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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
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We hypothesize that corticotropin-releasing
hormone (CRH), a regulator of the hypothalamic-pituitary-adrenal (HPA)
axis, is involved in sleep-wake regulation on the basis of observations that the CRH receptor antagonist astressin, after a delay of several hours, reduces waking and increases slow-wave sleep (SWS) in
rats. This delay suggests a cascade of events that begins with
the HPA axis and culminates with actions on sleep regulatory systems in the central nervous system. One candidate mediator in the brain for
these actions is interleukin (IL)-1. IL-1 promotes sleep, and
glucocorticoids inhibit IL-1 synthesis. In this study, central administration of 12.5 µg astressin into rats before dark onset reduced corticosterone 4 h after injection and increased mRNA expression for IL-1
and IL-1
but not for IL-6 or tumor necrosis factor- in the brain 6 h after injection. To determine directly whether IL-1 is involved in astressin-induced alterations in sleep-wake behavior, we then pretreated rats with 20 µg anti-IL-1 antibodies before injecting astressin. The increase in SWS and the reduction in
waking that occur after astressin are abolished when animals are
pretreated with anti-IL-1. These data indicate that IL-1 is a
mediator of astressin-induced alterations in sleep-wake behavior.
hypothalamic-pituitary-adrenal axis; cytokine; behavior; gene expression; interleukin
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CORTICOTROPIN-RELEASING HORMONE (CRH) is well documented as the major, although not only, mediator of responses to stressors. We have previously hypothesized that CRH also plays a role in the regulation/modulation of normal sleep-wake behavior in the absence of stressors (18). This hypothesis is on the basis of several observations. For example, genetically related rat strains that differ in the synthesis/secretion of CRH differ in the amount of time they spend sleeping and waking; reduced CRH synthesis is associated with reduced amounts of waking and increased sleep (19). In addition, central or peripheral administration of CRH increases waking and reduces slow-wave sleep (SWS) in several species (reviewed in Ref. 18). Furthermore, central or peripheral administration of CRH receptor antagonists reduces waking and increases SWS in the rat (6, 7). Responses to the CRH receptor antagonist astressin [cyclo(30-33)[D-Phe12,Nle21,38, Glu30,Lys33]-rat/human-CRH-12---41] occur with a relatively long delay; astressin reduces waking and enhances SWS beginning 6 or 7 h after either central (intracerebroventricular) or peripheral (intravenous) administration (6, 7). Because these responses to astressin occur after a long delay and CRH is a regulator of the hypothalamic-pituitary-adrenal (HPA) axis, we previously hypothesized that these alterations in sleep-wake behavior may be mediated by mechanisms that include the HPA axis.
Although results of our previous studies suggested a role for the HPA
axis in mediating these responses, we were unable on the basis of those
experiments to determine which sleep regulatory mechanism(s) in the
brain were ultimately responsible for astressin-induced alterations in
sleep-wake behavior. One potential candidate for this role is the
somnogenic cytokine interleukin-1 (IL-1). The somnogenic properties of
IL-1 are well documented (reviewed in Refs. 14, 21). IL-1 administered
centrally into rats during their active period (dark period) is
particularly effective in increasing SWS and reducing waking. IL-1
mRNA expression in the rat brain is highest during the light period of
the light-dark cycle, the period when rats sleep the most, and lowest
during the dark period, when rats are most active. CRH, by acting on the HPA axis to increase glucocorticoid synthesis and release from the
adrenal gland, is an important component of negative feedback
mechanisms for IL-1 actions because glucocorticoids inhibit IL-1
synthesis/release in peripheral tissue (25,
30) and in the central nervous system [CNS;
(11)]. We conducted two sets of experiments to determine
if astressin-induced alterations in sleep-wake behavior are mediated by
the HPA axis and glucocorticoid actions on IL-1 synthesis and secretion
in the brain. First, we determined the effects of central
administration of astressin on circulating corticosterone and IL-1
mRNA expression in the brain. We then pretreated rats with anti-IL-1
antibodies before central administration of astressin. We now report
that astressin injected centrally reduces circulating corticosterone
concentrations before IL-1 mRNA expression increases in the rat
brain and that anti-IL-1 abolishes the astressin-induced increase in SWS.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Substances
Stock solutions of astressin (Bachem, Torrance, CA), rabbit IgG (Sigma, St. Louis, MO), and rabbit anti-rat IL-1 (anti-IL-1) polyclonal antibody (Endogen, Woburn, MA) were prepared in pyrogen-free saline (PFS). The anti-rat IL-1 polyclonal antibody preparation was
purified from rabbit serum, is specific for natural and recombinant rat
IL-1, and does not cross-react with rat IL-1 (manufacturer's specifications). Aliquots of these stock solutions were stored at
80°C until use, when they were then brought to an appropriate injection volume (3 µl). The dose of astressin used in these
experiments was 3.5 nmol (12.5 µg), a dose that enhances SWS and
reduces waking (6). Doses of IgG and anti-IL-1 used in
these experiments were 20 µg · 3 µl
1 · rat1.
