![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 289, Issue 3, 1211-1219, June 1999
Department of Psychiatry, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Corticotropin-releasing factor (CRF) acts as a putative neurotransmitter in the locus ceruleus (LC) to mediate its activation by certain stressors. In this study, we quantified LC sensitivity to CRF 24 h after swim stress, at a time when behavioral depression that is sensitive to antidepressants is apparent. Rats were placed in a tank with 30 cm (swim stress) or 4 cm water and 24 h later, either behavior was monitored in a forced swim test or LC discharge was recorded. Swim stress rats were more immobile than control animals in the swim test. LC neurons of swim stress rats were sensitized to low doses of CRF (0.1-0.3 µg i.c.v.) that were ineffective in control animals and were desensitized to higher doses. Swim stress selectively altered LC sensitivity to CRF because neither LC spontaneous discharge nor responses to other agents (e.g., carbachol, vasoactive intestinal peptide) were altered. Finally, the mechanism for sensitization was localized to the LC because neuronal activation by low doses of CRF was prevented by the intracerulear administration of a CRF antagonist. CRF dose-response curves were consistent with a two-site model with similar dissociation constants under control conditions but divergent dissociation constants after swim stress. The results suggest that swim stress (and perhaps other stressors) functionally alters CRF receptors that have an impact on LC activity. Stress-induced regulation of LC sensitivity to CRF may underlie behavioral aspects of stress-related psychiatric disorders.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Hypothalamic
corticotropin-releasing factor (CRF) acts as a neurohormone that
promotes the secretion of adrenocorticotropin from anterior pituitary
corticotrophs during stress (Vale et al., 1981
). The widespread
distribution of CRF-immunoreactive terminals and binding sites in brain
(Swanson et al., 1983; De Souza, 1987) and autonomic and behavioral
effects produced by central CRF administration suggest that it also
serves as a brain neurotransmitter (Owens and Nemeroff, 1991; Valentino
et al., 1993). Parallel actions of neurohormone and neurotransmitter
CRF may coordinate endocrine with autonomic and behavioral components
of the stress response.
The noradrenergic nucleus, locus ceruleus (LC), which is activated by
stressors, is one putative target of CRF neurotransmission (Valentino
et al., 1993). This is supported by ultrastructural evidence for
synaptic specializations between CRF-immunoreactive terminals and LC
dendrites (Van Bockstaele et al., 1996). Moreover, CRF administered
into the LC increases LC discharge rates (Curtis et al., 1997) and
norepinephrine release in LC target regions (Smagin et al., 1995;
Curtis et al., 1997). Finally, LC activation elicited by certain
physiological stimuli is prevented or attenuated by microinjection of
CRF antagonists into the LC (Curtis et al., 1994; Lechner et al.,
1997). Together, these findings suggest that CRF acts as a
neurotransmitter in the LC to mediate its activation by certain stressors.
The neurohormone action of CRF is regulated by glucocorticoids, which
exert an inhibitory influence via actions on CRF synthesis and release
(Paull and Gibbs, 1983; Suda et al., 1983; Jingami et al., 1985; Young
et al., 1986; Fink et al., 1988; Imaki et al., 1991; Dallman et al.,
1992). Prior stress also regulates neurohormone CRF, and this may play
a role in the phenomenon of stress-induced sensitization of the
hypothalamic-pituitary-adrenal axis (Imaki et al., 1991; Dallman et
al., 1992; Mamalaki et al., 1992). The identification of the conditions
that regulate neurohormone CRF is of interest because dysregulation of
CRF neurohormone function has been proposed to underlie
hypothalamic-pituitary-adrenal hyperactivity observed in melancholic
depression (Gold et al., 1988).
Studies from this laboratory suggested that like the neurohormone
actions of CRF, the putative neurotransmitter actions of CRF in the LC
are regulated by glucocorticoids and stress. For example,
electrophysiological evidence suggested that CRF is tonically hypersecreted within the LC in adrenalectomized rats, presumably as a
result of loss of inhibitory regulation by corticosteroids (Pavcovich
and Valentino, 1997). Additionally, prior exposure to footshock stress
selectively altered LC postsynaptic sensitivity to CRF (Curtis et al.,
1995; Lechner et al., 1997), with repeated stress sensitizing LC
neurons to low (typically inactive) doses of CRF (Curtis et al., 1995).
