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1 Neuroscience Graduate Program and 2 Department of Psychiatry and Behavioral Sciences, University of Texas Medical Branch, Galveston, Texas 77550-0431
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We have previously hypothesized that
corticotropin-releasing hormone (CRH) is involved in the regulation of
physiological waking. To further elucidate this role for CRH, we
administered intracerebroventricularly into rats two specific
CRH-receptor antagonists, 
-helical CRH-(9
41) (-hCRH) or
astressin, and determined changes in electroencephalogram-defined
waking and sleep. Our results indicate that both of these receptor
antagonists reduce the amount of time spent awake in a dose-related
manner when administered before the dark period of the light-dark
cycle. However, the time courses for these effects differ between
antagonists; effective doses of -hCRH reduce waking during the first
2 h postinjection, whereas effective doses of astressin reduce waking
during postinjection hours 7-12. In contrast to
dark-onset administrations, the amount of waking is not altered by
either CRH-receptor antagonist when administered before the light
period. These results support our hypothesis that CRH contributes to
the regulation of physiological waking, since interfering with the
binding of CRH to its receptor reduces spontaneous waking.
sleep; hypothalamic-pituitary-adrenal axis; astressin; antagonist; partial agonist; stress
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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CORTICOTROPIN-RELEASING HORMONE (CRH) is the major mediator of behavioral, physiological, and autonomic responses to a variety of stressors. CRH, expressed in widely distributed regions of the central nervous system (CNS), mediates the hypothalamic-pituitary-adrenal (HPA) axis and central autonomic components of responses to stressors (12, 38). Behavioral responses to most stressors include periods of cortical arousal and electroencephalogram (EEG)-defined waking. In addition to stressor-induced alterations in sleep, we have previously hypothesized that CRH may also contribute to the regulation of physiological waking in the absence of stressors. Much of the evidence supporting this hypothesis is indirect. For example, CRH is found in cerebrospinal fluid (27, 33, 34, 44), where it exhibits circadian rhythms in humans and other primates (20, 23, 27, 39). In addition, the circadian fluctuation of plasma concentrations of ACTH and cortisol or corticosterone, which are lowest before the major sleep time and peak around the beginning of the active period in humans (19, 28), rhesus monkeys (26), and rats (21, 29), is temporally associated with waking and sleep. Additional evidence supporting CRH acting within the CNS to modulate and/or regulate waking comes from studies in which central or systemic administration of CRH into rats (4, 13-15, 30), rabbits (37), and humans (2) increases EEG-defined waking. Furthermore, genetically related rat strains that differ in the synthesis and/or secretion of CRH differ in the amount of time spent awake; reduced CRH synthesis is associated with reduced amounts of waking (36).
To further test the hypothesis that CRH contributes to the regulation
and/or modulation of waking, we administered centrally before
both the light period and dark period of the light-dark cycle two
specific CRH-receptor antagonists, -helical CRH-(941) (-hCRH)
and
cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]-rat/human-CRH-(1241)
(astressin). We now report that these two CRH-receptor antagonists
reduce spontaneous waking in the rat during the dark period of the
light-dark cycle, the time when CRH concentrations and HPA axis
activity are greatest and rats are most active. During the light
period, however, when endogenous CRH concentrations and HPA axis
activity are low and rats sleep the most, these antagonists have no
effect on waking and sleep. In contrast, administration of CRH itself
increases waking regardless of the timing of administration. These
results support our hypothesis that CRH contributes to the regulation
and/or modulation of waking.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Substances
Stock solutions of-hCRH (Peninsula Laboratories, Belmont, CA),
astressin (Bachem, Torrance, CA), and CRH (Peninsula Laboratories) were
prepared in pyrogen-free saline (PFS). Aliquots of these stock
solutions were stored at 
70°C until use, when they were then
brought to an appropriate injection volume. The doses of the substances
used in these experiments were as follows: for -hCRH, 0.26, 1.3, 6.5, and 13 nmol (1, 5, 25, and 50 µg, respectively); for astressin,
0.14, 0.7, and 3.5 nmol (0.5, 2.5, and 12.5 µg, respectively); and
for CRH, 0.05 and 0.1 nmol (0.25 and 0.5 µg, respectively).