Animals
Five groups of male Sprague-Dawley rats (250-300 g; Harlan, Indianapolis, IN) were used in these experiments. All animals were housed and maintained under the same conditions in the same environmentally controlled chambers regardless of the specific experimental protocol in which they were used. These animals were anesthetized (ketamine/ xylazine, 87/13 mg/kg) and injected with an analgesic [butorphanol tartrate (Torbugesic)] and a broad spectrum antibiotic [penicillin G benzathine (Bicillin, LA)]. Animals used to determine effects of astressin on circulating corticosterone and cytokine mRNA expression were implanted with only a guide cannula directed into the lateral cerebral ventricle. Rats used to determine effects of astressin and anti-IL-1 on sleep-wake behavior were
surgically implanted with electroencephalogram (EEG) screw electrodes,
a ventricular guide cannula, 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
(19). The animals were allowed to recover for 7 days
before the initiation of experiments. The rats were housed in
individual recording cages, 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), and 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 United States Department of Agriculture Animal
Welfare Act and the United States Public Health Service Policy on
Humane Care and Use of Laboratory Animals.
On the second postsurgical day, the rats were connected to the recording apparatus via a flexible tether. Three days after surgery, the patency and free drainage of the intracerebroventricular cannulas were assessed by administering 200-400 ng ANG II (human ANG II octapeptide; Peninsula Laboratories); ANG elicits a drinking response mediated by structures in the preoptic area (8). At the end of each experimental protocol, all rats were again injected with ANG; only data from those rats that exhibited a positive drinking response were included in the subsequent analyses. For the next 4 to 5 days, the animals were habituated by daily handling and intracerebroventricular injections of PFS timed to coincide with scheduled experimental administrations.
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 bandpass filtered between 0.1 and 40 Hz (frequency response: ±3 dB; filter frequency roll off: 12 dB/octave). Gross body movements were detected by custom-built ultrasonic detectors (Biomedical Instrumentation, University of Tennessee, Memphis, TN). These conditioned signals (EEG, 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 with the use of custom software (ICELUS, M. R. Opp) written in LabView for Windows (National Instruments). The animal's behavior was classified as either SWS, rapid eye-movement sleep (REMS), or waking on the basis of previously defined criteria (19). Briefly, SWS is characterized by large amplitude EEG slow waves, high-power density values in the delta frequency band (0.5-4.0 Hz), lack of gross body movements, and declining Tbr before and during entry. During REMS, the amplitude of the EEG is reduced, the predominant EEG power density occurs within the theta frequency (6.0-9.0 Hz), Tbr increases rapidly at onset, and there are phasic body twitches. During waking, the rats are generally active, there are protracted body movements, Tbr gradually increases, the amplitude of the EEG is similar to that observed during REMS, but power density values in the delta frequency band are generally greater than those in theta frequency band.
Experimental Protocols
Circulating corticosterone and cytokine mRNA expression.