Parallel dysregulation of CRF neurohormone actions at the level of the
pituitary and neurotransmitter actions within the LC could underlie the
coexistence of endocrine and behavioral symptoms in depression.
In the present study, we examined the effects of forced swim stress on
CRF-LC interactions. Swim stress results in behavioral depression
24 h later that is sensitive to antidepressant treatment (Porsolt
et al., 1977; Borsini and Meli, 1988; Detke et al., 1995), suggesting
that it induces neurochemical changes that may be targets for
antidepressant drugs and are similar to those that occur in depression.
The hypothesis that swim stress increases tonic CRF secretion in the LC
or alters LC sensitivity to CRF 24 h later (i.e., at the same time
that antidepressant-sensitive behaviors are expressed) was tested. LC
spontaneous discharge and sensitivity to CRF were characterized in rats
24 h after a single swim stress. Additionally, LC responses to a
muscarinic agonist (carbachol), vasoactive intestinal peptide (VIP),
and an excitatory amino acid input were examined.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Animals. The study animals were adult male Sprague-Dawley rats (Taconic Farms, Inc., Germantown, NY) weighing approximately 300 g at the beginning of the experiments. Rats were initially housed three to a cage in a controlled environment (20°C, 12-h light/dark cycle, lights on at 7:00 AM). Food and water were available ad libitum. The care and use of animals were in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals."
Forced Swim.
The procedures used for the forced swim were
identical with those described previously (Detke et al., 1995). Rats
were placed in a cylindrical glass tank (46 cm height × 20 cm
diameter) filled with water (25 ± 1oC) to a
depth of either 30 (swim stress) or 4 cm (nonswim controls) for 15 min.
The 30-cm depth allowed rats to swim or float without having their
tails touch the bottom of the tank. Rats in 4-cm-deep water got wet but
were able to maintain all four paws on the floor of the tank and the
head above water without struggling. Immediately after the 15-min swim,
rats were removed from the tank, towel dried, and put in a warming cage
(37°C) that contained a heating pad covered with towels for 15 min.
Rats were then returned to their home cage. The forced swim occurred
between 10:00 AM and 2:00 PM. On the following day (24 h after exposure
to water), rats were either anesthetized with halothane and surgically
prepared for electrophysiological recording or were placed in the same tank filled with water to a depth of 30 cm for 5 min (forced swim test). LC recordings were also obtained from a third group of unhandled
(naive) rats.
Surgery.
The procedures used for recording LC discharge of
halothane-anesthetized rats were similar to those described previously
(Valentino et al., 1983; Curtis et al., 1997). Rats were anesthetized
with 2% halothane-in-air mixture administered through a nose cone. The
anesthetic was maintained at 1% through the experiment. Body temperature was maintained at 36-37°C by a feedback-controlled heating pad. Rats were positioned in a stereotaxic instrument using
blunt ear bars, and the head was oriented at a
15o angle to the horizontal plane (nose down).
The skull was exposed, and a hole (approximately 3 mm diameter),
centered at 1.1 mm lateral to the midline and 3.5 to 3.7 mm caudal to
the intersection of midline and the transverse sutures, was drilled
over the cerebellum for approaching the LC. The dura over the
cerebellum was carefully removed using fine iridectomy scissors. In
some experiments, another hole was drilled with its center at 1.0 mm
caudal to bregma and 1.5 mm lateral to the midline for the placement of
a 26-gauge cannula to be used for i.c.v. drug administration. The
cannula was positioned 5.6 mm ventral to the skull surface, placing its tip in the lateral ventricle.
Recording.
For most experiments, a glass micropipette pulled
to a 2- to 4-µm-diameter tip (4-7 M
) and filled with 2%
pontamine sky blue (PSB) dye in 0.5 M sodium acetate was used to record
LC discharge. This was advanced toward the LC with a micromanipulator.
Microelectrode signals were amplified and filtered. Impulse activity
was monitored with an oscilloscope and a loudspeaker to aid in
localizing the LC. LC neurons were tentatively identified during the
recording by their spontaneous discharge rates (0.5-5 Hz), entirely
positive, notched waveforms (2- to 3-ms duration) and biphasic
excitatory-inhibitory responses to contralateral hindpaw or tail pinch.