Animals
A total of 68 male Sprague-Dawley rats (250-300 g; Harlan, Indianapolis, IN) were used in these experiments. These animals were anesthetized (87 mg/kg ketamine and 13 mg/kg xylazine) and injected with an analgesic [butorphanol tartrate (Torbugesic)] and a broad-spectrum antibiotic [penicillin G benzathine (Bicillin LA)]. The rats were surgically implanted with EEG screw electrodes, a guide cannula directed into the lateral ventricle, and a calibrated 30-k
thermistor (model 44008, Omega Engineering,
Stamford, CT) to monitor brain temperature
(Tbr) at the surface of the
cortex as previously described (36). Insulated leads from the EEG
electrodes and the thermistor were routed to a Teflon pedestal
(Plastics One, Roanoke, VA) cemented to the skull with dental acrylic
(Isocryl, Lang Dental Supply, Wheeling, IL). The incision was treated
topically with polymixin B sulfate-bacitracin zinc (Polysporin), and
the animals were placed under heat lamps and monitored until recovery from anesthesia. The animals were allowed a minimum of 7 days of
recovery from the surgical procedures before the initiation of
experiments. The rats were housed in individual recording cages, with
two cages in each environmentally controlled chamber (model 352601, Hotpack, Philadelphia, PA). The chambers were maintained at 23 ± 1°C with a 12:12-h light-dark cycle (25-W incandescent bulb, ~200
lx at cage height). Food and water were available ad libitum. All
procedures performed in these studies were approved by the local Animal
Care and Use Committee in accordance with the US Department of
Agriculture Animal Welfare Act and the National Institutes of Health
policy on Humane Care and Use of Laboratory Animals.
Experimental Protocol
On the 2nd postsurgical day, the rats were connected to the recording apparatus (see below) via a flexible tether that was attached to the pedestal on the rat's head. As such, the animals were allowed relatively unrestricted movement within the cage. Three days after surgery, the patency and free drainage of the intracerebroventricular cannulas was assessed by administering 200-400 ng ANG II (human ANG II octapeptide, Peninsula Laboratories); angiotensin elicits a drinking response mediated by structures in the preoptic area. At the end of each experimental protocol, all rats were again injected with angiotensin; only data from those rats exhibiting a positive drinking response were included in the subsequent analyses. For the next 4-5 days, the animals were habituated by daily handling, and intracerebroventricular injections of PFS were timed to coincide with scheduled experimental administrations.Intracerebroventricular administration of -hCRH.
Three groups of animals were used to determine the effects of -hCRH
on waking: two groups for experiments beginning at dark onset and one
group for experiments beginning at light onset. Group
1 (n = 8) was
administered vehicle (PFS) intracerebroventricularly on the 1st
recording day and subsequently received 0.26 and 1.3 nmol of -hCRH
on the 2nd and 3rd recording days. These intracerebroventricular injections were administered as a 3-µl bolus over a 2-min period. Group 2 (n = 8) received PFS on the 1st
recording day and 6.5 nmol of -hCRH on the 2nd recording day. In
this experimental protocol, 6.5 nmol of -hCRH was the highest dose
used, since concentrations greater than this exhibit partial agonist
properties (e.g., Refs. 1, 42 and below). The injection volume used for
these animals was 5 µl over an ~3-min period, since it was difficult to get this dose of -hCRH into solution in a smaller volume. The intracerebroventricular injections given to
groups 1 and
2 were initiated 20 min before dark
onset, and recordings were initiated at dark onset and continued for
24 h.
-hCRH on the 2nd and 3rd recording days,
respectively. The injections were administered over a 2-min period.
Recordings began at light onset and continued for 24 h.
Intracerebroventricular administration of astressin.