Rats in group 1 (naive, n = 12) were used to
determine basal levels of circulating corticosterone and cytokine mRNA
expression in the brain. These 12 rats were naive in that they received
no surgical procedures, they were not handled during habituation to the
environmental chambers, and they were undisturbed before death. Rats in
group 2 (surgical controls, n = 12) had
intracerebroventricular guide cannulas surgically implanted. These
animals were undisturbed throughout the habituation period. They were
handled on the experimental day at the time injections were given to
rats in the remaining groups, but they were not injected. These animals
served as controls for surgical procedures and handling for injections.
Rats in group 3 (n = 32) were used to
determine effects of astressin on circulating corticosterone and
cytokine mRNA expression in the brain. These animals were surgically
implanted with an intracerebroventricular guide cannula, and they were
habituated by daily handling and intracerebroventricular injection of
vehicle (PFS). On the experimental day, these rats were injected with
either PFS or 12.5 µg astressin at the beginning of dark onset. All
animals in these three groups were decapitated either 4 (n = 28) or 6 (n = 28) h after dark onset. Trunk blood was collected into Vacutainer (Franklin Lakes, NJ)
tubes containing EDTA and centrifuged for 15 min at 1,500 rpm (
400
g) at 4°C. Plasma was then aliquoted and stored at
80°C until assay. The brains were rapidly removed from the skull
after decapitation and placed on an ice-cold surface. The meninges were removed, and the hypothalamus, hippocampus, brain stem, and a section
of the parietal cortex were dissected out. All tissues were immediately
placed in liquid nitrogen and then stored at 80°C until RNA
extraction and assay.
Sleep-Wake Behavior
The effects of 20-µg anti-IL-1 antibodies on spontaneous
sleep during the dark period of the light-dark cycle were determined in
a pilot study. After obtaining 24-h undisturbed baseline recordings, rats in group 4 (n = 4) were injected 15 min
before dark onset with either PFS, 20 µg IgG, or 20 µg
anti-IL-1. Injections were not given on consecutive days, and the
order of substance administration was varied. Recordings were begun at
dark onset and continued for 24 h. Rats in group 5 (n = 8) were used in a double-injection protocol
consisting of three manipulations to determine effects of anti-IL-1
antibodies on astressin-induced alterations in sleep. After obtaining
24-h baseline recordings, the rats were injected with either IgG (20 µg) + PFS, IgG (20 µg) + astressin (12.5 µg), or
anti-IL-1 (20 µg) + astressin (12.5 µg). The order in which the injections were administered varied, and injections were separated by 2 days. The first injection was given 30 min before dark onset and
was followed 15 min later with the second injection. All recordings began at dark onset and continued for the next 24 h. The volume for all injections was 3 µl, and each injection was given over approximately a 2-min period.
Corticosterone Radioimmunoassay
Total plasma corticosterone was measured by radioimmunoassay (ICN Biomedicals, Costa Mesa, CA). The antiserum has very low cross-reactivity with other glucocorticoids and their products (<0.4%). Intra-assay coefficients of variation are between 4.4 and 10.3%, and intra-assay coefficient of variations are between 6.5 and 7.2% (manufacturer's specifications).Ribonuclease Protection Assay for Cytokine mRNA Expression
The ribonuclease protection assay is a sensitive and specific method used to detect and quantitate mRNA species. Total RNA from each tissue sample was extracted with the use of Tri Reagent according to the protocol provided by the manufacturer (Molecular Research Center, Cincinnati, OH). We used a rat-specific cytokine multiprobe template kit (rCK-1; PharMingen, San Diego, CA) for the T7 polymerase-directed synthesis of high specific activity, 32P-labeled antisense RNA probes. Twenty micrograms of total RNA from each sample was hybridized with excess antisense, radiolabeled probes after which free probe and remaining single-stranded RNA were digested with RNase A/T1. The double-stranded RNase-protected fragments were purified and resolved on 5% denaturing polyacrylamide gels. The probe template included rat-specific sequences for IL-1, IL-1, tumor necrosis
factor (TNF)-, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-, IL-2, and
gamma interferon. A probe specific for the ribosomal protein L32 was
added to the template to serve as an internal standard. Two control
samples were included in each assay, yeast tRNA as a negative control
and a compilation of total rat mRNA from splenocytes stimulated with
Staphylococcus enterotoxin A, lipopolysaccharide, ionomycin,
and anti CD3 or anti CD28 as a positive control. Dried gels were placed
on a phosphorimaging screen for 16 to 18 h. The phosphorimaging
screen was subsequently scanned with a Molecular Dynamics
phosphorimager (Sunnyvale, CA), which generated a computerized image.
