injected animals to levels that
were no longer significantly different from those seen 5h after injection with saline.
AVP hnRNA
Levels of AVP hnRNA remained very low in all saline-injected animals. At no time were
these significantly increased in any animals injected with PEG compared to animals injected
with saline (positively labelled cells in the PVHmpd at 5h: saline, 4.0 ± 1.0; PEG. 3.0 ±
1.0)
c-fos
Figure 3.4A shows that levels of c-fos mRNA in PVHmpd were very low at Oh but
significantly increased in both saline (P < 0.0002) and PEG-treated (P < 0.005) groups one
hour after injection, most likely because of the brief halothane anesthesia and surgery used
for the injections. At 2h, c-fos mRNA had returned to levels not significantly different from
Oh controls in both treatment groups. The first significant increase in c-fos mRNA in the
PVHmpd compared to saline-injected controls was measured at 3h in hypovolemic rats
(P < 0.05). Levels continued to increase from 3h onwards, remaining elevated in all PEG-
injected animals for the duration of the experiment (P < 0.005 - 0.0001). Although mean
28
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CRH hnRNA
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(0.9% saline; open circles) or 40% PEG (closed circles) as a function of time after injections, and (B) individual CRH
hnRNA cell counts plotted as a function of plasma volume deficit. The verticle dashed line signifies the plasma volume
deficit threshold necessary to induce CRH gene transcription (CRH hnRNA). C, Mean (*SEM) MGL of CRH mRNA
hybridization in the PVHmpd expressed in arbitrary units of animals injected sc with S ml of either vehicle (0.9%
saline; open circles) or 40% PEG (closed circles) as a function of time after injection. See text for level of significance.
29
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levels in PEG-injected animals were lower at 6h compared to those at 5h, this was not
statistically significant.
pEN K mRNA
Figure 3.4B shows that the pENK mRNA response in the PVHmpd to hypovolemia was
delayed when compared to those of CRH or c-fos mRNAs. Significant increases of pENK
mRNA in hypovolemic rats were first detectable after 4 h when compared to animals Oh
(P < 0.005) and 3h saline-injected controls (P <0.01. These elevated levels were
maintained until the end of the experiment (5h, P < 0.0005; 6h, P < 0.0005 vs saline-
treated group at 5h).
30
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c-fos mRNA
B
Time after Injection (hours)
pENK mRNA
45
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scinjections of either vehicle (0.9% saline; open circles) or 40% PEG (closed circles) expressed in
arbitrary units. See text for level of significance.
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DISCUSSION
Our results show that sustained hypovolemia evoked by sc PEG injections is accompanied
by a specific, sequential, and coordinate response from the motor elements o f the HPA axis
(Fig. 3.5). The physiological structure of this response combined with our ability to
measure the levels of neuropeptide primary transcripts, mRNAs, and plasma hormone
concentrations in the same animals allows us to address three central issues concerning the
dynamics of secretion, gene expression, and feedback regulation in neuroendocrine motor
neurons. First, the relationship between the onsets of secretion and gene activation.
Second, the effects of corticosterone feedback. And finally, concurrent effects of the
stressor on neuropeptides colocalized with CRH.
However, before discussing the implications of our findings it is worthwhile considering
two points relating to the character of the stressor used. First, sustained hypovolemia
differs from ether anesthesia, restraint, or immobilization in that it is not accompanied by
increased prolactin secretion (Minamitani et al. 1987; Gray et al. 1993; Lasen & Mau,
1994). This observation shows that sustained hypovolemia activates a different set of
afferents to the neuroendocrine PVH than these other stressors. Second, sustained
hypovolemia is a hemodynamic stressor that in terms of sensory signalling, has some
similarities to hypotension and hemorrhage. In this context three sets of studies in the rat
suggest that CRH rather than AVP is the major ACTH secretogogue associated with these
other hemodynamic stressors. First, ACTH secretion after hemorrhage is abolished by
lesions of the PVH (Darlington et al., 1988) or by immunoneutralization o f CRH (Plotsky
et al., 1985). Second, hemorrhage or nitroprusside-induced hypotension induce CRH
release into hypophysial portal blood, whereas there is no detectable rise in AVP
concentrations after nitroprusside-induced hypotension (Plotsky & Vale, 1984; Plotsky et
32
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Expt. Time (h)
1
- 1 0 1 2 3 4 5 6
Clack Time 06.00h 07.00 08.00 09.00 10.00 11.00
l
12.00 13.00
PHYSIOLOGICAL Hematocrit
ACTH
Corticosterone
c-fos mRNA
CRH hnRNA
CRH mRNA
pENK mRNA
AVP hnRNA
GENE ACTIVATION
FIG 3.5. Schematic summary of the temporal response observed along the HPA axis as sustained
hypovolemia progresses. The light grey bar along the c-fos mRNA response sequence denotes the
increase in mRNA levels observed in both saline- and PEG-injected animals and is attributed to the
nonspecific stress of anesthesia and sc injections. Based upon the indexes measured, ACTH, and
CORT secretion (open bars) precede CRH gene transcription (CRH hnRNA) and increases in c-fos,
CRH, and pENK mRNA levels (black bars), with no AVP gene response (AVP hnRNA) measured
(see text for details).
33
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al., 1986). Finally, hemorrhage or sustained hypovolemia increase CRH, pENK, and
neurotensin, but not AVP mRNAs in the PVHmpd (Watts & Sanchez-Watts, 1995b;
Darlington et al., 1992).
The relationship between the onset o f secretion and the onset o f gene activation.
In models where the stressor can be quantified, a relatively simple relationship exists
between the intensity of stress and secretory response of neuroendocrine motor neurons. In
particular, a tight correlation has been shown between hemorrhage and ACTH secretion
(Gann et al. 1978; Darlington et al., 1986), and for oxytocin and vasopressin secretion to
sustained hypovolemia (Dunn et al., 1973; Strieker & Verbalis, 1986). Our data now show
a similar stimulus-secretion correlation between sustained hypovolemia and ACTH
secretion. Furthermore, like other sensori-motor interactions, significant ACTH secretion
only occurs once a physiologically-identifiable threshold is crossed: in this experiment, a
12% plasma volume deficit, which occurs between 1 and 2h after injection.
Identifying the onset of ACTH secretion is currently the only way to relate the onset of
secretogogue release with those processes regulating CRH synthesis. This is because
methods that provide direct estimates of ACTH secretogogue release into hypophysial
portal blood depend upon prolonged anesthesia and surgery (eg. Fink et al., 1988; Plotsky
& Vale, 1984), which would undoubtedly activate genes non-specifically in the PVH.
Considering this caveat, establishing that the onset of stimulus-dependent CRH release
occurs no later than 2h after PEG injection allows us to relate the timing of this event to
others occurring in CRH neurons.
34
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Two hours after PEG injection the data show that although the CRH neuron is actively
releasing secretogogue into hypophysial portal blood (as indicated by elevated plasma
ACTH concentrations) there is no measurable increase in either the CRH or AVP primary
transcripts in the PVHmpd. Increased transcription of the CRH gene above that of controls,
was first seen at least I hour after significant increases in ACTH secretion had occurred. In
this context, it is important to consider three points. First, our results cannot be explained
by the presence of a fixed lag period between the onset of gene activation and the
production of the primary transcript, as would be required for new protein synthesis; CRH
hnRNA levels increase as rapidly as 5 minutes of stimulus onset (Herman et al. 1992,
Kovacs & Sawchenko, 1996a; Imaki et al. 1995). Second, significantly elevated levels of
CRH hnRNA can still be measured up to one hour after a transient stressor (Kovacs &
Sawchenko, 1996a) indicating that the half-life of the hnRNA is long enough to detect
significant increases at each of our time points. Finally, preliminary experiments show that
our assay system was sufficiently sensitive to detect increases in CRH hnRNA 5 mins after
ether anesthesia demonstrating that a significant period of activated transcription is not
missing. Therefore, these data show that stimulus-induced CRH release and subsequent
gene activation possess distinct and separate thresholds suggesting some degree of
mechanistic dissociation.
The accumulation rate of CRH mRNA in the PVHmpd of unstimulated animals is not
constant over a 24h period (Watts & Swanson, 1989; Kwak et al., 1992), and any
perturbation to the HPA axis is superimposed upon this circadian pattern (Dallman et al.
1987). Thus, a decline in CRH mRNA levels, consistent with the circadian pattern of CRH
mRNA accumulation (Watts & Swanson, 1989; Cai & Wise, 1996), was seen in our
saline-injected animals. However, when the sustained stressor was presented, this decline
35
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was abolished so that CRH mRNA levels were significantly higher than equivalently timed
controls 3,4, and 5h after injection. Taken together with the CRH hnRNA data from the
same animals, the prevention of this decline can be accounted for, at least in part, by
steadily increased CRH gene transcription.
The effects o f corticosterone feedback.
At least three sets of afferent inputs convey sensory information to CRH neuronal cell
bodies and terminals at the median eminence during sustained hypovolemia. First,
decreased output from low-pressure baroreceptor signals reductions in blood volume
through the vagus and glossopharyngeal nerves to the nucleus of the solitary tract (NTS).
The NTS, along with other brainstem catecholaminergic afferents, sends this information
rostrally to the PVH (Sawchenko & Swanson, 1981; Cunningham & Sawchenko, 1988).
Second, increased plasma concentrations of angiotensin-II (AH) access the brain through
All-receptors in the subfornical organ SFO which in turn projects to the PVH.
The third afferent signal important for regulating the size of the CRH mRNA pool is
circulating corticosterone (Swanson & Simmons, 1989; Watts -Sanchez-Watts, 1995a).
Keller-Wood & Dallman (1984) proposed that the negative feedback action of
corticosterone acts on ACTH release in three time domains— rapid, delayed, and
slow—and it is valuable to utilize this same concept when considering how corticosterone
regulates the CRH gene. Here the data show that levels of CRH hnRNA, mRNA, and
plasma ACTH all remain elevated above control values for at least 3 hours despite sustained
corticosterone secretion. This suggests that negative feedback actions in the rapid and
perhaps the early delayed phase either do not operate or are overridden. This conclusion is
36
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consistent with data presented in Chapter 4 where the size of the CRH mRNA response to
PEG at 5h in intact animals, able to mount a robust corticosterone secretory response, was
not different from adrenalectomized animals given a low-dose corticosterone replacement
(sufficient in normalize thymus weights) but incapable of secreting corticosterone.