When stable, unitary action potentials were isolated, a window
discriminator was used to convert the occurrence of each action
potential into digital pulses, which were led into either a Gateway
computer via a CED 1401 Plus interface (Cambridge Electronic Design,
Cambridge, UK) using Spike 2 software or an Apple IIe for on-line
visualization and storage and off-line analysis.
; Curtis et al., 1997). These consisted of a
recording pipette glued using a photopolymerizing resin (Silux, 3M)
next to an infusion pipette (Fisher). The recording pipette had a 2- to
4-µm-diameter tip (4-7 M) and was filled with PSB. The infusion
pipette (20-50-µm-diameter tip) was angled at approximately 30 to 45 degrees with its tip adjacent to the tip of the recording pipette but
100 to 120 µm dorsal. This was filled with a solution of
[D-Phe12,Nle21,38,C
MeLeu37]r/hCRF(12-41)
(DPheCRF12-41; 0.33 mg/ml) and connected by PE
tubing to a source of solenoid-activated pneumatic pressure (Picospritzer; General Valve, Inc.). This infusion pipette was calibrated such that known volumes could be administered (1 mm displacement = 60 nl). Intracerulear infusions were made by
applying small pulses of pressure (5-25 psi, 10-30 ms in duration) to
the peptide-containing barrel at a frequency of 0.2 to 1 Hz to deliver a volume of 30 nl (10 ng of peptide).
Protocol.
Once an action potential was isolated, spontaneous
discharge rate was recorded for at least 9 min before i.c.v. drug or
peptide administration. CRF [0.03-3 µg in 3 µl of artificial
cerebrospinal fluid (aCSF)], carbachol (0.1 µg in 3 µl of aCSF),
or VIP (0.02-0.2 µg in 3 µl of aCSF) was injected i.c.v. over a
period of 30 to 45 s, and LC discharge rate was recorded for at
least 15 min after i.c.v. administration. The dose of carbachol was one
that was previously determined to be submaximal for activating LC
neurons (Valentino and Aulisi, 1987). The doses of VIP were calculated to be equimolar with doses of CRF (0.03/0.3 µg), respectively.
). Discharge was
recorded for at least 12 min. The cannula was replaced with one
containing CRF, and CRF (0.1-3.0 µg in 3.0 µl of aCSF) was injected.
For experiments involving the intracerulear infusion of
DPheCRF12-41, LC discharge rate was recorded for
at least 9 min before the microinfusion. The movement of the solution
through the calibrated pipette was observed through a microscope
throughout the infusion. Injection of the entire volume (10 ng in 30 nl) usually required 1 to 2 min. CRF (0.1 µg in 3.0 µl of aCSF) was injected through the i.c.v. cannula at least 9 min after
DPheCRF12-41. The dose of intracerulear
administered DPheCRF12-41 was one that was
previously shown to prevent the effects of a maximally effective dose
of i.c.v. administered CRF (Curtis et al., 1997).
For all experiments involving the administration of drug or peptide,
only one cell from an individual rat was tested, and only one dose was
administered. In a few cases in which the only measurements taken were
spontaneous and sensory-evoked discharge rates, more than one cell (but
no more than three) from an individual rat was used.
Histology.
The recording site was marked by iontophoresis
(
15 µA, 10 min) of PSB at the end of the experiment. Neutral red (5 µl) was injected through the i.c.v. cannula to ensure placement in
the lateral ventricle. Rats were anesthetized with pentobarbital (100 mg/kg i.p.) and perfused with a 10% solution of paraformaldehyde in
phosphate buffer. Brains were removed and cut to visualize neutral red
in the ventricular system. They were then stored for at least 24 h
in this solution. Frozen 40-µm-thick coronal sections cut on a
cryostat were mounted onto gelatinized glass slides and stained with
neutral red for localization of the PSB spot. The data presented are
from neurons that were histologically identified as being within the
nucleus LC (Valentino et al., 1983).
Forced Swim Test.
To verify that the swim stress used in the
present study could produce behaviors comparable to those previously
reported (Porsolt et al., 1977; Borsini and Meli, 1988; Detke et al.,
1995), behavior in the forced swim test was recorded in rats during the
first 5 min of an initial exposure to 30 cm water during a 5-min
reexposure 24 h later, and during a 5-min exposure to 30 cm water
24 h after exposure to water of a depth of 4 cm. These rats were a
separate group from those used for electrophysiological analysis.
Behavior was videotaped and later scored as described below by an
observer blind to the condition.