The images generated by the phosphorimager were processed with the use of ImageQuant software (Molecular Dynamics). The intensity of a band in the computer-generated image is directly proportional to the amount of radioactivity within the band. The software parameters were set to automatically detect bands within individual lanes on the image thus eliminating subjectivity; the same parameters were used for the entire study. The optic density (OD) values obtained from each band were normalized against the OD obtained from the L32 band in the same sample by the following expression: [(OD of the sample band/OD of the L32 band) × 100].
Statistical Analyses
Circulating corticosterone concentrations and cytokine mRNA
expression.
Circulating corticosterone concentrations are presented as the
means ± SE in units of nanograms per milliliter. Values for cytokine mRNA expression are depicted as OD in arbitrary units. Independent sample t-tests were performed to determine if
values differed between control manipulations (either naive animals, surgical controls, or injection of PFS) and those obtained after injection of astressin. An -level of P 
0.05 was taken
as indicating a statistically significant difference between the two manipulations.
Behavioral Experiments
All values are presented as the means ± SE. One-way ANOVA for the duration of each vigilance state (SWS, REMS, waking) and for Tbr values were performed across the two 12-h time blocks comprising the 24-h recording period. The main effect consisted of manipulation [substance(s) administered]. If statistically significant differences were detected, post hoc multiple comparisons were made to determine which condition(s) contributed to the effect. An-level of
P 0.05 was taken as indicating a statistically significant departure from control values.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Changes in Circulating Corticosterone Concentrations After Central Administration of Astressin
Circulating corticosterone did not differ statistically among rats of groups 1, 2, or 3 at either time point sampled (Fig. 1). Relative to naive rats of group 1, however, variability was greater in samples taken 4 h after dark onset from the surgical control animals (group 2) and in the animals that were implanted with an intracerebroventricular guide cannula, handled daily, and injected with PFS before death (Fig. 1). However, there were no differences in circulating corticosterone in samples from surgical control animals and those injected with PFS. The increases in corticosterone in these two groups thus reflect handling and injection procedures given before dark onset. When rats were injected with 12.5 µg astressin and killed 4 h later, circulating corticosterone was reduced by ~44%, from 654.08 ± 61.86 ng/ml after PFS to 369.05 ± 51.60 ng/ml. Concentrations of circulating corticosterone did not differ between samples taken from animals 6 h after dark onset, regardless of condition (Fig. 1).
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Astressin-Induced Alterations of Cytokine mRNA Expression
Low levels of mRNA expression for IL-1, IL-1, IL-6, and
TNF- were detected in tissue samples taken from naive rats both 4 and 6 h after dark onset. These values represent basal levels of
cytokine mRNA expression detectable by this technique during the first
half of the dark period of the light-dark cycle. The values did not
differ between the two time points and were thus pooled for
presentation (Table 1). Similarly,
cytokine mRNA expression in the surgical control animals did not differ
between samples collected at the 4- or 6-h time points, so these values
were also pooled (Table 1). Although the level of mRNA expression for
IL-1 and -1 was in some cases slightly increased in samples taken from the surgical control animals, there were no statistical
differences between the two groups, and the pattern of expression did
not change (Table 1). IL-6 mRNA was not detected in naive animals, but
it was detected in two samples from the hypothalamus and one sample
from the brain stem of animals that served as surgical controls.