Essentially the same protocol outlined above for -hCRH was used to
determine effects of astressin on spontaneous waking. Group 4 (n = 8) was administered PFS on the
1st recording day and subsequently received 0.14 nmol of astressin on
the 2nd recording day. Group 5 (n = 12) received PFS on the 1st
recording day and 0.7 and 3.5 nmol of astressin on the 2nd and 3rd
recording days, respectively. The intracerebroventricular injections
for groups 4 and
5 consisted of a 3-µl bolus
administered over a 2-min period. These injections started 20 min
before dark onset; recordings were initiated at dark onset and
continued for 24 h.
Intracerebroventricular administration of CRH and a high dose of
-hCRH.
Groups of rats were also used to determine the effects of CRH peptide
itself on waking. Group 7 (n = 8) received 3 µl of PFS on the
1st recording day. Two days later, these animals were injected with
0.05 nmol of CRH. After another 2-day interval, 0.1 nmol of CRH was
injected intracerebroventricularly into these animals. These injections
of CRH were given as a 3-µl bolus beginning 20 min before dark onset,
at which time 24-h recordings were initiated. Two days after the
completion of the final recordings of responses to CRH, these same rats
were injected with a high dose (13 nmol) of the CRH-receptor
antagonist, -hCRH. As previously mentioned, -hCRH at doses above
6.5 nmol is a partial agonist. We elected to administer a dose of
-hCRH at twice the reported threshold dose for partial agonist
actions when administered intracerebroventricularly. In this way, we
could directly compare responses of rats to the agonist (CRH) with
responses in the same animals to a high dose of -hCRH exhibiting
mixed agonist-antagonist actions to evaluate the degree of agonist
actions of -hCRH in our rat sleep-assay system.
Intracerebroventricular administration of this high dose of -hCRH
was given before dark onset. Recordings were initiated at dark onset
and continued for 24 h.
-hCRH administered before light
onset, group 8 was also injected
intracerebroventricularly with 13 nmol of -hCRH 2 days after the
last administration of CRH.
Apparatus and Recording
Signals from the EEG electrodes and thermistors were fed into amplifiers (Colbourn Instruments, Lehigh Valley, PA, models S75-01 and S71-20, respectively). The EEG was amplified (factor of 3,000) and analog band-pass filtered between 0.1 and 40 Hz (frequency response ±3 dB; filter frequency roll off 12 dB/octave). The EEG amplifiers were calibrated before the initiation of these experiments by use of a Bio-Systems Calibrator (Colbourn Instruments). Similarly, the amplifiers used to record Tbr were calibrated with a thermistor and water bath. Gross body movements were detected by custom-built ultrasonic detectors (Biomedical Instrumentation, University of Tennessee, Memphis, TN). These conditioned signals (EEG and Tbr) as well as those from the movement detectors were subjected to analog-to-digital conversion with 12-bit precision at a sampling rate of 128 Hz (AT-MIO-64F5, National Instruments, Austin, TX). The digitized EEG waveform, Tbr samples, and integrated values for body movements were stored as binary computer files until subsequent analyses.Postacquisition determination of vigilance state was done by visual scoring of 12-s epochs using custom software written in LabView for Windows (National Instruments) as previously described. In addition to the amount of time spent in each vigilance state, the number and duration of individual bouts of behavior were determined using criteria modified from those of Tobler and colleagues (7, 18), as previously described (36). Briefly, a wakefulness (Wake) bout is considered initiated if three consecutive 12-s epochs are scored as Wake (36-s) and terminated if three consecutive epochs are scored as slow-wave sleep (SWS). Wake bouts with a duration of >60-min are eliminated from subsequent statistical analyses because such bouts encompass parts of either two or three individual hourly time blocks and therefore skew subsequent determination of hourly bout duration. This exclusion criterion has previously been applied to studies of rat sleep-wake architecture (36, 45). A SWS bout is considered initiated if three consecutive 12-s epochs (36 s) are scored as SWS and terminated if two consecutive 12-s epochs (24 s) are scored as either Wake or rapid-eye-movement sleep (REMS). A REMS bout is initiated by two consecutive 12-s epochs (24 s) being scored as REMS and terminated if two consecutive 12-s epochs (24 s) are scored as either SWS or Wake. Finally, transitions from one state to another are determined from consecutive 12-s epochs without regard to criteria for episodes of vigilance state defined above. Therefore a transition is considered to occur if two consecutive 12-s epochs are scored dissimilarly.