The data presented here show that increasing plasma corticosterone concentrations do not
significantly inhibit ACTH secretion for at least 5h after adrenocortical activation has
occurred and are intriguingly similar to those of Keller-Wood & Dallman (1984). These
workers pointed out that there were two types of stressor with respect to how
corticosterone rapid feedback impacts ACTH secretion: corticosterone-sensitive (eg. ether
anesthesia) and corticosterone-insensitive (eg. hemorrhage). Our results now show that a
similar difference may also occur regarding corticosterone feedback action on CRH gene
regulation, and make the critical point that feedback mechanisms operating during one
stressor may not act on CRH gene expression in the same manner as another.
The rather abrupt and significant reduction in CRH mRNA seen between 5 and 6h is
striking and may reflect a corticosterone feedback inhibitory component acting in the
delayed time domain, particularly since it occurred at a time when ACTH secretion was also
beginning to decline. This decline in mRNA seems unlikely to have resulted from non
specific influences on CRH neurons because pENK and c-fos mRNAs— mRNAs
previously shown to be colocalized with CRH in these circumstances (Watts & Sanchez-
Watts, 1995b; Kovacs & Sawchenko, 1996a)— as well as CRH hnRNA were all still
significantly elevated at this time. One explanation for the reduction in CRH mRNA is that
corticosterone is interacting with mechanisms responsible for processing of cytoplasmic
mRNAs. Thus, at this point of the stress event, corticosterone begins either to inhibit
37
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afferent signalling to CRH neurons (perhaps as reflected in the downward trend in CRH
hnRNA and c-fos mRNA levels seen at this time), compromise CRH mRNA stability, or
both. Iredale & Duman (1997) recently showed that corticosterone reduced CRH-R1
receptor mRNA stability by 50% in pituitary derived AtT-20 cells by a process dependent
upon de novo protein synthesis; a similar mechanism may operate as the stress event
progresses.
Concurrent effects o f the stressor on neuropeptides colocalized with CRH.
That AVP gene transcription did not increase at any time during the response to sustained
hypovolemia confirm previous results with colocalized neuropeptide mRNAs (Watts &
Sanchez-Watts, 1995b). They are consistent with the notion that, like in other
hemodynamic stressors, AVP does not play a role in activating the HPA axis in this model.
Interestingly, elevated levels of pENK mRNA were not seen until lh after the CRH gene
had been activated. This suggests that there is significant divergence in the signal
transduction mechanisms regulating pENK, CRH, and AVP genes. Those responsible for
controlling pENK mRNA may require a higher stimulus threshold or a longer synthesis
period than the CRH gene, as is the case for the AVP gene (Kovacs & Sawchenko,
1996a). However, it should be noted that levels of pENK mRNA are measured and not the
primary transcript, and it is possible that activation of both CRH and pENK do occur
simultaneously but, at this time of day, significant time is required for measurable amounts
of pENK to become detectable. Taken together these data are consistent with the notion that
distinct cellular signalling pathways control the AVP, pENK, and CRH genes during
stress.
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Chapter 4
INTRODUCTION
Despite the general appreciation for the structure of glucocorticoid feedback, the cellular
mechanisms involved with corticosterone’s actions on CRH gene expression are unclear.
For example, some abrupt transient stressors increase CRH mRNA levels (eg. Harbuz &
Lightman, 1988; Imaki et al., 1991; Darlington et al. 1992) even though coincidentally
increasing plasma corticosterone concentrations attain levels that would reduce its
accumulation if persistently maintained in the unstimulated animal (Swanson & Simmons,
1989). This suggests that during abrupt stress the suppression of CRH mRNA in the
PVHmp seen with chronically elevated corticosterone either does not occur because of the
short duration of the corticosterone surge, or is inhibited by other processes.
In intact animals, low levels of plasma corticosterone found in the early morning are
sufficient to maintain levels of CRH mRNA in the PVHmp during the mid-point of the light
phase (Watts & Sanchez-Watt, 1995a) and these, presumably, position the transcriptional
machinery in the CRH neuron to respond to ensuing stress at this time. Here, the
manipulation of the antecedent corticosterone environment was performed to reveal the
nature of its interaction with CRH gene regulatory mechanisms operating during a
subsequent stress event.
In chapter 3, the data showed that a sustained viscerosensory stressor, colloid-induced
hypovolemia, is accompanied by a temporally-ordered sequence of events in CRH
neuroendocrine neurons, corticotropes, and adrenal cortical cells: first, stimulus onset;
second, release of ACTH secretogogue, ACTH, and corticosterone; and finally, activation
of CRH gene expression in the PVHmpd. CRH gene transcription, ACTH and
39
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corticosterone secretion reach peak values 4h after stimulus onset (ie. 5h after injection).
Here the same viscerosensory stressor was used to investigate how the corticosterone
environment preceding the stressor affects the subsequent response of CRH gene
expression.
To this end, in situ hybridization was used to investigate how CRH gene expression
responds to sustained hypovolemia in intact, adrenalectomized, or adrenalectomized
animals with corticosterone replacement. Different doses of corticosterone were given to
three different groups of adrenalectomized animals 6 days before the stressor. First, a low
dose which was not adequate to normalize thymus weights or CRH mRNA levels in the
PVHmp; second, a dose which resulted in the same thymus weights and PVH CRH
mRNA as intact animals; and Finally, a higher dose which reduced thymus weights and
PVH CRH mRNA to levels below those seen in intact animals at the time of maximum
activation.
40
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MATERIALS AND METHODS
Five groups of rats were anesthetized with halothane and either bilaterally adrenalectomized
(four groups) or sham adrenalectomized (one group) using flank incisions. While still
under anesthesia, adrenalectomized (ADX) animals were implanted s.c. with either Omg
(ADX/0), 25 mg (ADX/25), 50mg (ADX/50), or lOOmg (ADX/100) slow-release
corticosterone pellets (Innovative Research of America, Sarasota Beach, FL) and allowed
to recover for 6 days. The corticosterone pellets provide stable plasma concentrations of
corticosterone for up to 21 days.
Following surgery, animals were provided with unrestricted access to water, 0.9% saline
and rat chow. On the morning of day 7 water, saline, and food were removed and rats
given s.c. injections as stated in the Materials and Methods section.
Perfusion, tissue handling, in situ hybridization, radioimmunoassays, and semi
quantitation of 35S-UTP-cRNA Hybridization were performed as noted in the Materials and
Methods section (Chapter 2).
Statistical analysis. The significance of differences between dependent variables across
treatment groups were determined using multifactorial ANOVA, followed by Tukey or
Dunnetts two-tailed post-hoc test with intact values taken as control. P < 0.05 regarded as
being statistically significant for all tests. The significance of differences in dependent
variables between saline-injected and PEG-treated animals within each steroid treatment
group were determined using Student’s t test assuming unequal variances. All statistical
analyses were performed using Excel (Macintosh version 4.0; Microsoft) and Systat
(Macintosh version 5.2).
41
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RESULTS
Hematocrits were not significantly different between saline-injected animals of any group
(Table 4.1); however, hematocrits were significantly elevated in all PEG-treated groups as
compared to their respective controls (P < 0.001 in all cases). Table 4.1 also shows that
plasma concentrations of corticosterone were markedly elevated 5h after PEG-treatment in
intact animals {P <0.0001), whereas there was no significant increase after PEG injection
in ADX animals given exogenous corticosterone. There were no significant differences in
thymus weights between animals injected with vehicle or PEG (data not shown). Values
from vehicle and PEG-injected animals in each steroid treatment group were pooled for
both plasma corticosterone concentrations and thymus weights for subsequent comparisons
across groups. Except for the ADX/25 and ADX/50 groups mean pooled plasma
corticosterone concentrations in each adrenalectomized group were all significantly different
from each other (Fig. 4.1 A; P < 0.01 or greater). Figure 4. IB shows that plasma
corticosterone from the ADX/50 animals reduced mean thymus weights of
adrenalectomized animals to that seen in intact animals. Thymus weights in ADX/0
animals, or ADX/25 or ADX/100 animals were significantly different from any other group
(Fig. 4 .IB, P < 0.025 or greater).
CRH mRNA levels in the PVH mpd
Saline-injected groups showed the anticipated inverse relationship between circulating
corticosterone and basal levels of CRH mRNA in the PVHmpd: CRH mRNA increased in
adrenalectomized rats, and decreased as plasma corticosterone concentrations were
increased (Fig. 4.2A). All treatment groups except the 50mg corticosterone pellet were
significantly different from intact animals (Fig. 4.2A, ADX/0, P < 0.0001; ADX/25,
P < 0.001; ADX/100, P < 0.05 vs intact group). CRH mRNA levels in the PVHmpd of
42
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TABLE 4.1
TREATMENT Hematocrit n Corticosterone n
(%) (ng/ml)
Intact Saline 47.1±0.3 8 188±27 7
PEG 56.9±0.7 12 906±38 12
Omg Saline 48.8±0.5 10 15±2 10
PEG 64.6±1.0 12 14±1 12
25mg Saline 48.2±0.4 6 75±5 6
PEG 64.4±1.4 5 111 ±20 5
50mg Saline 47.6±1.2 5 1 09±13 5
PEG 68.1±1.7 5 16 1±19 5
1 OOmg Saline 48.5+0.8 6 227±2l 6
PEG 57.5±1.6 5 303±70 5
TABLE 4.1. Mean (±SEM) hematocrits, plasma volume deficit, and plasma corticosterone
concentrations in intact and each corticosterone treatment group injected sc with either 0.9%
saline or 40% PEG (see text for significance).
43
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B
E
o h
300 i
J 250
1 3
c
o
u
D
e
o
u
a
B
2
U J
c/i
c
s
s
200
150 •
100
50 •
500
450
400
350
§ 300 J
"S i
| 250
•y j
I 200 1
JZ
150 -
100
50
0 i
0 25 50 100
Corticosterone capsule
size (mg)
Intact 0 25 50 100
Corticosterone capsule
size (mg)
FIG 4.1. Mean (+SEM) plasma corticosterone concentrations (nanograms per ml) in each
steroid replacement group (A) and thymus weights (milligrams) in intact and steroid treatment
groups (B). See test for levels of significance.
44
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adrenalectomized animals given placebo or exogenous corticosterone were linearily
correlated to the log1 0 plasma corticosterone concentration in a significant maimer
(R2=0.7105, F=50.1, P < 0.0001).