TNF- mRNA was consistently detected in samples from the brain stem
and cortex from both groups of animals (Table 1). TNF- mRNA was not
detected in the hypothalamus or hippocampus of naive animals under
these conditions, but it was detected in a total of three samples from
the surgical control animals (Table 1).
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IL-1 and -1 and TNF- mRNA expression was consistently detected
in tissue samples taken from rats injected with PFS and killed 4 h
later (data not shown). The values from animals killed 4 h after
PFS injections did not differ statistically from those animals injected
with PFS and killed 6 h later; these 6-h values are shown in Fig.
2. IL-6 mRNA was variably expressed in
low levels in animals injected with PFS, being detected in two brain
stem and two cortex samples from animals injected with PFS and killed 4 h later (data not shown) and in one brain stem sample from
animals killed 6 h after PFS (Fig. 2). TNF- mRNA was detected
in about half the samples taken from animals injected with PFS and
killed 4 h later, but the levels of expression were low (data not
shown). When injected with PFS and killed 6 h later, TNF- mRNA
was detected in between 50 and 60% of samples taken from the
hypothalamus, hippocampus, or brain stem, but in only one sample taken
from the cortex (Fig. 2).
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IL-1 and -1 mRNA expression in tissue samples taken from animals
killed 4 h after administration of 12.5 µg astressin did not
differ from values in samples obtained from animals injected with PFS
and killed at the same time point (data not shown). However, IL-1
and -1 mRNA expression increased two and threefold in the brain stem
and cortex 6 h after intracerebroventricular administration of
astressin (Figs. 2 and 3). IL-1 mRNA expression doubled in the hypothalamus after astressin relative to values obtained from animals injected with PFS (Fig. 2). Although there was a strong tendency for an increase in IL-1 mRNA expression in the hypothalamus and hippocampus 6 h after astressin administration, this increase was variable and did not achieve statistical significance (Fig. 2).
IL-6 mRNA expression was not consistently altered by astressin. Similarly, although levels of TNF- mRNA expression were elevated in
all brain regions 6 h after astressin, these increases did not
achieve statistical significance (Fig. 2).
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Effects of Anti-IL-1 on Astressin-Induced
Alterations in Sleep-Wake Behavior and Tbr
at this dose altered sleep-wake behavior did
not reveal differences in the amount of time rats spent any vigilance
state under baseline conditions, when injected with PFS, or when
injected with 20 µg anti-IL-1. Under these three conditions,
waking ranged from 59.7 ± 3.1 to 64.0 ± 4.7%, SWS from
28.4 ± 3.7 to 33.8 ± 2.5%, and REMS from 6.5 ± 0.7 to 7.6 ± 1.2% recording time of the 12-h dark period immediately
after the injections. When these rats were injected with 20 µg IgG, there were moderate increases in the percentage of time spent in SWS
(to 40.3 ± 2.5%) concomitant with slight reductions in waking
(to 54.9 ± 2.9%) and REMS (to 4.8 ± 0.8%). None of these changes were statistically different from the PFS or anti-IL-1 conditions, although they differed from baseline values.
Values obtained after control injections in the double-injection
protocol (IgG + PFS) did not differ from baseline values obtained
from undisturbed animals for any behavioral state (Table 2). SWS increased, and waking decreased
relative to values obtained after control injections when the double
injection of IgG + astressin was given (Table 2, Fig.
4). There was a moderate increase in SWS
and a reduction in waking during the first postinjection hour, but the
major portion of astressin-induced changes in SWS and waking occurred
during postinjection hours 6-10 (Fig. 4). The time
course and magnitude of astressin-induced alterations in sleep-wake
behavior during postinjection hours 1-12 in this study are virtually identical to those we previously reported after a single
intracerebroventricular administration of this dose of astressin
(6). As previously reported, REMS was not altered after
intracerebroventricular administration of this dose of astressin (6;
Table 2, Fig. 4).