Statistical Analyses
All values are presented as means ± SE for the indicated sample sizes. One-way ANOVA for the duration of each vigilance state (SWS, REMS, and Wake) for Tbr values and for sleep architecture parameters were performed across the two 6-h time blocks comprising either the 12-h light period or the 12-h dark period. The main effect consisted of manipulation (vehicle,-hCRH,
astressin, or CRH peptide doses). If statistically significant
differences were detected, post hoc comparisons were made to determine
which hourly intervals during experimental conditions deviated from values obtained from the same animals during control conditions. An
-level of P < 0.05 was taken as
indicating a statistically significant difference between vehicle and
active substances.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Effects of Intracerebroventricular Administration of
-hCRH
-hCRH before dark onset
transiently reduced spontaneous waking in a dose-related manner.
One-way ANOVA across 6-h time blocks did not reveal consistent alterations in waking or REMS after intracerebroventricular
administration of any dose of -hCRH (Table
1). However, the amount of time spent in
SWS increased significantly after the 6.5-nmol dose of -hCRH (Table
1). Visual inspection of the data revealed that there were consistent
and reproducible reductions in waking and increases in SWS during the
first 2-h postinjection (Fig. 1). Therefore
subsequent statistical analyses were confined to this 2-h postinjection
period. The two lowest doses of -hCRH (0.26 and 1.3 nmol) tended to
reduce waking and enhance SWS during the first 2-h postinjection,
although these changes did not achieve statistical significance (data
not shown). The amount of time spent awake during this 2-h period after
the 6.5-nmol dose was reduced from 75.1 ± 3.7% of recording time
after vehicle to 58.9 ± 4.2% of recording time after -hCRH
(P < 0.01). This reduction in waking
was mirrored by increases in the amount of SWS (Fig. 1). Although there
was a slight increase in REMS during the first 2-h postinjection, this
difference did not achieve statistical significance.
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The reduction in waking during the 2-h postinjection period after 6.5 nmol of -hCRH was primarily due to a decrease in Wake bout duration.
Analyses of sleep architecture parameters across postinjection
hours 1-6 did not reveal statistically significant alterations in sleep-wake architecture, although the numbers of transitions from one state of vigilance to another was increased significantly after administration of 6.5 nmol of -hCRH, indicating that waking was fragmented (Table 2).
However, analyses restricted to the 2-h postinjection period when the
amount of time spent awake was reduced indicated that the duration of
Wake bouts reduced from 7.1 ± 2.1 min after vehicle to 2.3 ± 0.7 min after -hCRH (P < 0.05).
The number and duration of SWS bouts were not statistically altered by
this manipulation.
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In contrast to intracerebroventricular administration before dark
onset, the 0.26- to 6.5-nmol doses of -hCRH when injected before
light onset failed to alter any aspect of sleep-wake activity determined in these experiments during any time point (Fig. 1 for
responses to 6.5-nmol dose; other data not shown). Similarly, Tbr was not altered by any of the
doses of -hCRH used in these experiments, regardless of the timing
of administration (data not shown).
Responses to Intracerebroventricular Administration of Astressin
Intracerebroventricular administration of astressin also reduced spontaneous waking in a dose- and time-related manner. When injected intracerebroventricularly before the dark period, each dose of astressin used in this experiment tended to slightly reduce waking and increase SWS during postinjection hours 1-6; these minor alterations in behavior did not deviate statistically from control values (Table 1). During postinjection hours 7-12, however, the amount of time spent in waking was reduced, but the amount of time spent in SWS was increased in a dose-related manner (Table 1, Fig. 2); most of these effects were confined to postinjection hours 7-10 (Fig. 2). During this 4-h period, the amount of time spent awake was reduced from 77.0 ± 3.3% of recording time after vehicle to 63.4 ± 3.1% ofrecording time after 3.5 nmol of astressin (P < 0.005; Fig. 2). This reduction in waking was accompanied by increases in SWS, whereas REMS was not consistently altered.