Data Analysis. LC discharge was recorded on-line on either an Apple IIe computer or Cambridge Electronics Design 1401 data analysis system using Spike-2 software. The mean baseline LC spontaneous discharge rate was calculated from at least three 3-min intervals. Mean baseline LC spontaneous discharge rates of various treatment groups were compared with the use of a one-way factorial ANOVA.
Peristimulus time histogram data were quantified as previously described (Curtis et al., 1994); the histogram was divided into
different time components, and the discharge rate in each component was
calculated and compared between groups with the use of Student's
t test for independent samples.
In experiments involving CRF, VIP, or carbachol administration, the
mean preinjection discharge rate was calculated over at least three
3-min intervals, and postinjection discharge rates were expressed as a
percentage of this baseline value. A one-way ANOVA with repeated
measures was used to assess the statistical significance of effects in
an individual treatment group, and Scheffé's F test
was used post hoc to determine statistically significant differences at
individual time points. A two-way ANOVA with repeated measures was used
to compare differences between time course data of different treatment groups.
The maximum percentage increase in LC discharge rate produced by a dose
of CRF during the 15 min after injection was used to generate the
dose-response curve. CRF dose-response curves were analyzed by a
one-way factorial ANOVA. CRF dose-response curves in different
treatment groups were compared with the use of a two-way factorial
ANOVA. Responses of different treatment groups to a given dose of CRF
were compared with the use of a one-way factorial ANOVA.
In antagonist experiments, the mean LC discharge rate determined after
DPheCRF12-41 and before CRF injection was used as a baseline rate, and post-CRF rates were expressed as a percentage of this baseline. For experiments comparing responses of only two
treatment groups, significant difference was determined by Student's
t test for independent samples. Statistical significance was
considered at p < .05.
Behavior in the forced swim test was scored using a time-sampling
procedure identical to that previously reported (Detke et al., 1995).
Behaviors were scored at the end of each 5-s epoch as 1) immobility,
defined by floating without struggling (i.e., the rat makes only those
movements necessary to keep its head above the water); 2) swimming,
defined as swimming movements that result in a change in position of at
least one fourth of the cylinder circumference; 3) diving, in which the
entire body is submerged; and 4) climbing, which was defined as making
active movements with the forepaws in and out of the water (usually
directed against the cylinder walls). Climbing could be discriminated
from swimming because it did not result in a significant change in the
relative position in the cylinder. Although diving was scored
separately, it occurred relatively infrequently and therefore was added
to swimming scores in the final analysis. Finally, as another measure, the incidences of swimming and climbing were added together to make an
"active behavior" score that was compared between groups.
Behavior was scored at least two times by an observer who was blind to
the condition, and test-retest reliability was determined by Pearson's
correlation. The mean incidence of a particular behavior in a group of
rats was determined and compared between groups using a one-way
factorial ANOVA with Dunnett's t test for individual comparisons.
Drugs.
Ovine CRF and DPheCRF12-41
were generously supplied by Dr. Jean Rivier (Clayton Foundation
Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA).
VIP was purchased from Bachem (Torrance, CA). The peptides were
dissolved in water to make a 1 mg/ml solution. Aliquots (10 µl) of
this solution were concentrated using a Savant Speed Vac concentrator.
The 10-µg aliquots were stored at 70°C and dissolved in aCSF on
the day of the experiment. Carbachol (Sigma Chemical Co., St. Louis,
MO) was dissolved in aCSF (0.03 mg/ml) and administered i.c.v. in a
volume of 3 µl.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Effects of Prior Swim Stress on Behavior in Forced Swim Test.
As previously reported (Porsolt et al., 1977; Detke et al., 1995),
prior experience with swim stress significantly increased the incidence
of immobility on retest 24 h later (Fig.
1). In contrast, the incidence of
immobility in nonswim control animals was not different than that
observed in naive rats (Fig. 1A). Consistent with this, the incidence
of active behaviors (swimming plus climbing) was lower in rats with
prior experience with swim stress compared with either nonswim control
animals or naive rats (Fig. 1A). Comparative analysis of individual
active behaviors (swimming and climbing) indicated that the incidence
of swimming tended to be lower in rats previously exposed to swim
compared with nonswim control animals or naive rats (Fig. 1B). Although this effect did not reach statistical significance using the factorial ANOVA (p = .07), a comparison between swim stress rats
and nonswim control animals revealed a statistically significant
difference (p < .05, Student's t test for
independent samples). Climbing behavior was similar across all groups
of rats. The test-retest correlations for immobility, swimming, and
climbing scores were 0.92, 0.88, and 0.85, respectively.
|
Effects of Prior Swim Stress on LC Spontaneous Discharge.