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The amount of time spent in SWS after astressin (IgG + astressin)
during the subsequent 12-h light period (postinjection hours 13-24) was reduced relative to values obtained during control conditions (baseline or IgG + PFS) and after administration of anti-IL-1 + astressin (Table 2, Fig. 4). This reduction in SWS was limited to the first 4 h of the light period, was mirrored by
increases in waking, and appears to represent a flattening of the
normal diurnal rhythm of sleep. When animals were pretreated with
anti-IL-1 and then injected with astressin (anti-IL-1 + astressin), all astressin-induced changes in SWS and waking during postinjection hours 1-12 and postinjection hours
13-24 were abolished (Table 2, Fig. 4).
Tbr was moderately elevated during postinjection hours
1-12 after control injections of IgG + PFS, relative to
values obtained during undisturbed baseline recordings (Table 2). Tbr
did not differ from control values (IgG + PFS) after
intracerebroventricular administration of astressin (IgG + astressin). There was, however, a slight increase in Tbr values
relative to double-injection control conditions when the rats were
pretreated with anti-IL-1 and then injected with astressin (Table
2). Tbr was also slightly elevated during postinjection hours
13-24 after both IgG + astressin and anti-IL-1 + astressin manipulations (Table 2).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We have previously reported that SWS is enhanced and waking is
reduced after central or peripheral administration of the CRH receptor
antagonist astressin into rats (6, 7). The
present results indicate IL-1 is a mediator of these responses to
astressin and that the mechanisms responsible for these actions are
likely to include HPA axis feedback. The finding that IL-1 mRNA
expression in the brain increases only after circulating corticosterone
is reduced suggests that transcriptional regulation is important in
these responses rather than simply a biological antagonism.
The present results confirm previous observations that blockade of CRH
receptors induces subsequent alterations in sleep (6, 7) and extends them by indicating these responses are
attributable to IL-1 actions within the CNS. IL-1 mRNA in the rat
brain exhibits diurnal variation (32, 33)
with lower levels during the dark period, when rats are most active,
and higher levels during the light period, when rats sleep the most.
HPA axis activity of the rat, as indexed by circulating corticosterone,
is lowest during the light period and highest shortly after dark onset.
The diurnal variation in IL-1 message is thus out of phase with HPA
axis activity due to the inhibitory actions of glucocorticoids on
IL-1 synthesis (13, 15). Our results
indicate that central administration of astressin reduces circulating
corticosterone concentrations 4 h later, whereas cytokine mRNA
expression in tissue samples from the brain taken at this same time is
not altered. However, IL-1 mRNA expression is upregulated in discrete
brain regions 6 h after astressin administration, by which time
circulating corticosterone has returned to control values. These data
are in agreement with observations that hypophysectomy or adrenalectomy increases IL-1 gene expression in the rodent brain (11,
17). The time course of changes in corticosterone and
cytokine mRNA expression in response to central administration of
astressin is appropriate when viewed in the context of glucocorticoid
inhibition of IL-1 synthesis in the brain; first circulating
corticosterone is reduced, then IL-1 mRNA expression increases.
Astressin-induced alterations in sleep-wake behavior under the
conditions of this study are apparent beginning in postinjection hour 6, the same time at which we reported responses to
intracerebroventricular administration of astressin in our previous
study (6). Data in this study suggest that sometime
between 4 and 6 h after central administration of astressin,
IL-1 mRNA expression begins to increase. The time points at which
rats were killed in this study were selected to precede changes in
behavior previously observed and not to generate a detailed time course
of responses. It is possible that HPA axis activity is reduced in
response to central administration of astressin before the 4-h time
point used in this study, which could conceivably increase IL-1 mRNA
before the 6-h time point sampled. The temporal pattern of
astressin-induced alterations in circulating corticosterone and IL-1
mRNA expression provides indirect evidence that IL-1 mediates the
SWS-enhancing effects of astressin. However, the finding that
pretreatment with antibodies directed against IL-1 abolishes
astressin-induced alterations in sleep-wake behavior provides direct
evidence that IL-1 is a mediator of these responses. There is a large
body of evidence indicating a role for IL-1 in the regulation of sleep
(reviewed in Ref. 14). IL-1 is a particularly potent enhancer of SWS
when administered centrally into rats before the dark period of the light-dark cycle (e.g., Ref. 24). On the basis of our data and extensive literature, it is reasonable to conclude that in response to
central administration of astressin, HPA axis activity is reduced at a
time point when it would normally be at or near its peak. The reduction
in circulating corticosterone may then release the inhibitory tone
exerted in the brain by glucocorticoids on IL-1 synthesis.