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During postinjection hours 1-6, there were no
consistent alterations in sleep-wake architecture, although the number
of transitions from one state to another were increased after the
3.5-nmol dose (Table 3). There were,
however, statistically significant alterations in sleep-wake
architecture parameters during postinjection hours 7-12. During this 6-h time period, bout durations for Wake
were reduced (Table 3). During postinjection hours
7-10, when waking was significantly reduced, the Wake bout
duration decreased from 11.3 ± 2.3 to 6.7 ± 1.3 min after 0.7 nmol of astressin and 5.7 ± 1.4 after 3.5 nmol of astressin. In
addition, during this same 4-h time period, the number of transitions
from one state of vigilance to another increased from 30.7 ± 3.4 after administration of vehicle to 39.7 ± 2.6 after the 3.5-nmol
doses of astressin, indicating a fragmentation of normal sleep-wake
activity. As with -hCRH, astressin when injected before light onset
failed to alter any aspect of sleep-wake activity determined in these
experiments (Fig. 2 for 3.5-nmol dose; other data not shown).
Similarly, Tbr values were not
altered by any dose of astressin regardless of time of administration
(data not shown).
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Intracerebroventricular Administration of CRH and High Doses of
-hCRH
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The groups of rats that received CRH also received a high dose (13 nmol) of the CRH-receptor antagonist -hCRH. No statistically significant departures from controls were revealed after analyses of
6-h time blocks when 13 nmol of -hCRH was administered before dark
onset (Table 1). However, visual inspection of the data revealed a
consistent and reproducible reduction in waking concurrent with
reductions in SWS and REMS during the 2nd postinjection hour after this
dose of -hCRH (Fig. 3). When confined to hourly time blocks,
statistical analyses indicated a significant departure from control
values during this time point. In marked contrast, however,
intracerebroventricular administration of this dose of -hCRH before
light onset increased waking and reduced SWS during the 1st
postinjection hour (Fig. 3). During postinjection hour 1 after -hCRH, waking was increased from 28.5 ± 3.0% of recording time after vehicle to 67.7 ± 10.1% of recording time after
-hCRH. This increase in waking was at the expense of SWS; REMS was
not affected (Fig. 3). During the 1st postinjection hour after the low
dose (0.05 nmol) of CRH, waking increased from 28.5 ± 3.0% after
vehicle to 81.4 ± 12.2%, a value statistically indistinguishable from the 67.7 ± 10.1% observed after 13 nmol of -hCRH during this same time period in the same animals (Fig. 3); i.e., the increase
in waking induced by 13 nmol of the CRH antagonist -hCRH and a low
dose (0.05 nmol) CRH were identical. The 1-h increase in Wake after
this dose of -hCRH during the light period was due to an increase in
Wake bout duration, which increased from 3.6 ± 0.4 min after
vehicle to 27.5 ± 9.3 min after -hCRH.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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There is ample indirect evidence to suggest that CRH may be involved in the regulation of physiological waking in addition to its role as a mediator of responses to stressors (reviewed in Ref. 35). We now extend these observations and provide direct evidence that CRH may be involved in the regulation of waking by reporting that two separate and specific CRH-receptor antagonists reduce waking in a dose- and time-dependent manner in freely behaving animals.