Five hours after PEG injection the mean CRH mRNA level in the PVHmpd was
significantly different to that of respective saline injected controls in all treatment groups
(intact, P < 0.02; ADX/0, P < 0.01; ADX/25, P < 0.001; ADX/50, P < 0.05; ADX/100,
P < 0.025). However, in distinction to increased CRH mRNA levels seen in intact animals
and all corticosterone replaced groups, CRH mRNA levels decreased 5h after PEG
injections in adrenalectomized animals not given corticosterone. Furthermore, figure 4.2B
shows that the size of the increment in CRH mRNA levels in adrenalectomized animals
given 25mg corticosterone pellets was significantly greater than that in any other group
(P < 0.05 vs intact and ADX/50, P < 0.02 vs ADX/100, P < 0.0001 vs ADX/0). The
increment in CRH mRNA accumulation in ADX/50mg or ADX/100 was no different to that
seen in intact animals.
CRH hnRNA and pENK mRNA levels in the PVH mpd
To investigate the mechanisms responsible for the regulation of CRH mRNA seen in
adrenalectomized animals with and without low replacement doses of corticosterone, CRH
hnRNA and pENK mRNA levels were determined in the PVHmpd of intact, ADX/0 and
ADX/25 animals (Fig. 4.3). In all cases, the group trends in the CRH mRNA response to
saline and PEG (Figs. 4.3A & 4.4) were paralleled by those of CRH hnRNA (Figs. 4.3B
& 4.4). Thus, in saline-injected animals there was a significant increase in CRH hnRNA in
the PVHmpd of ADX/0 when compared to intact (P < 0.005) and ADX/25 (both P <
0.02). Although not significantly different from intact animals, values for ADX/25 animals
45
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J
o
s
30
20
1 0
0
Intact 0 25 50 100
Corticosterone capsule
size (mg)
= i - 2 0
-30
-40
-50
Intact 0 25 50 100
Corticosterone capsule
size (mg)
FIG 4.2. Mean (+SEM) MGL expressed in arbitrary units of CRH mRNA hybridization in the
PVHmpd of saline-injected groups and each corticosterone treatment group. B, Mean (+SEM)
changes in the MGL of the CRH mRNA response to sc injection of 40% PEG in intact and
replacement groups. See text for levels of significance.
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were intermediate between those from intact and ADX/0 animals. In response to PEG
injections both intact and ADX/25 animals showed significant increases in CRH hnRNA
signal when compare to saline-injected animals (intact, P < 0.005; ADX/25, P < 0.02).
However, in the ADX/0, CRH hnRNA was significantly lower after PEG injection than the
corresponding saline-injected controls (p<0.02).
pENK mRNA in the PVHmpd was significantly elevated 5 hours after PEG injection in all
groups. (Fig.4.3C; P < 0.0005 for intact animals; P < 0.0001 for ADX/25; P < 0.025 for
ADX/0 animals).
47
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CRH mRNA
300 i
200 ■
100 -
Q jj
B
700
.S 500 '
X)
X
0
1
ui
vi
+,
c
C 3
< U
300 -
100
0
400
300
100
200 -
0
Intact
CRH hnRNA
I
I
I
ENK mRNA
X
25 0
Corticosterone capsule
size (mg)
FIG 4.3. Mean (+SEM) MGL of CRH mRNA (A), CRH hnRNA (B), and pENK mRNA (C)
hybridization signal of the dorsal aspect of the medial parvicellular part of the hypothalamic
paraventricular nucleus seen 5h after sc injections of either vehicle (0.9% saline: open bars)
or 40% PEG (black bars), expressed as a percentage of the mean intact control (saline-
injected) value. The number of animals per group is shown in Table 1. See text for levels of
significance.
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INTACT ADEX
saline PEG saline PEG
FIG 4.4. CRH mRNA, CRH hnRNA, or pENK mRNA response to sustained hypovolemia.
Images from Cronex microvision x-ray film of three serial sections hybridized for
CRH mRNA, CRH hnRNA, or pENK mRNA in the PVHmpd of representative intact and
ADX animals 5h after sc injection of either 0.9% saline or 40% PEG.
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DISCUSSION
Colloid-induced hypovolemia in intact animals leads to sustained activation of CRH gene
transcription and consequent elevations in CRH mRNA in PVHmpd neurons (Chapter 3).
The data presented here show that the magnitude of the CRH gene response seen 5h after
PEG injection in intact animals is dependent on the corticosterone environment preceding
the stressor. This dependency has three aspects each related to prevailing circulating
corticosterone concentrations. First, in the absence of corticosterone the CRH gene
response is severely compromised to the extent that mRNA levels are now reduced 5h after
injection; second, low doses of corticosterone apparently facilitate the CRH gene response
beyond that seen in intact animals; and third, the response is normalized by plasma
corticosterone concentrations which also normalize thymus weights and basal levels of
CRH mRNA (Watts & Sanchez-Watts, 1995a).
The CRH hnRNA data indicate these differences in the accumulation rate of CRH mRNA
most likely involve alterations in gene transcription. In the absence of corticosterone,
decreases in CRH mRNA accumulation seen 5h after PEG injection is paralleled by
decreases hnRNA levels. These data suggest that a substantial modification of the
mechanisms associated with stress-induced CRH gene activation occurs in the absence of
corticosterone. This modification cannot be accounted for by a radical alteration in afferent
signals because there were still significant increases in hematocrit, c-fos and pENK
mRNAs in the same PVH neuronal cell group that expressed CRH mRNA 5h after PEG-
injection in adrenalectomized groups. Thus, PVHmpd neurons in adrenalectomized animals
still apparently receive afferent signals conveying stimulus information, and the
transcriptional machinery of these neurons can still respond to the stressor. When
adrenalectomized animals are given a dose of exogenous corticosterone which is
insufficient to normalize thymus weights or CRH mRNA levels in the PVHmpd, the ability
50
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of the CRH neuron to accumulate mRNA in response to the stressor not only returns but
actually appears to be facilitated, since rates of mRNA accumulation exceed those seen in
intact animals. At least part of this increase in mRNA accumulation is likely due to
facilitated transcription because there is now an significant elevation in CRH hnRNA levels
at 5h in ADX/25 animals. In this manner, corticosterone-dependent facilitated CRH gene
expression is perhaps analogous to the corticosterone-dependent facilitated ACTH secretory
responses to novel stressors recently reported by Akana & Dallman (1997) and by
Murakami et al. (1997).
Although the nature of this facilitatory process is currently unclear, two possibilities can
account for our data. First, in the absence of corticosterone, gene transcription is never
activated by hypovolemia; second, gene activation occurs initially but cannot be maintained.
Data from other groups suggest that corticosterone modifies the CRH neuron using the
second mechanism; corticosterone allows CRH gene transcription to be maintained in the
event that a particular stressor becomes prolonged. However, it should be noted that this
interpretation derives from stressors that undoubtedly use different afferent mechanisms
from those activated in the present study by sustained hypovolemia. After abrupt transient
stressors many responses of the HPA axis are augmented by adrenalectomy. For example
at the pituitary, adrenalectomy, or adrenalectomy with low-level corticosterone replacement
produces hypersecretion of ACTH in response to a stressor (Buckingham & Hodges,
1974; Akana et al. 1988; Jacobson & Sapolsky, 1993). In PVHmp CRH neurons of
adrenalectomized rats, Lightman and Young (1989) reported increased CRH mRNA levels
4h after intraperitoneal injections of hypertonic saline. Furthermore, Imaki et al. (1995)
reported an exaggerated response in CRH hnRNA 30 minutes after restraint stress in
adrenalectomized rats. Our data now show that if gene transcription is activated by PEG in
ADX/0 animals, it cannot be maintained in the absence of corticosterone ultimately leaving
51
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the animal with reduced ability to cope with the stress event. The results suggest that in the
presence of a maintained stressor of high intensity, low levels of plasma corticosterone
before the stress event (as would occur in intact animals) can facilitate the mechanisms of
CRH gene transcription and prolong the ability of the CRH neuron to respond and maintain
secretion. The reason CRH hnRNA and mRNA levels actually decline in the absence of
corticosterone is currently unclear, but may well be related to a subsequent alteration in
turnover rates of these components. Faced with stress-induced CRH secretion, the
increased rates of CRH translation coupled with a reduced transcription rate, may well
result in the reduced accumulation rates of hnRNA and mRNA we observed. This does not
occur in intact animals where CRH hnRNA levels are elevated above controls from 3h until
at least 6h after PEG injection (Chapter 3).
Two observations suggest that the amount of corticosterone required to facilitate CRH gene
transcription during hypovolemia is rather low. Thus, both thymus weights and levels of
CRH mRNA in the PVHmpd in saline-injected ADX/25 animals were significantly greater
than those of intact animals.In the present study, thymus weights of the ADX/25 animals
were 82% of those measured in saline-treated ADX/0 group. This indicates that these
animals had plasma corticosterone levels within or below the lower part of this range
required for normalization of these variables (Dallman et al., 1987; Watts, 1996).
The fact that only low plasma concentrations of corticosterone are required to facilitate
CRH gene expression suggests that it is possibly a predominantly MR-mediated event. In
support of this assertion it is significant to note that adrenalectomized rats given aldosterone
(a MR agonist) alone, at a dose adequate to normalize sodium appetite, had significantly
increased CRH mRNA levels compared to adrenalectomized animals with no steroid
replacement (Watts & Sanchez-Watts, 1995a), showing that in some circumstances MR
occupation can be facilitatory to CRH gene expression.
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The reduction in the magnitude of the stress-induced accumulation of CRH mRNA seen as
plasma corticosterone concentrations increase from ADX/25 to ADX/50 and ADX/100
PEG-injected animals is consistent with the data of Kovacs & Sawchenko (1996b). Here
dexamethasone or corticosterone 5 day pre-treatment inhibits accumulation of CRH hnRNA
after a brief transient stressor. However, it should be noted that differences in this stress
model (ether anesthesia) caution against more detailed comparisons with the data generated
in response to sustained hypovolemia at this time. Considering the present and previously
published data ( Watts & Sanchez-Watts, 1995a), it is tempting to speculate that MR
receptor occupation facilitates, while GR receptor occupation inhibits CRH mRNA
accumulation in the PVHmpd of both unstressed and stressed animals. This dual nature of
corticosterone action on CRH gene expression is consistent with the coordinate action of
MR and GR receptors in regulating common sets of genes first suggested by Evans &
Arriza (1989). However, it is important to emphasize that these data do not provide
information about the neural circuits and mechanisms through which these events occur.