Subsequently, IL-1 increases and alters sleep-wake activity due to its
actions within the CNS.
Although the present study focused on IL-1 as a mediator of
astressin-induced alterations in sleep, the actions of other cytokines implicated in sleep regulation cannot be entirely discounted. TNF, for
example, exhibits many of the biologic properties of IL-1, including
the ability to increase sleep (12, 29). As with IL-1, inhibition of endogenous TNF reduces spontaneous sleep (35), and blocking IL-1 actions attenuates TNF-induced
sleep responses (34). Furthermore, TNF- mRNA exhibits
diurnal variations similar to those of IL-1 (5). These and
numerous other observations indicate a role for TNF in sleep regulation
(reviewed in Ref. 14). TNF- mRNA expression increases modestly in
each of the brain regions assayed after administration of astressin.
Although glucocorticoids regulate TNF- gene expression both
transcriptionally and posttranscriptionally, their effects are far
stronger posttranscriptionally (4). As such, reductions in
glucocorticoid actions in the brain may not influence the accumulation
of TNF- mRNA to the same extent as that of other cytokines such as
IL-1.
The astressin-induced alterations in HPA axis activity may result in relatively prolonged alterations in sleep-wake behavior. This conclusion is on the basis of the observation that SWS is reduced during the first 4 h of the light period some 13 to 16 h after administration of astressin. One plausible explanation for this observation is that astressin-induced reductions in HPA axis activity at a time when such activity would normally be high (i.e., the dark period) are followed by a "rebound" increase in HPA axis activity at a time when it would normally be low (i.e., the light period). Because components of the HPA axis (CRH, adrenocorticotrophic hormone, glucocorticoids) generally increase waking (9), such alterations may reduce SWS and increase waking. Experiments to determine if this is indeed the case remain to be conducted.
Most studies to date of interactions between IL-1 and CRH have focused on the role of CRH in modulating responses to IL-1. A large body of evidence indicates that CRH and the HPA axis are critical components of feedback mechanisms for IL-1 actions (see for example Refs. 3, 16, 28; and reviewed in Refs. 26, 36), including sleep (20). IL-1 acts in the brain at multiple levels to activate CRH-producing neurons (2), increase CRH secretion in the paraventricular nucleus of the hypothalamus and in the median eminence (1, 28, 37), and stimulate CRH gene expression (31). These actions result in adrenocorticotrophic hormone synthesis and release from the pituitary (27), which then stimulates glucocorticoid release from the adrenal gland. Circulating glucocorticoids act in the brain to inhibit subsequent synthesis of IL-1. Such feedback mechanisms have been well documented and have functional consequences for behavioral responses to IL-1. Our present study, however, focused on interactions between CRH and IL-1 from a different perspective, i.e., targeting the CRH system in otherwise normal animals and determining subsequent effects on IL-1-mediated behavior. The results presented herein in conjunction with previous observations and data in the literature support the hypotheses that CRH and IL-1 are involved in the regulation of physiological sleep-wake activity. The present study indicates that blocking IL-1 actions abolishes the effects of CRH receptor blockade on sleep-wake behavior. The interactions between CRH and IL-1 as they pertain to sleep appear to be mediated by the HPA axis, specifically by the actions of glucocorticoids on IL-1 synthesis in the brain.