-hCRH is a selective and competitive CRH-receptor antagonist that
was initially characterized for its ability to inhibit CRH-induced ACTH
secretion in vitro (43). -hCRH binds to CRH receptors with ~10
times less potency than does CRH itself (5). Although it is not very
potent, -hCRH has been widely used to elucidate mechanisms whereby
CRH contributes to responses to stressors and is more effective when
administered into the CNS than when administered systemically (17). For
example, -hCRH administered intracerebroventricularly into rats
inhibits CRH- or ether stress-induced ACTH secretion (43), blocks
CRH-induced changes in locomotor activity and conflict test responding
(3), reverses the stress-induced inhibition of exploratory behavior
(24), reverses the "anxiogenic" responses to ethanol withdrawal
(1), and reduces measures of "emotionality" in socially defeated
animals (25). These and many other similar studies in which -hCRH
has been used as a CRH antagonist provide much of the evidence that
indicates a pivotal role for CRH as a mediator of responses to
stressors.
Our present study extends the aforementioned observations of the
involvement of CRH in responses to stressors to include behavior of
normal, nonstressed rats freely behaving in their home cages. Rats that
are well habituated to handling and injection procedures respond to
-hCRH with a transient reduction in the amount of physiological
waking. The reduction in waking induced by 6.5 nmol (25 µg) of
-hCRH lasts only 2 h. This relatively short effect on sleep-wake
behavior is similar in duration to that reported in other studies using
-hCRH and may be due to a relatively short half-life. For example,
intravenous administration of -hCRH into adrenalectomized rats
reduces plasma ACTH concentrations for 2 h (43). The ability of
-hCRH to reduce waking is dose related; low doses [0.26 and
1.3 nmol (1 and 5 µg, respectively) in our study] tend to
reduce waking in the 2nd postinjection hour, whereas a statistically
significant reduction in waking in the first 2-h postinjection period
is apparent after administration of 3.5 nmol (25 µg). This effective
dose of -hCRH in our rat sleep assay of freely behaving animals is
generally similar to doses reported to be effective in blocking or
attenuating behavioral responses to stressors. However, in some models
of stressor-induced alterations in behavior, -hCRH may effectively
attenuate responses at doses in the range of 1-5 µg (e.g., see
Ref. 24, 25). That -hCRH is effective at lower doses in interfering
with behavioral responses to stressors is probably due to the fact that
CRH concentrations are elevated well above basal values in response to
stressors.
Our observation that spontaneous waking in the rat is reduced when
-hCRH is administered intracerebroventricularly into freely behaving
rats is in contrast to that recently reported by Gonzalez and Valatx
(20a).These authors report that a single 100-µg (~26-nmol) dose of
-hCRH administered before dark onset does not alter spontaneous waking in the rat, although it is effective in blocking alterations in
REMS induced by immobilization stress. These authors conclude that CRH
is not involved in the regulation or modulation of spontaneous waking.
This conclusion is problematic, however, because -hCRH is widely
reported to exhibit partial agonist activity (1, 10, 24, 25, 32, 41,
42, 47), particularly at doses of >25 µg. The partial agonist
effect of high doses of -hCRH is clearly observed in the results
obtained in our experiments when 13 nmol (50 µg) of -hCRH is
administered before the light period, the time when endogenous
concentrations of CRH in the rat are at their lowest. In the 1st
postinjection hour, waking increases after this dose of the antagonist
by an amount statistically indistinguishable from the increase in
waking that occurs during this same time period after administration of
a low dose (0.05 nmol) of CRH itself. As such, high doses of the CRH
antagonist -hCRH may be functionally equivalent to low doses of CRH.
It is therefore not surprising that when 100 µg of -hCRH is given before the dark period of the light-dark cycle, when endogenous concentrations of CRH in the rat are at their greatest, there is no
detectable alteration in CRH-mediated behavior, since, in effect, a
functional low dose of CRH has been added to a system in which
endogenous CRH is already high. When in our studies this 13-nmol
(50-µg) dose of -hCRH was administered before the dark period,
some antagonist actions were apparent. However, the magnitude of these
alterations more closely resembled the middle dose (1.3 nmol or 5 µg)
than the highest effective dose (6.5 nmol or 25 µg). The fact that
-hCRH possesses partial agonist actions is one of several factors
that has hampered the use of this antagonist in basic and preclinical
research. As such, the development of specific and potent CRH
antagonists devoid of partial agonist properties continues to be a
priority, and several new CRH antagonists have recently been developed
and characterized that meet this particular criterion (22, 31, 32, 42).