That plasma corticosterone values taken 5h after injections from adrenalectomized
corticosterone-replaced animals were higher than previously reported (Watts & Sanchez-
Watts, 1995a) is somewhat puzzling, but may be a consequence of the halothane anesthesia
at the time of injection on the hepatic clearance of corticosterone; halothane anesthesia is
known to affect some aspects of liver metabolism in this manner (Wood & Wood, 1984;
Debaene, et al., 1990; Hartman et al., 1992). Nonetheless, the values of the other
measured indicators of corticosterone bioactivity in saline injected controls—thymus weight
and CRH mRNA levels in the PVHmpd— show that exposure to low levels of plasma
corticosterone before the stress event is all that is required to restore CRH gene activation to
that seen in intact animals.
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Finally, two other points supported by our data are worthy of mention. First, the
significant differences in the levels of CRH hnRNA in saline injected intact, ADX/0, and
ADX/25 animals are consistent with the notion that CRH gene transcription is suppressed
by corticosterone acting in the slow feedback time domain (Keller-Wood & Dallman,
1984), and is thus an important component of normal negative feedback inhibition.
Second, the fact that CRH mRNA accumulation in ADX/50 animals was indistinguishable
from intact animals shows that the significant elevation of plasma corticosterone
concentrations which occurs as a consequence of hypovolemia does not modify
concomitant CRH gene activation because there was no stress-induced increase in plasma
corticosterone in the pellet treated animals. These data show that in intact hypovolemic rats
the pronounced stress-activation of corticosterone secretion in itself does not compromise
CRH gene transcription and mRNA accumulation during the stress event (Chapter three); a
conclusion that confirms previous findings from more abrupt stressors (Lightman &
Young, 1989). Taken together with these data, the results presented here suggest that the
continued presence of low levels of plasma corticosterone before the stress but not the
stress-mediated elevation in plasma corticosterone is required to maintain CRH gene
transcription during a prolonged viscerosensory stress event. If corticosterone is absent
before the stress event, mechanisms antecedent to transcription are altered in such a manner
that activated CRH gene expression is either never initiated or cannot be maintained.
In summary, by removing corticosterone and then presenting a sustained stressor a subtle
property that has not been recognized previously at the CRH neuroendocrine neuron has
been revealed; that of a facilitatory agent in the regulation of CRH gene transcription and
mRNA accumulation. The facilitatory nature of these low levels of plasma corticosterone
appears to allow the formulation of a normal stress event, and signifies that reduced adrenal
54
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function will have profound consequences on mechanisms of CRH gene expression, and
the subsequent ability of an animal to mount an adequate stress response.
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Chapter 5
INTRODUCTION
Sustained hypovolemia in the intact rat provokes robust and sustained ACTH secretion and
elicits measurable increases in CRH but not AVP gene transcription in the dorsal aspect of
the medial parvicellular subdivision of the PVH (PVHmpd). Furthermore, corticosterone
secreted during the first 3h of sustained hypovolemia does not inhibit ongoing CRH gene
expression or ACTH secretion (Chapter 3). Other data from this model point to the fact that
corticosterone may have functions beyond its more familiar role as a negative feedback
signal on secretogogue gene expression. Specifically, in adrenalectomized (ADX) rats the
numbers of CRH hnRNA positive cells and mRNA levels are significantly decreased in
hypovolemic animals 5h after stress onset compared with saline-treated controls. However,
ADX rats treated for six days with doses of corticosterone that are insufficient to normalize
thymus weights, display an augmented mRNA response at this time compared with intact
animals, while higher doses of corticosterone (those able to normalize thymus weights)
produce a CRH mRNA response that is not significantly different from that of intact rats.
These data strongly suggest that the antecedent corticosterone environment determines the
dynamics of the CRH gene response during stress, with normal blood levels providing the
environment for the optimal response of the CRH gene in the PVH (Chapter 4).
With regard to potential mechanisms, the data from ADX rats with no corticosterone
replacement are particularly intriguing because they provide an experimental basis for
considering how corticosterone mediates the CRH gene response to stress. However,
before considering this question further it is important to determine which of two possible
explanations for the data presented in chapter 4 is correct. First, is CRH gene transcription
56
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never initiated during sustained hypovolemia, and levels of CRH hnRNA and mRNA are
subsequently reduced by the stressor; or, second, is it first activated by the stressor, but
then (unlike what happens in the presence of corticosterone) cannot be maintained? A final
question here is the behavior of the AVP gene response. In rats, AVP gene expression is
strongly suppressed by circulating corticosterone (Davis et al. 1986; Swanson & Simmons
1989), and it is not activated by sustained hypovolemia (Chapter 3). However, it is
conceivable that in ADX animals (where restraining effects of corticosterone are lacking)
parvicellular AVP may play a more prominent role in the response to sustained
hypovolemia, in a manner comparable to what occurs with more chronic stressors
(Bartanusz et al., 1993; Makino et al., 1995).
These issues have been addressed in the present study which was designed to answer the
following questions. First, does CRH gene transcription occur during sustained
hypovolemia in ADX animals, and if so, how is it’s temporal response organized; second,
what is the expression pattern of other genes that are colocalized with CRH in the
PVHmpd, such as pENK and AVP; and third, what is the accompanying temporal profile
of ACTH secretion? Secretory and synthetic responses to sustained hypovolemia in ADX
rats were measured every hour for 6h after PEG injection given during the early part of the
light period. Using in situ hybridization, cellular levels of CRH, c-fos, and pENK mRNAs
were measured, along with the primary transcripts of CRH and AVP in the PVHmpd.
Additionally, plasma concentrations of ACTH and corticosterone were measured using
radioimmunoassays.
57
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MATERIALS AND METHODS
Animal weight, housing and acclimation period were performed as stated in the Materials
and Methods (Chapter 2). Six animals were assigned for each treatment group at every time
point. Seven groups of rats were anesthetized with halothane and bilaterally
adrenalectomized (ADX) using flank incisions and allowed to recover for 6 days. After
ADX, animals were provided with unrestricted access to water, 0.9% saline, and rat chow.
On the morning (0700-0800h) of day 7, animals were either injected subcutaneously with
0.9% saline or 40% PEG and left undisturbed as stated in the materials and methods
(Chapter 2). One group remained intact (INT: n=l2 animals) and was sacrificed 5h
following sc injection of either 0.9% saline (INT/SAL) or 40% PEG (INT/PEG).
Perfusion or decapitation, tissue handling, in situ hybridization, radioimmunoassays, and
semi-quantitation of S-UTP-cRNA Hybridization were performed as noted in the
Materials and Methods section (Chapter 2).
Statistical analysis
Significant differences between dependent variables in 0.9% saline- and PEG-treated
animals at different times in the experiment were determined using single factor ANOVA,
followed by least square differences post-hoc test. The significance of differences between
saline- and PEG-injected animals at any one time was determined using Student’s test.
P < 0.05 was regarded as being statistically significant for all tests. All statistical analyses
were performed using Excel (Macintosh version 4.0; Microsoft, Redmond, WA) and
Systat (Macintosh version 5.2, Systat, Evanston EL).
58
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RESULTS
Thymus weights Mean thymus weights between all ADX groups were identical and
significantly increased compared with intact animals (ADX: 778.1±14.2 mg, INT:
597.5±16.l mg: P < 0.0001)
Hematocrit Fig. 5.1A shows that the hematocrit is significantly increased lh after PEG
injection (P < 0.05) compared with that of saline-injected rats and remained significantly
increased for the duration of the experiment compared with their respective ADX/SAL
groups (P < 0.0002-0.0001). The hematocrit remained virtually identical in all ADX/SAL
groups. In the INT/PEG group at 5h, the hematocrit significantly increased (P < 0.001)
compared with INT/SAL controls. Fig 5. IB shows that the rate of change in the plasma
volume deficit of INT/PEG and ADX/PEG rats were virtually identical, as determined from
the hematocrit (Chapter 3). Data for INT/PEG rats were taken from chapter 3 for ease of
comparison.
CRH Gene Expression
CRH hnRNA. The mean number of CRH hnRNA positive cells significantly increased lh
and 2h after PEG-injection compared with corresponding saline-injected controls (Fig.
5.2A). But then at 3h, the mean number of CRH hnRNA positive cells declined to values
that were no longer significantly different from saline-injected controls. At 4h, 5h and 6h
following injection, CRH hnRNA cell numbers were now significantly increased in saline-
injected animals (P < 0.001) compared with the corresponding PEG-injected group.
Moreover, in comparing differences in saline-injected groups only, from 3h to 4h CRH
hnRNA cell numbers were significantly increased in ADX/SAL animals (P < 0.001), and
59
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Hematocril (%)
55
50
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FIG. 5.1 A, Mean (+SEM) hematocrits of rats injected sc with either 5ml vehicle (0.9% saline; open
circles) or 40% PEG (black circles). At 5h, the mean (+SEM) vehicle injected inject animals are
depicted as an unfilled square and the PEG-injected intact group as a filled square. See text for signficance.
(B) Mean (+SEM) relative plasma volume deficit as determined by the hematocrit in intact (closed
circles) and ADX (open circles) rats as a function of time after sc PEG injection. See text for
significance.
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CRH hnRNA B CRH mRNA
1 2 0
> 100
2 40
& 20
0 1 2 3 4 5 6
120
100
80
60
40
20
0
0 1 3 4 5 6
Time (Hours) Time (Hours)
FIG. 5.2 A. Mean (+SEM) number of CRH hnRNA-labeled cells in rats injected sc with either
5ml vehicle (0.9% saline; open circles: 5h intact; unfilled square) or 40% PEG (closed circles: 5h intact;
filled square). (B) Mean (+SEM) MGL of CRH mRNA hybridization in the PVHmpd expressed in
arbitrary units of rats injected sc with 5ml of either vehicle (0.9% saline; open circles; 5h intact;
open square) or 40% PEG (closed circles: 5h intact; closed square). See text for significance.
61
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remained elevated for the remainder of the experiment (P < 0.02-0.05) when compared to
3h ADX/SAL group. In the intact group, PEG-injected animals CRH hnRNA cell numbers
were significantly increased (P < 0.001) compared with respective control group.