LC
spontaneous discharge activity was recorded from 125 neurons in 114 swim stress rats and 75 neurons in 65 nonswim control animals 24 h
after the manipulation and 79 neurons in 79 naive rats. The mean and
range of LC spontaneous discharge rates for the three groups were
1.6 ± 0.1 Hz (0.6-3.6 Hz; swim stress), 1.5 ± 0.1 Hz
(0.5-3.7 Hz; nonswim control animals), and 1.5 ± 0.1 Hz
(0.5-3.8 Hz; naive rats). The mean LC discharge rates were not
different (F2,247 = 0.6, p > .1) among the three groups and were similar to
those reported in previous studies (Curtis et al., 1994, 1997).
Effects of Prior Swim Stress on LC Activation by CRF.
CRF
produced a dose-dependent increase in LC discharge rates in all groups
of rats (F3,35 = 21.5, p < .001; F3,30 = 17.0, p < .001; and
F4,36 = 6.0, p < .001 for naive rats, nonswim control animals, and swim stress rats,
respectively) (Fig. 2). CRF dose-response curves generated in naive rats and nonswim control animals were not
different (F1,66 = 1.1, p > .1) and exhibited the characteristic sigmoidal
shape when plotted as response versus log dose (Fig. 2, compare filled
and open circles). In contrast, the shape of the CRF dose-response
curve generated in swim stress rats was markedly different, consisting
of two components, separated by an inflection. A relatively
high-potency, low-efficacy component was defined by the effects of 0.03 to 0.3 µg of CRF, and a second component was apparent at doses
greater than or equal to 1.0 µg (Fig. 2, filled triangles).
Importantly, low doses of CRF (0.1 and 0.3 µg) that had little effect
in either naive rats or nonswim control animals increased the LC
discharge rate of swim stress rats. LC activation produced by 0.1 and
0.3 µg of CRF was significantly greater in swim stress rats than in
the other groups (F2,23 = 8.5, p < .002 for 0.1 µg, and
F2,26 = 5.3, p < .02 for 0.3 µg). In contrast, 1.0 µg of CRF was significantly less
effective in swim stress rats compared with the other groups
(F2,25 = 5.2, p < .02). Table 1 shows the mean basal
discharge rates before administration of each dose of CRF in the three
treatment groups tested. There were no significant differences in basal
discharge rate among the three groups.
|
|
Effects of a CRF Antagonist on LC Responses to CRF.
The i.c.v.
administration of DPheCRF12-41 (3 µg) to swim
stress rats 24 h after the swim did not alter LC discharge rate.
The mean LC discharge rates before and 12 min after antagonist administration were identical (1.6 ± 0.1 Hz; n = 12), and the rates during the pretreatment interval were stable
(F4,59 = .9, p > .1).
However, i.c.v. administered DPheCRF12-41
prevented LC activation by 0.1 and 0.3 µg of CRF in swim stress rats
when administered 12 to 15 min before CRF (p > .06 and
.1, respectively; t test for matched samples) (Fig.
3A). A comparison between the effects
produced by 0.1 and 0.3 µg of CRF in rats pretreated with the CRF
antagonist versus rats not pretreated with the antagonist revealed a
statistically significant difference (Fig. 3A).
DPheCRF12-41 significantly attenuated
(p < .05, t test for independent samples) but did not completely block (p < .002, t
test for matched samples) LC activation by 3.0 µg of CRF (Fig. 3A).
In contrast, DPheCRF12-41 pretreatment did not
alter the LC response to 1.0 µg of CRF (Fig. 3A). The mean LC
discharge rates before CRF administration were similar in rats
pretreated with the CRF antagonist versus nonpretreated rats [i.e.,
1.7 ± 0.3 Hz (n = 5) versus 1.8 ± 0.3 Hz
(n = 7) before 0.1 µg of CRF, 2.1 ± 0.4 (n = 6) versus 1.6 ± 0.2 (n = 10)
before 0.3 µg of CRF, 1.4 ± 0.2 Hz (n = 6)
versus 1.6 ± 0.1 Hz (n = 8) before 1.0 µg of
CRF, and 1.5 ± 0.2 Hz (n = 10) versus 1.8 ± 0.2 Hz (n = 8) before 3.0 µg of CRF].
|
Effects of Swim Stress on LC Activation by Non-CRF Inputs.