One of the members of this new generation of CRH-receptor antagonists
is astressin. Astressin is ~100 times more potent than -hCRH at
inhibiting ACTH secretion in vitro and is extremely effective in
blocking ACTH release in stressed or adrenalectomized rats in vivo
(22). Furthermore, astressin exhibits little if any intrinsic agonist
activity. Astressin is effective in blocking responses to a variety of
stressors. For example, astressin blocks CRH- and alcohol-induced
increases in ACTH (42) and stress-induced alterations in gastric and
colonic motor function (31) with ~30 times more potency than does
-hCRH. We extend these observations of biological actions of
astressin to include a reduction of spontaneous waking in freely
behaving rats.
To date, three CRH-receptor subtypes that differ in anatomical
distribution and pharmacological profile have been described (reviewed
in Refs. 6, 11); they are termed
CRH-R1,
CRH-R2
, and
CRH-R2
. In pituitary,
CRH-R1 mRNA is expressed in both the anterior and intermediate lobes (11, 40, 46). In contrast, the
expression of CRH-R2 mRNA in the
anterior pituitary is either undetectable (46) or only detected in
scattered cells (11). Therefore
CRH-R1 appears to be the major
receptor isoform regulating pituitary ACTH secretion. Within the rat
brain, the distribution of hybridization signals for
CRH-R1 mRNA is widespread,
including neo-, olfactory, and hippocampal cortices, cerebellum,
septum, amygdala, and brain stem sensory relay structures, with only
low levels of expression detected in thalamic and hypothalamic nuclei (11, 40, 46). In contrast, the expression of
CRH-R2 mRNA within rat brain is
generally confined to subcortical structures, in the lateral septal
nucleus, ventromedial hypothalamic nucleus, olfactory bulb, amygdala,
and the choroid plexus (11, 46). CRH-R2 is the predominant
CRH-R2 isoform in neuronal
systems, whereas CRH-R2 is
localized in nonneuronal tissues of the CNS (e.g., choroid plexus and
cerebral arterioles; Ref. 11) but is primarily expressed in the
periphery in heart, skeletal muscle, lung, kidney, and intestine (11,
46). Of relevance to our experiments, -hCRH is a more effective
antagonist of CRH-R2 than
CRH-R1 (11). As such, -hCRH is
more effective in blocking brain CRH receptors than pituitary CRH
receptors (42), which may explain why in most models -hCRH is more
effective when administered centrally than peripherally (e.g., Ref.
17). Astressin on the other hand, effectively blocks both
CRH-R1 and
CRH-R2 in brain and pituitary
(42). These differences in CRH-receptor distributions and affinities
for -hCRH and astressin may account for the different time courses
of alterations in waking previously described. We hypothesize that, in
our rat sleep-assay system, the relatively quick reduction in waking
after intracerebroventricular administration of -hCRH is due to
central actions mediated by subcortical
CRH-R2. In contrast, the
behavioral effects of astressin in our system may be primarily mediated
by the HPA axis by blocking CRH-R2
in the paraventricular nucleus of the hypothalamus and/or the
CRH-R1 in the anterior pituitary.
Such potential sites or mechanisms of action may explain the relatively
long delay from the administration of astressin until behavior is
altered. Experiments to further elucidate the respective roles for
these CRH-receptor subtypes in the modulation of waking are currently
in progress.