CRH mRNA. Fig. 5.2B illustrates that CRH mRNA levels showed a small but significant
increase 2h after PEG injection (P < 0.02). At 3h, CRH mRNA levels were identical
between treatment groups, whereas from 4h to 6h, CRH mRNA levels in the ADX/SAL
rats were significantly elevated above corresponding ADX/PEG animals (P < 0.001-
0.0001). CRH mRNA levels in INT/PEG animals were significantly increased (P < 0.01)
compared with INT/SAL rats.
AVP Gene Expression
AVP hnRNA. AVP hnRNA positive cells were significantly increased lh after PEG
injection (Fig. 5.3A, P < 0.001) compared with saline-injected controls and remained
significantly increased through the duration of the experiment compared with respective
saline-injected controls (P < 0.01-0.0001). In the intact group, AVP hnRNA positive cells
were not significantly different 5h after injection. Fig. 5.3B shows significantly increased
AVP hnRNA levels in the supraoptic nucleus (SON) from 2h to 6h in ADX/PEG groups
compared with their respective ADX/SAL counterparts (P < 0.01-0.001). AVP hnRNA
levels between saline- and PEG-injected groups in the intact animals at 5h were not
significantly different.
Other Patterns of Gene Expression
c-fos mRNA. Fig. 5.4A shows that 2h after PEG injection and through the duration of the
experiment, c-fos mRNA levels were significantly increased compared with saline-injected
animals (P < 0.02-0.0001). In the intact group, c-fos mRNA levels were significantly
62
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Number o f Labeled Cells i n th e PVHmpd
AVP hnRNA (PVHmpd)
B
AVP hnRNA (SON)
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120
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FIG. 5.3 A, Mean (+SEM)number cf AVP hnRNA-labeled parvicellular cells in the PVHmpd in rats
injected sc with either vehicle (0.9% saline; open circles: 5h intact; unfilled square) or 40% PEG
(closed circles: 5h intact; filled square). (B) Mean (+SEM) MGL of AVP hnRNA hybridization in
the SON expressed in arbitrary units of rats injected sc with either vehicle (0.9% saline; open circles:
5h intact; open square) or 40% PEG (closed circles: 5h intact; closed squares). See text for
significance.
63
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increased in PEG treated rats compared with INT/SAL animals (P < 0.002) and the
ADX/PEG group was significantly higher than c-fos mRNA levels in the INT/PEG group
( P < 0.0001).
pENK mRNA. Fig. 5.4B shows that pENK mRNA levels were significantly increased 2h
after PEG injection (P < 0.002) as compared with saline-injected animals. At 3h pENK
mRNA levels were not significantly different most likely because of the large variances
apparent in both treatment groups. Hypovolemic animals in groups 4h through 6h (P <
0.05-0.01) were significantly increased pENK mRNA levels when compared with their
respective control groups.
Plasma Hormone Concentrations
Plasma ACTH. Fig 5.5 shows that a significant increase in the plasma ACTH concentration
occurred 2h after PEG injection (P < 0.001) compared with the 2h saline injected group.
Plasma ACTH levels continued to rise and remain significantly increased up to the 5h time
point (P < 0.001-0.0001) compared with their respective controls. At 6h, the two treatment
groups were not significantly different. The intact PEG-injected group had significantly
increased plasma ACTH concentrations compared with the respective saline-injected group.
Note that the plasma ACTH concentration measured in the INT/SAL group at 5h was
significantly lower than any ADX/SAL groups (P < 0.0001).
Plasma corticosterone. Plasma corticosterone concentrations in all ADX rats were below
the limit of sensitivity of the RIA. In the intact group, the PEG-injected group was
significantly higher than the saline-injected animals ( PEG: 738.1±25.6 ng/ml, SAL:
36.0±14.9 ng/ml: P<0.0001)
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c-fos mRNA
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FIG. 5.4 A, Mean (+SEM) MGL of c-fos (A) or pENK mRNA (B) hybridization in the PVHmpd
expressed in arbitrary units of rats injected sc with either vehicle (0.9% saline; open circles: 5h intact;
unfilled square) or 40% PEG (closed circles: 5h intact; filled square). See text for significance.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3 5 0 0 i
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FIG. 5.5 Mean (+SEM)plasma ACTH concentrations in rats injected sc with either vehicle (0.9%
saline; open circles: 5h intact; unfilled square) or 40% PEG (closed circles: 5h intact; filled square).
See text for significance.
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DISCUSSION
The data presented here show that the ADX rat responds to sustained hypovolemia with
activation of the hypothalamo-pituitary limb of the HPA axis. However, following ADX,
ACTH secretogogue gene expression differs significantly both in magnitude and dynamics
to what occurs in the intact rat (Chapter 4). These results show that during stress and
without the prior exposure to CORT, mechanisms operating within the neuroendocrine
CRH neuron, its afferents. or both are modified in a way that results in earlier activation of
CRH gene transcription, which is associated with a small but significant increase in CRH
mRNA levels not seen this early in INT/PEG rats (Chapter 3). But then, as the stressor
increases in intensity, ADX animals are unable to sustain this initial episode of CRH gene
transcription beyond 2h post-injection, and from 3 to 6h the numbers of CRH hnRNA
positive cells markedly decline. Interestingly, during this time frame, levels of CRH
hnRNA and mRNA in the PVHmp of ADX/SAL animals increase, so contributing to an
overall temporal profile consistent with a circadian pattern reported previously in ADX rats
(Kwak et al., 1993). It is worth emphasizing that in ADX/SAL animals CRH hnRNA and
mRNA levels are beginning to increase 3h from the start of the experiment suggesting that
CRH gene expression is actually being stimulated. However, the fact that CRH hnRNA
and mRNA levels are falling coincidentally in ADX/PEG animals suggests that, as a
consequence of the stress, either CRH gene transcription is actively inhibited, alterations in
post-transcriptional processing occur, or both. Collectively, these data support our
hypothesis that those levels of circulating corticosterone normally present across the
circadian day act to facilitate the CRH response to a subsequent sustained stressor (Chapter
4).
In the intact rat, AVP gene transcription in parvicellular positive neurons in the PVHmpd is
not activated in response to sustained hypovolemia, at least within 5h of onset. In contrast
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however, the data presented here, now show a robust increase in AVP hnRNA cell
numbers in the PVHmpd of ADX/PEG animals that begins as early as lh post-injection and
is maintained for the duration of the experiment. This shows that while CRH hnRNA cell
counts and mRNA levels decline from 3h to 6h in ADX/PEG animals, AVP gene
transcription and plasma ACTH concentrations remain markedly elevated. Furthermore,
AVP hnRNA levels in the supraoptic nucleus (SON) do not significantly increase until one
hour after those m the PVHmpd of the same animals. Although the methods used for
semiquantitative analysis for AVP hnRNA levels in the PVHmpd and SON differed, the
data suggest that during sustained hypovolemia, mechanisms activating AVP gene
transcription in magnocellular and parvicellular neuroendocrine neurons have distinct and
separate thresholds. This finding also corroborates that our methods can differentiate
between AVP gene transcription in the magnocellular and parvicellular populations of the
PVH.
Components of the hypothalamo-pituitary limb of the HPA axis show increased activity in
response to ADX: upregulation of CRH and AVP mRNAs in the PVHmp (Wolfson et al.,
1985; Young et al., 1986; Swanson & Simmons, 1989; Watts & Sanchez-Watts, 1995b),
their cognate peptides (Sawchenko et al., 1984; Kiss et al., 1984), and basal plasma ACTH
concentrations (Dallman et al., 1987). Therefore, when a stressor of sufficient magnitude is
presented, it seems likely that the subsequent elevation of AVP synthesis (and possibly
release) helps sustain the augmented ACTH secretion during sustained hypovolemia. Part
of this altered responsiveness may also be due to the recruitment of previously unstimulated
corticotropes. Jia et al. (1991) have reported three functionally distinct corticotropes that
show responsiveness to either CRH but not AVP, CRH or AVP, or CRH and AVP.
Therefore, it is likely that the altered activities of AVP and CRH gene transcription in
ADX/PEG rats may not only modify secretogogue packaging in CRH neuroendocrine
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neurons, but may also modify the AVP/CRH ratio in hypophysial portal blood, ultimately
increasing the number of corticotropes that secrete ACTH (Childs et al., 1987; Neill et al.,
1987; Jia et al., 1991). With this in mind, our data collectively suggest that in the absence
of CORT, the AVP gene is now transcribed in preference to the CRH gene, possibly as
part of an overall strategy to try and maintain pituitary responsiveness in the presence of
sustained CRH release (Scaccionoce et al. 1991).
The shift of synthesis from CRH to AVP (and most likely the production of their cognate
peptides) in the PVHmpd of ADX/PEG rats bears striking resemblance to what is seen in
intact rats exposed to repeated stress. Here, repeated immobilization initially increases CRH
rather than AVP mRNA levels (Makino et al., 1995; Ma & Lightman, 1998) which is
consistent with data from INT/PEG animals (Chapter 3). However, as the stressor is
repeated CRH gene transcription is suppressed in favor of AVP (Ma & Lightman, 1998);
events that are likely associated with the increased ratio of copackaged AVP/CRH
immunoreactivity seen in CRH terminals (De Goeij et al., 1991). De Goeij and colleagues
(1991) have hypothesized that the shift observed during repeated immobilization may be a
physiological response of the PVH neurosecretory neurons to sustained stimuli, and that
AVP may play a pivotal role in adjusting ACTH release in response to increasing demand
(Aguilera, 1994). The data presented here are consistent with a similar interpretation in the
ADX rat, again emphasizing the role that the preceding corticosterone environment plays in
determining how the transcriptional machinery in the neuroendocrine CRH neuron
responds to stress.