In
contrast to the alterations in LC sensitivity to CRF, LC sensitivity to
other agents was unaffected by swim stress. For example, LC discharge
evoked by repeated sciatic nerve stimulation, which is mediated by
excitatory amino acid inputs to LC (Ennis and Aston-Jones, 1988), was
similar in swim stress rats and nonswim control animals. The magnitude
of this evoked response, as measured by discharge rate during the
response, was 10.0 ± 1.4 Hz (n = 15) and
10.4 ± 1.1 Hz (n = 15) for swim stress rats
versus nonswim control animals, respectively. Similarly, there was no
difference in the duration of the evoked response: 68 ± 5 and
68 ± 6 ms for swim stress rats versus nonswim control animals,
respectively. The magnitude and duration of LC responses to sciatic
nerve stimulation were comparable to those reported in naive rats in
previous studies (Curtis et al., 1994).
) were not altered in swim stress rats (Fig.
4B). LC discharge rates before carbachol
administration were similar for swim stress rats versus nonswim control
animals: 1.7 ± 0.2 Hz (n = 6) and 1.5 ± 0.3 Hz (n = 6), respectively. Finally, in contrast to CRF,
there was no apparent change in LC sensitivity to VIP in swim stress
rats (Fig. 4A). The mean LC discharge rates before VIP administration
were not different for swim stress rats compared with naive rats
[i.e., 1.5 ± 0.2 Hz (n = 5) versus 1.6 ± 0.4 Hz (n = 5) before 0.02 µg of VIP, 1.8 ± 0.2 Hz (n = 5) versus 1.4 ± 0.1 Hz (n = 5) before 0.07 µg of VIP, and 1.4 ± 0.3 (n = 7) versus 1.1 ± 0.1 (n = 7) before 0.2 µg of
VIP].
|
Modeling of CRF-LC Interactions.
The shift in the CRF
dose-response curve produced by swim stress was indicative of a change
in CRF receptor-binding kinetics. The shape of the CRF dose-response
curve in swim stress rats resembled the theoretical curve for a ligand
binding to two independent sites described by the equation:
|
).
Figure 5A shows hypothetical curves
describing ligand binding to two sites that are in present in equal
amounts (i.e., A = B = 0.5) (Fig. 5A). When
K1 and
K2 are sufficiently similar, the curve
exhibits the characteristic sigmoidal shape and two sites cannot be
discriminated (e.g., Fig. 5A, filled circles). As the dissociation
constants diverge, the slope becomes more shallow, and when they are
sufficiently different (e.g., K1 = 0.001 K2; triangles), an inflection
becomes apparent that separates the sites at a point representing the
proportion of each site. In the case of the theoretical curve shown in
Fig. 5A, the inflection appears at 50% because the two sites are
present in equal amounts.
|
), the CRF ED50 value in
naive rats was 0.9 µg, similar to the value previously reported by
this laboratory (Curtis et al., 1997). The theoretical dose-response curve in naive rats was generated by substituting this value into the
equation E/Emax = [CRF]/[CRF] + ED50 (Fig. 5B, open circles). The hypothetical curve in swim stress rats (open squares) was generated
based on the assumptions that 1) CRF binds to two sites that exist in a
40:60 ratio (determined by the point of inflection of the experimental
dose-response curve), 2) the dissociation constant of the high-affinity
site is proportional to the ED50 value of the
first component of the dose-response curve (determined to be 0.06 µg), 3) the dissociation constant of the low-affinity site is
proportional to an ED50 value of 10 µg, and 4)
the response is proportional to the fraction of receptors occupied (Y).
Substitution of 0.4 and 0.6 for A and B, respectively, and 0.06 and 10 for K1 and
K2, respectively, in the above
equation describing a two-site model yielded the hypothetical
dose-response curve in swim stress rats (open squares). Thus, the
hypothetical curve predicts the response to CRF acting at two
independent binding sites, with one site making up 40% of total sites
and having a dissociation constant 166 times lower than the second
site, which consists of 60% of total sites. Although the hypothetical
dose-response curve based on this model was not completely superimposed
on the actual data, there was a considerable overlap (three of five
points) between the hypothetical and experimental curves.