The circadian rhythmicity of HPA axis activity in the rat is
particularly striking, with peak activity occurring during the dark
period of the light-dark cycle (e.g., Refs. 19, 21). Changes in CRH in
hypothalamus parallel those in the periphery and generally reflect the
status of the HPA axis (48). Thus endogenous CRH concentrations are low
during the light (rest) period and high during the dark (active) period
in rats. As such, neither of the CRH-receptor antagonists used in these
experiments affected sleep-wake behavior during the light period, with
the exception of the partial agonist actions described above for high doses of -hCRH. Our observations that CRH antagonists are
ineffective in altering spontaneous behavior of rats during the light
period are not surprising and, in fact, are expected on the basis that low endogenous CRH concentrations indicate a system with relatively "little" to antagonize. Previously published observations that -hCRH (at low doses) does not affect "nonstressed" animals are probably due to this circadian rhythmicity, since most studies using
rats are conducted during the light (rest) period. In contrast to light
period administration, if CRH antagonists are administered during the
dark (active) period when CRH concentrations are high, they are
effective in altering spontaneous behavior (this study). The circadian
modulation of responses to CRH antagonists is also observed when CRH
itself is administered. Intracerebroventricular administration of CRH
increases waking (e.g., Refs. 15, 37) regardless of timing of
administration (this study); however, the magnitude and duration of the
response are time dependent. Administration of CRH during the light
period, when endogenous CRH is normally low, results in an increase in
waking of a larger magnitude than when the same dose is administered
during the dark period, when endogenous CRH concentrations are already
high. Similarly, when animals are subjected to stressors during the
light period, the normally low concentrations of CRH are greatly
elevated in response to the stressor, and CRH-receptor antagonists are
effective in blocking or attenuating responses mediated by this CRH
surge. As with administration of CRH itself, the magnitude of responses of rats to stressors may not be as great during the dark (active) period as those observed to the same manipulation conducted during the
light (rest) period, since the activity of the system is already high.
In conclusion, we have shown that central administration of two specific CRH-receptor antagonists reduces, in a time- and dose-related manner, spontaneous waking in freely behaving rats. The time courses of responses to these antagonists differ, an effect that may be due to tissue-specific distribution patterns for CRH-receptor subtypes. Although additional experiments must be conducted before definitive conclusions can be made concerning the involvement of specific CRH-receptor subtypes in these responses, our results provide direct evidence that CRH may be involved in the regulation or modulation of sleep-wake activity because interfering with the binding of CRH to its receptors reduces waking.
Perspectives
Animals respond to stressors with a variety of complex behavioral, physiological, and autonomic processes that act in concert to eliminate the stressor. There has been extensive effort to determine mechanisms that mediate responses to stressors, and in fact most studies focusing on CRH are fundamentally "stress" studies. As a result, there is now a large body of knowledge indicating a major role for CRH in responses to stressors. We have previously suggested, for example, that the involvement of CRH in negative-feedback mechanisms for immune activation is a critical feature in the complex alterations in sleep that occur throughout the course of an acute infection. Indeed, CRH through multiple pathways is able to modulate behavioral responses to immune active substances. Although determining mechanisms responsible for changes in behavior during pathology is important, it is equally important to understand the regulation of normal behavior in the absence of overt stressors. In our opinion, this is one aspect of research concerning CRH that is generally lacking. How will we be able to fully understand the role of CRH in psychiatric illness, immune challenge, or responses to "psychological stressors" if we do not know what CRH does in brain in the absence of these conditions? Studies such as this current one are important because they form the basis for understanding more completely the basic neurobiology of CRH. With the development of new tools, e.g., more selective receptor antagonists, we will be able to explore in greater detail the role of CRH in normal behavior.| |
ACKNOWLEDGEMENTS |
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The technical assistance of Kristi Overgaard and William Dalmeida is gratefully acknowledged.
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FOOTNOTES |
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This work was supported in part by National Institute of Mental Health Grant MH-52275.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: M. R. Opp, Dept. of Psychiatry and Behavioral Sciences, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0431.
Received 19 March 1998; accepted in final form 14 May 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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] or 
). * Hourly periods in which
values obtained after 
).
Values are expressed as differences from vehicle (PFS), depicted by
dotted zero lines. One group of rats
(n = 8) had substances injected before
beginning of light period of light-dark cycle, whereas a separate group
of rats (n = 8) was used for
experiments initiated at beginning of the dark period. Symbols
designate statistical differences as follows:
* P < 0.05 vs. vehicle (dotted
zero lines); # P < 0.05 vs.