In this context, although both the pre-stress values in all ADX animals and the plasma
ACTH concentrations in ADX/SAL animals were higher than INT animals (as would be
expected), both the time of onset and the profile of stress-induced ACTH secretion in
69
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ADX/PEG animals were remarkably similar to those already reported in INT/PEG animals
(Chapter 3). However, here, the data show that in ADX/PEG animals the onset of CRH
gene activation occurs earlier than in INT/PEG animals and that AVP gene transcription is
recruited at the same time. This shows that during sustained hypovolemia, CORT does not
alter the stimulus threshold required to elicit secretory responses (CRH, AVP, and ACTH)
but, critically, it does modify the stimulus threshold required to activate the appropriate
secretogogue genes in the PVHmpd. Furthermore, that this hypersensitivity of
secretogogue gene activation in the PVHmpd of ADX/PEG animals also extends to the c-
fos and pENK mRNA profiles (gene products that have previously been reported to be
upregulated during sustained hypovolemia: chapter three) is supported by two
observations. First, both c-fos and pENK mRNA levels have earlier onsets (2h) in
ADX/PEG rats compared with INT/PEG animals (3h and 4h, respectively; chapter 3).
Second, in the absence of corticosterone, c-fos mRNA levels were dramatically augmented
at 5h in ADX/PEG rats compared with ENT/PEG animals, a finding previously reported in
ADX rats exposed to acute restraint (Imaki et al., 1995). Taken together, it appears that the
temporal dynamics of all the gene products measured in the PVHmpd suggests that during
sustained hypovolemia CORT regulates either the sensitivity of the afferent input to the
PVHmpd, the signal transduction mechanisms within the neurons themselves, or both.
It is important to note that ADX produces very well-defined disturbances in basal activity of
various physiological functions, particularly alterations in neural and humoral components
involved in fluid homeostasis. Several studies have shown that various acute stressors in
the ADX rat induce augmented ACTH responses (Buckingham & Hodges, 1974; Akana et
al., 1988; Jacobson & Sapolsky, 1993) and it may be the upregulation of its two
secretogogues, particularly AVP, and their concerted release facilitates this response.
Despite these changes, it is clear that compensatory mechanisms exist to increase the
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likelihood of the survival of an animal. For example, after hemorrhage, Darlington et al.
(1989) showed that the recovery of mean arterial blood pressure was very similar in ADX
compared with intact rats, and that this was most likely due to augmented vasoconstriction
and cardiac output through compensatory responses from other vasoactive systems (i.e.
renin-angiotensin system). With this in mind, it is perhaps not surprising that the kinetic
response (rate of decreased plasma volume as measured by the hematocrit) observed in
ADX/PEG rats were virtually identical to that seen in the INT/PEG animals.
In conclusion, using this model it appears that the absence of corticosterone before the
stressor appears to reduce CRH gene transcription in the PVHmpd in favor of AVP gene
transcription, most likely as part of the mechanisms that ultimately maintain pituitary
responsiveness. The results presented here strongly support our hypothesis that
corticosterone acts to facilitate and direct the CRH gene response, at least to hemodynamic
stressors.
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Chapter 6
The ability to monitor aspects of each limb of the HPA axis while stressor-specific circuits
are activated has provided much insight into corticosterone modulation of ACTH
secretagogue gene expression. The response of the HPA axis to stress can best be
described within three temporal components: onset, maintenance, and termination - all of
which are influenced by corticosterone-dependent mechanisms. Specifically, the onset
refers to the threshold necessary to induce transcription of specific genes in the PVHmpd as
well as the hormone secretion. Second, the maintenance of the stress response refers to the
magnitude and duration of neuropeptide gene activation, and ACTH and corticosterone
secretion. Lastly, the termination is associated with HPA axis responses once the stressor
has been removed. The nature of the stimulus used in this thesis, sustained hypovolemia,
has provided the opportunity to generate data that address the onset and maintenance of
ACTH secretagogue gene transcription and its modulation by corticosterone in response to
a sustained stress event in the intact and ADX rat (with and without corticosterone
replacement).
However, before discussing the CRH gene response during sustained hypovolemia, it is
important to reiterate that any stress event is superimposed upon the circadian rhythm. This
underlying rhythm becomes even more relevant when the stressor is sustained. The design
of the time course experiments has allowed for comparison between treatment groups at or
near the same time point, and the possible interactions between the temporal response and
the diurnal rhythm. As such, it provides further insight regarding corticosterone modulation
of CRH gene transcription in the basal and stressed states. With this in mind,
corticosterone-dependent modulation of CRH gene expression in saline-injected controls
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occur during the first half of the light period. Specifically, intact saline-injected rats show a
decline in CRH gene transcription (as indicated by hnRNA cell counts) and mRNA levels
during the mid-point of the light period (10.00-12.00) whereas ADX saline-injected rats
display the inverse profile with CRH hnRNA cell numbers and mRNA levels increasing
during this period, similar to the profile in CRH mRNA levels by Kwak and coworkers
(1993). The differences in CRH profiles suggests that corticosterone is required for
maintenance of the circadian rhythm during lights-on. Although these data are drawn from
two separate experiments (Chapters 3 and 5) and therefore absolute values cannot be
directly compared, the change in temporal profiles is dramatic and therefore worth noting.
Corticosterone-dependent regulation of CRH gene expression is also observed in saline-
injected animals where corticosterone is clamped (ie ADX + corticosterone pellet) for six
days. The accumulation rate of CRH hnRNA levels exhibits a dose dependency on low
maintained levels of CORT similar to that previously reported for mRNA levels in the
medial parvicellular PVH (PVHmp: Swanson & Simmons, 1989; Watts & Sanchez-Watts,
1995a). That CRH hnRNA cell numbers and mRNA levels decline as plasma
corticosterone increases suggests that at least one aspect of the response is due to altered
gene transcription. It also appears that low levels of corticosterone are necessary to
maintain a level of secretory activity during this period of lights-on since administration of
the MR antagonist RU28318 in the intact, conscious rat has been shown to increase plasma
corticosterone levels within 15 minutes when introduced intraventricularly or within 1 hour
when give subcutaneously (Ratka et al., 1989). Together, these data suggests that MR
activation is necessary to maintain tonic inhibition of HPA axis activity, at least during the
light period.
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INTACT RAT
In the intact rat, sustained hypovolemia is accompanied by a specific, sequential, and
coordinate set of responses from the HPA axis (Chapter 3). ACTH (and therefore, CRH)
secretion occurs approximately one hour prior to CRH gene transcription indicating that the
threshold for secretion of CRH into portal blood is distinct and lower than that for CRH
gene activation. This also suggests that stimulation of CRH secretion is not sufficient to
produce secretogogue transcription. Additionally, neuropeptides that are commonly
colocalized with CRH in the PVHmpd are differentially regulated by sustained
hypovolemia. Specifically, pENK mRNA levels showed a slower temporal onset compared
to CRH, while AVP gene transcription is not activated during the time course of the
experiment which is consistent with reports that AVP plays a minor role in maintaining
ACTH secretion during hemodynamic stressors (Plotsky & Vale, 1984; Plotsky et al.,
1986; Watts & Sanchez-Watts, 1995b; Darlington et al., 1992).
At least three models can account for the observed temporal dissociation of CRH secretion
and gene transcription. First, secretion and gene expression are stimulated by separate
receptor and transduction mechanisms. Second, they share a common receptor but
divergent signal transduction mechanisms. Third, the CRH gene is activated using some
component of the transduction pathway associated with CRH release, perhaps through a
process allied to peptide release at the terminal. One possibility here is that the influx of
Ca2+ seen during depolarization and action potential generation induces gene activation (eg.
Guardiola-Diaz et al., 1994). However, our data from intact, hypovolemic animals would
argue against this possibility since significant depolarization of CRH neurons must coincide
with the increased ACTH release seen before gene activation. In this context, Luckman,
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Dyball & Leng (1994) have shown that in magnocellular neuroendocrine neurons the
presence of action potentials and thus, the ionic membrane fluxes per se do not activate c-
fos gene expression. Our data now suggest that this also applies for CRH gene activation
during sustained hypovolemia, in that depolarization and the propagation of action
potentials by the CRH neuron in itself does not immediately lead to the initiation of CRH
gene expression. In the absence of specific data on the receptor types responsible for
stimulating CRH secretion and gene expression during hemodynamic stress, we currently
are unable to distinguish between these models.
In Chapter 3, the temporal profile of the CRH gene response and plasma ACTH clearly
show that the stress-induced increase in circulating corticosterone present during the first 3
hours after onset of secretion does not act rapidly to inhibit CRH neuron or corticotrope
function, such that maximal activation of all indices measured within the HPA axis occurs
at the 5h time point. However, since CRH gene transcription remains activated between 5
and 6h, but mRNA levels markedly decline, corticosterone may act in a delayed time frame
to compromise mRNA stability. During this time frame, corticosterone may also begin to
suppress excitatory input to neuroendocrine neurons of the PVHmpd, which is evidenced
by the decline in CRH hnRNA cells numbers and the downward trend in c-fos mRNA
levels.
CORTICOSTERONE MANIPULATED RAT
To investigate corticosterone-mediated regulation of CRH gene expression in response to
stress, a common manipulation is to adrenalectomize rats and implant a corticosterone pellet
such that plasma levels remain relatively constant. In this way, aspects of MR- and/or GR-
mediated influences on CRH gene regulation may be studied. In ADX rats that have had
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corticosterone clamped for 6 days, sustained hypovolemia produces dose-dependent
changes in CRH gene transcription at the ‘peak’ time point (5h - in intact rat). Specifically,
ADX hypovolemic rats with low doses of corticosterone (insufficient to normalize thymus
weights) show an augmented CRH mRNA response compared with intact animals. When
corticosterone replacement is sufficient to normalize thymus weights, the magnitude of the
mRNA response is reduced to that seen in intact rats. Interestingly, the absence of
corticosterone compromises the ability of CRH transcriptional mechanisms to mount the
activated response seen in intact animals. In contrast to the CRH gene response, pENK
mRNA levels in the PVHmpd were unaffected by CORT concentrations. These results
suggest that corticosterone affects CRH gene transcription in the PVHmpd using two
mechanisms: first, facilitation, which is seen at low plasma concentrations and supports
gene transcription in the presence of sustained stress, possibly using MR-mediated
mechanisms; and second, inhibition which probably uses GR-dependent mechanisms and
contributes to classic negative feedback.
Corticosterone’s facilitatory nature is further supported by the ADX time course data
(Chapter 5) where a brief episode of CRH gene transcription was seen in hypovolemic rats,
however, was not maintained. This suggests that low levels of corticosterone (where MR
occupation predominates) are required to support CRH gene transcription during a
sustained stress event. Additionally, CRH gene transcription was upregulated in saline-
injected controls during the time when gene activation was reduced in the hypovolemic
groups. This suggests that two factors prevail during this period; (1) an activation of CRH
gene transcription in saline-injected controls, and (2) an inhibition of both the circadian
drive and excitatory input associated with sustained hypovolemia on CRH gene expression
in hypovolemia animals. Corticosterone may also participate in the overall excitability of
neurons in the PVHmpd since neuropeptides (CRH and pENK) and c-fos gene expression
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increased at earlier time points compared to intact rats.