Responses produced by the combination of the CRF antagonist
DPheCRF12-41 (3 µg i.c.v.) and different doses
of CRF in swim stress rats were also well predicted by the two-binding
site model described above, factoring in the predicted effect of a competitive antagonist, which is to increase the agonist
ED50 value by a factor of [1 + I/IC50]. Figure 5C shows the hypothetical dose-response curve that would be generated in the presence of DPheCRF
(3 µg) based on the equation:
![<UP>Y</UP>=<FR><NU><UP>A </UP>[<UP>CRF</UP>]</NU><DE>[<UP>CRF</UP>]+<UP>ED<SUB>50a</SUB> </UP>(<UP>1</UP>+<UP>I/IC</UP><SUB><UP>50</UP></SUB>)</DE></FR>+<FR><NU><UP>B </UP>[<UP>CRF</UP>]</NU><DE><UP>CRF</UP>+<UP>ED<SUB>50b</SUB> </UP>(<UP>1</UP>+<UP>I/IC</UP><SUB><UP>50</UP></SUB>)</DE></FR>](/content/vol289/issue3/fulltext/1211/img002.gif)
|
). Note that the hypothetical curve for the combination of CRF
and DPheCRF12-41 in swim stress rats is shifted
to the right in a parallel manner from the hypothetical CRF
dose-response curve and that the experimental data obtained in
antagonist-pretreated rats fit the model curve well.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The present results demonstrate that swim stress resulting in
behavioral depression 24 h later profoundly alters LC responses to
CRF at the same time, sensitizing LC neurons to low doses of CRF and
desensitizing LC neurons to high doses. Alterations in LC sensitivity
produced by swim stress were selective to CRF. Additionally,
sensitization to CRF was apparent at the level of the LC. These
findings, taken with the results of pharmacological modeling, suggest
that swim stress alters the binding kinetics of CRF receptors that
mediate LC activation. The findings that repeated shock (Curtis et al.,
1995), which also produces behavioral deficits 24 h later (Maier
and Seligman, 1976), has similar effects on LC sensitivity to CRF and
that exposure to a stress (4 cm water) that does not produce behavioral
depression does not alter LC sensitivity to CRF suggest that changes in
LC sensitivity to CRF play a role in the behavioral depression. Because
this behavior is sensitive to antidepressant treatment (Porsolt et al.,
1977; Borsini and Meli, 1988; Detke et al., 1995), the neuronal changes reported here may be involved in the pathophysiology of depression and/or other stress-related psychiatric disorders.
CRF-LC interactions were demonstrated to be regulated at a presynaptic
level by adrenalectomy, which increases tonic CRF secretion in the LC
(Pavcovich and Valentino, 1997). This effect is of interest in light of
hypotheses implicating CRF hypersecretion in the pathophysiology of
depression (Nemeroff et al., 1984; Gold et al., 1988). In contrast to
adrenalectomy, swim stress did not produce hypersecretion of CRF in the
LC because LC spontaneous discharge rates were comparable in swim
stress rats versus control animals and were unaffected by the
administration of a CRF antagonist. The inability of swim stress to
alter CRF-LC interactions on a presynaptic level is shared by prior
footshock stress (Curtis et al., 1995; Lechner et al., 1997). These
findings argue against the possibility that a history of prior stress
induces CRF hypersecretion within the LC.
Alterations in LC Sensitivity to CRF: Putative Mechanisms.
In
contrast to its lack of effect on LC spontaneous discharge, swim stress
profoundly altered LC sensitivity to CRF. Certain explanations for
altered LC sensitivity to CRF can be ruled out. It is unlikely that
swim stress affected the overall excitability of LC neurons, as
spontaneous discharge and responses to an excitatory amino acid input,
a muscarinic agonist, and VIP were comparable to control rats. These
results underscore the specificity of the response. The results with
VIP are of particular interest because both VIP (Wang and Aghajanian,
1990) and CRF (Grigoriadis et al., 1996) receptors are positively
coupled to adenyl cyclase. The finding that swim stress differentially
affects sensitivity to two peptides that activate a common second
messenger system via distinct receptors argues against a nonselective
change at the level of this second messenger system.
).