There are several caveats associated with studies such as this in which
substances are administered directly into the brain. First, surgical
manipulations are necessary that by their very nature induce tissue
injury. Proinflammatory cytokines such as IL-1, IL-6, and TNF are
upregulated in response to such insult, and surgeries could thus
provide a potential confound. However, in our experience, the increases
in cytokine mRNA in the brains of animals subjected to surgical
manipulations relative to expression levels in naive, unoperated
animals in most cases are modest [this study (10)]. If
substances do not readily cross the blood-brain barrier, there is no
method currently available to get substances into the brain without
creating some tissue damage. Given that animals injected with vehicle
and animals injected with the substance of interest are both subjected
to the same surgical manipulation, any surgically induced increases in
cytokine mRNA expression should be present in both groups and may be
accounted for by appropriate controls. Secondly, administration of
antibodies requires that an appropriate IgG preparation be used for
vehicle controls. We have previously reported that high doses of IgG
may induce nonspecific responses that include fever and/or enhanced
sleep (22, 23). Therefore, multiple doses
must be tested to determine whether the presence of foreign protein per
se alters sleep-wake behavior. In the present experiments, we conducted
a pilot study to determine if a dose of anti-IL-1 we previously
administered into rats before the light period of the light-dark cycle
(22) could be used successfully before the dark period.
The four rats used in our pilot study exhibited a modest increase in
SWS after a 20-µg dose of IgG under these conditions. It is important
to note, however, that when administered before dark onset, 20 µg of
anti-IL-1 itself did not alter SWS compared with values obtained
under baseline conditions or after administration of PFS. Most
importantly, sleep-wake behavior of rats after control injections in
the double-injection protocol used in this study (IgG + PFS) was
identical to that determined during undisturbed baseline conditions. As
such, we are confident that the blockade of astressin-induced
reductions in SWS are due to the anti-IL-1 itself, rather than to
nonspecific responses of the brain to foreign protein. Finally, the
present study and others ongoing in our laboratory have revealed one
aspect of HPA axis activity not often referred to in the literature, namely differential responses to handling and injections on the basis
of the timing of the procedure. When handled and injected at the
beginning of the light period, there is a surge in circulating corticosterone and a period of increased waking; both subside relatively quickly and within 30 to 45 min, the rats return to sleep
(unpublished observations). When the same procedures are conducted
before dark onset, elevated corticosterone does not subside for several
hours. We speculate this is due to the fact that during the dark
period, the animals are active, and the elevated HPA axis activity
remains so due to increased behavioral activity. These observations are
being explored further.
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ACKNOWLEDGEMENTS |
|---|
The technical assistance of Dr. Carmelina Gemma, William Dalmeida, and Kristi Overgaard is appreciated.
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FOOTNOTES |
|---|
This work was supported in part by National Institute of Mental Health Grant MH-52275.
Address for reprint requests and other correspondence: M. R. Opp, Dept. of Psychiatry and Behavioral Sciences, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-0431 (E-mail: mark.opp{at}utmb.edu).
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.
Received 3 November 1999; accepted in final form 27 March 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) were animals that had not been subjected to surgical procedures
nor been handled during the habituation period before death. Surgical
control rats (
) were implanted with chronic guide
cannulas. These animals were not handled during the recovery or
habituation period. They were handled immediately before the dark
period in which they were killed to simulate an injection, but they
were not injected. The other groups of rats were implanted with chronic
guide cannulas and handled daily during the recovery and habituation
periods. These animals were then injected intracerebroventricularly
with either vehicle (pyrogen-free saline, PFS; 
) or
12.5 µg astressin (
) immediately before dark onset on the day
of death. Values are the means ± SE; numbers in parentheses
indicate sample sizes. One sample was lost from a naive rat killed
4 h after dark onset. * Statistically significant difference
between values for circulating corticosterone obtained from animals
injected with vehicle and those injected with astressin
(P < 0.05).
, 129 nucleotides). Cont. RNA, control RNA obtained from
stimulated rat splenocytes (positive control); Temp. RNA, template; Hy,
hypothalamus; Hi, hippocampus; Bs, brain stem; Ct, cortex.