Interestingly, in the PVHmpd, AVP and CRH gene transcription occurred concurrently in
ADX, PEG-injected animals while plasma ACTH levels showed a trend toward increase at
lh. That a level of significance was not reached at lh may be due to the extremely high
levels of plasma ACTH at the onset of the experiment (ie = 550 ng/ml), thus necessitating a
relatively larger increment to produce significant differences between treatment groups.
Considered together with earlier increases in c-fos and pENK mRNA, the data suggest that
one of the effects of corticosterone is to modulate HPA sensitivity to stress by establishing
separate thresholds for gene activation and most likely secretion by either regulating the
excitability of afferent input, signal transduction mechanisms, or both. The up-regulation of
AVP gene transcription in response to sustained hypovolemia is not surprising since AVP
expression is known to be more sensitive to circulating levels of corticosterone (Swanson
& Simmons, 1989; Ma et al., 1997; Kovacs 1998), regulated at least in part by a
glucorticoid response element (GRE) 592 bp upstream from the start site (Mohr & Richter,
1990). Additionally, AVP hnRNA levels have been shown to increase in response to ADX
alone (Wolfson et al., 1985; Albeck et al., 1994) and display an enhanced response to ether
stress compared to intact rats (Kovacs, 1998). However, the absence of corticosterone may
simply remove the mechanism(s) suppressing AVP gene transcription, lending the AVP
gene more susceptible to excitatory transduction signals. The maintenance of AVP gene
transcription (and most likely its release into the hypophysial portal vasculature) in the face
of declining CRH gene activation, but is sustained increase in ACTH levels suggest that
AVP is generated to maintain ACTH secretion in the face of prolonged activation of
neuroendocrine neurons in the PVHmpd. This apparent shift from CRH to AVP gene
transcription is similar to what is seen in intact, chronic stress models, where this change is
attributed to a maintenance of pituitary responsiveness.
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POTENTIAL MECHANISMS FOR CORTICOSTERONE-MEDIATED REGULATION
OF THE CRH GENE
There are at least three fundamental routes by which corticosterone may modulate negative
feedback on CRH gene expression: (1) alterations in cellular properties of CRH neuron, (2)
influence on steroid-sensitive afferent inputs, or (3) a combination of both.
Action on CRH gene transcription
Although CRH neurons express GRs (Aronsson et al., 1988; Swanson & Simmons,
1989), the CRH gene of the rat does not possess an obvious inhibitory GRE (iGRE:
Guardiola-Diaz et al., 1996), indicating that corticosterone negative regulation must be
indirect. Much information regarding GR function on CRH gene expression has been
gathered recently using transgenic mice. Functional GRs appear to be required for CORT-
dependent suppression since GR _ /‘ transgenic mice, lacking the GR, show increased
expression of CRH (Reichardt & Schiitz, 1996). Moreover, transgenic mice expressing
antisense RNA against GRs show decreased CRH peptide in the PVH, CRH and AVP
stores in the external zone of the median eminence (Dijkstra et al., 1998), as well as
augmented stimulus evoked ACTH responses and impaired glucocorticoid negative
feedback at the level of the hypothalamus (Karanth et al., 1997). Critically, direct DNA
binding does not appear to be necessary for CORT-mediated suppression, since GR^'m /y‘ m
mutant mice that possess GRs lacking the ability to bind to DNA show normal CRH
immunoreactivity in the median eminence (Reichardt et al. 1998). These transgenic mice
experiments support the idea that GR-dependent mechanisms negatively regulate CRH gene
transcription through indirect mechanisms that do not include direct DNA binding.
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With this in mind, a likely candidate mechanism for corticosterone negative regulation is
through GR interaction with signal transduction pathways. To this end, Legradi et al.
(1997) recently demonstrated that systemic administration of dexamethasone inhibited
phosphorylation of c-AMP regulatory element binding protein (CREB) induced by ether or
handling stressors in CRH neurons, suggesting that corticosterone may inhibit CRE-
dependent promoter activity. Although the CRH gene does not appear to have an AP-1 site,
forskolin and phorbol esters can up-regulate CRH gene expression in AtT-20 (Dorin et al.,
1989) and NPLC cells (Adler et al., 1990), respectively. A potential AP-2 binding site,
which may mediate responses to both PKA and PKC signaling systems, is located
approximately 150bp upstream from the initiation site (Majzoub et al., 1993) in the human
and rat CRH genes. This site may be susceptible to indirect GR-mediated inhibition via
protein-protein interactions (ie: ‘cross talk’ between AP-1 proteins and GRs: Diamond et
al., 1990; Konig et al., 1992; Guardiola-Dia et al., 1996; Heck et al., 1994).
Although the data presented in Chapter 3 show that secretion and gene transcription are
dissociable events, this does not necessary indicate that the two responses are under
separate signal transduction mechanisms. Therefore, it is worth considering aspects of
secretory stimulation as they will affect membrane properties of the CRH neuron and
potentially affect signal transduction pathways regulating gene transcription. With this in
mind, corticosterone is likely to interact with catecholaminergic transduction pathways,
particularly in viscerosensory stressors where brainstem afferents are most likely involved.
The general consensus is that catecholaminergic influence conveyed by the ventral
noradrenergic bundle (VNAB) stimulates CRH secretion (Pacak, 1995) and possibly CRH
gene expression since NA microinjection into PVH increase CRH mRNA levels (Itoi et al.,
1994). Moreover, dexamethasone (GR agonist) has been shown to reduce basal-, K+, or
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noradrenergic- stimulated CRH release in vitro (Hu et al., 1992; Bertini et al., 1993). In
organotypic hypothalamic slices, Szafarczyk et al. (1995) showed that noradrenaline (NA)
acts in a time- and dose-dependent manner to release CRH when corticosterone is present.
However, in the absence of corticosterone, NA stimulation is reversed and strongly inhibits
CRH release. Interestingly, VNAB lesions that deplete catecholamines, inhibited ether-
induced ACTH and corticosterone surges in rats. These results were reversed when
animals were also adrenalectomized (Gaillet et al., 1993) and restored when oral
corticosterone supplement was provided, suggesting that ascending catecholaminergic
pathways are modulated in opposing directions by the presence of plasma corticosterone.
Additionally, it has been shown that corticosterone levels can affect the expression of
catecholaminergic receptor subtypes in the PVHmp (ie a -, B-receptors: Jhanwar-Uniyal &
Leibowitz, 1986; Kiem et al., 1995) which in turn could feasibly affect signal transduction
pathways within the CRH neuron. Recently Feuvrier et al., (1998) reported that in anterior
hypothalamic slices, glucocorticoids provoked a shift from a -2 to a -1 adrenoreceptor
activity that led to opposite NA effect on CRH release suggesting that the ratio of the two
receptor subtypes direct the effects of noradrenergic modulation during stimulation.
Because sustained hypovolemia progresses over a course of hours, these types of changes
may be pertinent to the maintenance of neuropeptide gene expression in the PVHmpd.
Action on afferent input to the CRH neuron
Neuronally mediated inhibition has been proposed as a major contributor for feedback
regulation on the HPA axis. GR-mediated events may act via three primary routes: (1)
suppression of neural excitability, (2) increase inhibitory drive, (3) or combination of both.
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Suppress excitatory input
There is evidence to suggest that corticosterone can act rapidly to suppress the firing
activity of many neurons, including the cells in the PVH, both in vitro and in vivo (Kasai &
Yamashita, 1988; Saphier & Feldman, 1988; Chen et al., 1991). It is important to note that
direct application of corticosteroids to PVH neurons does not mimic what occurs naturally
in the intact rat and therefore, its physiological relevance remains to be determined. With
this in mine, iontophoretically applied cortisol or corticosterone (Joels & de Kloet, 1989;
Feldman & Davney, 1970) has been shown to alter the ratio of facilitatory-.inhibitory
hypothalamic neuronal responses to sensory stimuli. Electrophysiological studies in vivo
have demonstrated a suppression in firing activity of PVH neurons with application of
cortisol, effects that could be blocked with the GR antagonist, RU38486 (Saphier &
Feldman, 1988; Chen et al., 1991). These inhibitory effects of corticosterone may be
related to suppression of CRH release rather than to alterations in synthesis (Jones &
Hillhouse, 1976; Jones et al., 1977) since corticosterone can rapidly inhibit stimulated
CRH secretion (Vermes et al., 1977) and stimulated ACTH secretion (Buckingham &
Hodges, 1977; Vale & Rivier, 1977). Since the majority of studies have concentrated on
CRH secretion, little is known about the actual mechanisms regulating corticosterone
modulation on CRH gene transcription and mRNA accumulation. What has been reported
indicates that corticosterone does not act rapidly to suppress CRH gene expression
(Lightman & Young, 1989). Recently, Ma et al. (1997) showed that a high concentration
of corticosterone injected into the ADX rat does not reduce CRH hnRNA levels for 2h and
CRH mRNA levels for 4h. Interestingly, they also reported that AVP hnRNA levels were
decreased within 15 minutes after the administration of corticosterone. These data suggest
that corticosterone negative regulation acts rapidly on AVP gene transcription, whereas
influences on CRH gene transcription occurs within a delayed time frame.
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Increase inhibitory drive
Corticosterone may also act to enhance GABAergic tone. Sutanto et al. (1989, 1993)
demonstrated that corticosterone can enhance GAB A turnover in the hypothalamus,
suggesting that increased GABAergic influence may govern inhibitory control over PVH
activity. The hippocampus is considered the primary candidate for negative regulation of
the HPA axis both in the basal and stressed states (Herman & Cullinan, 1997). Herman
and colleagues have proposed that the ventral hippocampus provides a tonic inhibitory tone
on CRH neurons in the PVHmp; where the excitatory outflow from the ventral
hippocampus to the ventral subiculum maintains inhibition on CRH neurons in the
PVHpmd by way of the BST. This concept is supported by studies demonstrating
hippocampal ablation or transection of the fornix increase CRH and AVP gene expression
in the PVHmp (Herman et al., 1989; 1992b), secretion of CRH and AVP into portal blood
(Sapolsky et al., 1989), and hypersecretion of ACTH and corticosterone in the nonstressed
rat (Feldman & Confronti, 1980; Wilson et al., 1980; Herman et al., 1989). Conversely,
stimulation of the hippocampal formation has been shown to inhibit neurosecretory neurons
in the PVH (Saphier & Feldman, 1987) and diminish circulating corticosterone (Dunn &
Orr, 1984). It is Herman’s contention that stress-activated circuits increase the excitability
of hippocampal neurons along with other GABAergic projections to the PVH and increase
inhibitory tone on CRH neuroendocrine neurons (Herman & Cullinan, 1997). Sapolsky et
al. (1991) showed that hippocampal damage augments and prolongs stress-induced
corticosterone secretion in rat and primate.
Roland & Sawchenko (1993) have provided neuroanatomical data demonstrating PVH-
projecting GABAergic neurons in the perinuclear zone. They postulated that this projection
may serve as a relay from which limbic structures may exert inhibitory influence on the
PVH since the perinucleur zone receives afferents from amygdala, ventral subiculum and
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infralimic cortex (Swanson, 1983). Recently, Boudaba and coworkers (1996) rendered a
three dimensional model depicting a ring-like area of GAB A containing neurons
surrounding and projecting to the PVHp. This group also provided electrophysiological
evidence to support an inhibitory role for this region.Specifically, by using intracellular and
whole-cell patch-clamp recordings, they demonstrated that glutamatergic microstimulation
within the area surrounding the PVH, produces a marked suppression in firing rates in both
parvicellular and magonocellular neurons. Together, these data support the contention that
the perinuclear zone is in a position to exert an inhibitory influence on PVH function
originating from limbic structures.
MODEL FOR CORTICOSTERONE-MEDIATED REGULATION OF CRH NEURON
The current dogma of steroid receptor mediated effects on HPA axis activity is that MR-
dependent actions maintain basal activity; whereas, higher levels of corticosterone seen
during the circadian peak and at times of stress, act via GR-mediated events to constrain
HPA activity (Dallman, et al., 1987; de Kloet & Reul, 1987; de Kloet et al., 1993;
Herman, 1997). The model presented here does not dispute current thinking; rather, it
offers a complementary mechanism by which the CRH neuron can maintain responsiveness
in the presence of high levels of corticosterone.The concept relies heavily upon
considering the stress response as a dynamic event, and therefore requires reevaluation by
the brain on a moment to moment basis. Where corticosterone modulation of cellular
properties of many neurons occur in a time and state dependent manner and are not
considered soley to bring about suppressive effects on HPA function. Thus, the
responsiveness of the CRH neuron is dependent upon the presence or absence of excitatory
drive, its intensity and GR dependent modulation at multiple sites.
In the basal state during the circadian trough, MRs are predominantly occupied and
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subsequently maintain tonic inhibition on the CRH neuron (Fig 6.1 A). If a viscerosensory
stressor exceeds the threshold necessary to activate the HPA axis, stress-induced increases
of corticosterone produce widespread occupation of GRs throughout the brain (Fig. 6. IB).
If the stressor is removed (ie acute stress), the decrease in excitatory drive and GR-
mediated suppression work together to terminate the response (Fig 6.1C). If the stressor is
maintained for a prolonged period of time (> 30 mins), GR-mediated events act to maintain
HPA responsiveness by reducing excitatory output from the ventral hippocampus which in
turn, decreases inhibitory tone from the BST to the PVH (Fig. 6. ID). This disinhibitory
effect would increase the sensitivity of CRH neuroendocrine neurons to steroid-sensitive
and insensitive afferent input.
Mechanism by which hippocampal outflow is affected by corticosterone receptor
occupation
de Kloet and Joels have played a critical role in differentiating the properties of MR- and
GR-mediated effects on neuronal excitability of hippocampal neurons, focusing on CA1
cells. They have shown that in most cases, the two receptors work in opposition and are
critically dependent upon the ratio of receptor subtype occupation (Vidal et al., 1986; Talmi
et al., 1992; Joels & Frenhout, 1993; Joels et al., 1994). Interestingly, GR dependent
actions are conditional in that they are not apparent under resting conditions (Joels & de
Kloet, 1989; Kerr et al., 1989). It is only after cells are shifted away from the resting
potential (ie depolarization, hyperpolarization) that steroid-mediated ionic conductances
emerge (Karst et al., 1993). Specifically, predominant MR occupation is associated with a
stable flow of fast synaptic transmission (Joels & de Kloet, 1989); whereas, concurrent
activation of MRs and GRs produce an opposite pattern that requires a minimum of 30
minutes to 1 hours to develop (Joels & de Kloet, 1989, 1990; Kerr et al., 1989) and de
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FIG. 6.1 Model for corticosterone-mediated regulation of the CRH neuron. A, In the basal
state, during the circadian trough, MR occupation predominates and maintains tonic
inhibition of the CRH neuron. B, Once the stressor exceeds the secretory threshold,
secretion of CRH, ACTH, and corticosterone is stimulated. C, If the stressor is removed,
the decrease in the excitatory drive and GR-mediated suppression work together to teminate
the response. D, If the stressor is sustained for a prolonged period of time (> 30 minutes),
GR-mediated events act to maintain HPA responsiveness by reducing excitatory output
from the ventral hippocampus which in turn, decreases inhibitory tone from the BST to the
PVH. This disinhibition would increase the sensitivity of the CRH neuroendocrine neurons
to steroid-sensitive and insensitive afferent input.
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a
l
<
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STRESSOR STRESSOR
novo protein synthesis (Karst, & Joels, 1991). As such, the synaptic transmission is not
initially different, but the neuronal responsiveness starts to decline with repeated
stimulation. Thus, exposure to high levels corticosterone, in the presence of sustined
stimulation, help to attenuate the synaptic response through activation of the slow Ca-
dependent K-conductance.
Depolarizing current pulses have been shown to activate a slow calcium dependent K-
conductances (/A H P) which is slowly deactivated at the end of the pulse (Hotson & Prince,
1980; Gustaffson & Wigstrom, 1981; Madison & Nicoll, 1984). The continuous activation
of the /A H p slowly attenuates cell firing (accommodation) and produces a transient
afterhyperpolarization (AHP). With selective MR-occupation, there is an increase in action
potentials (ie decrease in AHP/accommodation amplitude and AHP duration: Joels & de
Kloet, 1989). Additionally, with predominant MR occupation but also partial GR
occupation, the activation of /A H p produces only a transient increase in the number of action
potentials. However, simultaneous MR and GR binding results in a suppression of action
potential (ie increase in AHP/accommodation: Joels & de Kloet, 1989, 1990; Kerr et al.,
1989). This decrease in excitability only developed after a delay of approximately lh (Joels
& de Kloet, 1990, 1993) and depended upon de novo synthesis (Karst & Joels, 1991).
Thus, it would appear that MRs serve to maintain a level of excitatory outflow from CA1
area, while concurrent GR-mediated activation would lead to suppression of local activity
and result in decreased transmission of excitability from the CA1 area (Joels & de Kloet,
1990).
If MR- and GR-mediated events occur as proposed and the ventral hippocampus provides
an inhibitory influence on CRH neuroendocrine neurons of the PVHmpd as hypothesized it
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is conceivable to see how GR-dependent disinhibition would enable the CRH neuron to
remain responsive during a sustained stress event and elevated levels of circulating
corticosterone. In the case of sustained hypovolemia in the intact rat, it is clear that
excitatory input to the CRH neuron is sufficient to stimulate secretion but does not initially
induce CRH gene transcription. As the intensity of the stressor increases along with
markedly elevated levels of corticosterone and therefore concurrent GR occupation, it is
possible that one aspect of GR-mediated events is to produce disinhibition from the ventral
hippocampus to facilitate CRH responsiveness to stressor-specific afferents (ie
baroreceptor and AH). This would explain how CRH gene transcription could be
maintained for a prolonged period of time in the face of elevated corticosterone levels.
Most certainly, this pathway is but one manner in which varying levels of corticosterone
modulate CRH gene expression in the basal or stressed state. This is emphasized by the
presence of both receptor subtypes in many stress related nuclei (ie NTS, locus coeruleus,
amygdala, lateral septum) that undoubtedly can influence afferent information to the CRH
neuron in a time and state-dependent manner. Again, it appears necessary to consider the
stress response as a dynamic event that is being evaluated by the CRH neuroendocrine
neuron on a moment to moment basis to determine the onset, maintenance and termination
of the HPA axis response to stress.
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Appendix
Abstracts
Sanchez-Watts G, Tanimura SM, Watts AG Hypovolemia increases the levels and the
extent of co-localization of neurotensin and enkephalin in hypothalamic CRH-containg
neurons. 23rd Annual Meeting of the Society for Neuroscience,Washington DC 1996, p
392.17
Tanimura SM, Sanchez-Watts G, Watts AG Corticosterone modulates corticotropin-
releasing hormone gene expression associated with hypovolemia in the paraventricular
nucleus of the hypothalamus. 26th Annual Meeting of the Society for Neuroscience,
Washington DC 1996, p 335.9
Tanimura SM, Watts AG Corticosterone facilitates as well as inhibits CRH gene expression
in the rat hypothalamic paraventricular nucleus. 27th Annual Meeting of the Society for
Neuroscience, New Orleans, LA 1197, p 392.4
Articles
Tanimura SM, Sanchez-Watts G, Watts AG (1998) Peptide gene activation, secretion, and
expression in the rat hypothalmic paraventricular nucleus. Endocrinol 139: 3822-3829
Tanimura SM, Watts AG (1998) Corticosterone can facilitate as well as inhibit
corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular
nucleus. Endocrinol 139: 3830-3836
Tanimura SM, Watts AG (1999) Adrenalectomy dramatically modifies the dynamics of
neuropeptide and c-fos gene responses to stress in the hypothalamic paraventricular
nucleus. (Submitted)
108
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Creator Tanimura, Susan Mariko (author)

Core Title Corticosterone modulation of stress-induced neuropeptide gene expression in the paraventricular nucleus

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Neuroscience

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,biology, neuroscience,OAI-PMH Harvest

Language English

Advisor Watts, Alan G. (committee chair), Baudry, Michael (committee member), McClure, William (committee member), Stallcup, Michael (committee member), Swanson, Larry (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-592303

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