Ruling out the above possibilities, the shift in the CRF dose-response
curve produced by swim stress could be explained by a change in CRF
receptor-binding kinetics. The shape of the CRF dose-response curve
generated in swim stress rats resembled that of a ligand binding to two
independent sites with different dissociation constants (Pratt and
Taylor, 1990), and pharmacological modeling was consistent with this. A
recent study demonstrated that recombinant CRF-R1 receptors expressed
in Sf9 cells have two binding sites with dissociation constants that
differ by a factor of 10 (Primus et al., 1997), a difference that would
make the sites indistinguishable on dose-response analysis (Fig. 5A).
The experimental dose-response curve obtained in swim stress rats could
be produced if swim stress caused the dissociation constants of the two
sites to diverge, by decreasing the dissociation constant of one site
and increasing that of the second, thus allowing the two sites to be
discriminated on the dose-response curve. The response produced by the
1.0 µg dose of CRF was the most discrepant from the model curve.
Because the model assumes two independent sites on the same receptor, this discrepancy could arise as a result of negative cooperativity between the two sites that occurs when the first site becomes saturated. It is noteworthy that the results obtained with the CRF
antagonist DPheCRF12-41 were also consistent
with the two-site model described above.
Although the two-site model described above could account for the
effects of swim stress on the CRF dose-response curve, alternative mechanisms have not been discounted. For example, the existence of an
uncoupled high-affinity receptor in the naive rat that becomes coupled
to cellular effectors after swim stress could explain the sensitization
observed. Similarly, the expression of new, high-affinity receptors to
account for sensitization has not been ruled out.
Alterations in LC Sensitivity to CRF: Functional Implications.
Sensitization of LC neurons to low doses of CRF and desensitization to
high doses of CRF produced by swim stress are reminiscent of the
effects produced by repeated sessions of footshock (Curtis et al.,
1995). Interestingly, only desensitization was apparent after a single
session of shock (Curtis et al., 1995). Desensitization to CRF has also
been reported after the repeated administration of CRF and auditory
stress (Conti and Foote, 1995, 1996). The studies of Conti and Foote
(1995) examined only the effects of a relatively high dose (3 µg
i.c.v.) of CRF, and it is unknown whether sensitization was also
produced by their manipulations (Conti and Foote, 1995, 1996). Taken
together, these studies underscore the ability of CRF-LC interactions
to be regulated at a postsynaptic level by prior stress.
; Curtis et al., 1997), a predicted consequence of LC sensitization
to CRF would be hyperarousal or hyperreactivity to certain stimuli. It
is noteworthy that these are hallmark symptoms of post-traumatic stress
disorder, which occurs after uncontrollable trauma. It is tempting to
speculate that LC sensitization to CRF initiated by uncontrollable
stress underlies these symptoms of post-traumatic stress disorder. LC sensitization to CRF may also play a role in irritable bowel syndrome, a prevalent functional bowel disorder that often coexists with anxiety
or depression and is characterized by heightened central responses to
bowel distention (Mayer et al., 1995). Because LC activation by low
magnitudes of bowel distention is mediated by CRF release in the LC
(Lechner et al., 1997), LC sensitization to CRF would be expected to
facilitate LC and forebrain activation by colonic stimuli.
Finally, correlations between the neuronal effects reported here and
behavioral deficits implicate LC sensitization to CRF in the
pathophysiology of depression. Thus, two different manipulations that
sensitize LC neurons to CRF, swim stress (this study) and repeated
shock (Curtis et al., 1995), also produce behavioral deficits that are
sensitive to antidepressant drugs (Maier and Seligman, 1976; Porsolt et
al., 1977). In contrast, a stress that did not sensitize LC neurons to
CRF (exposure to 4 cm water) did not produce behavioral deficits.
Consistent with this, increased responsiveness of the noradrenergic
system to a subthreshold shock in rats previously exposed to repeated
shock was correlated to the development of learned helplessness (Petty
et al., 1994) (i.e., it was not observed in rats that did not develop
the behavioral deficit). Together, these findings support the
compelling hypothesis that LC sensitization to CRF is involved in the
pathology of depression and/or is a target of action of antidepressant drugs.
| |
Acknowledgments |
|---|
We thank Dr. Jean Rivier for the generous gifts of oCRF and DPheCRF12-41 and Dr. Paul McGonigle for comments on data presentation and interpretation. The expert technical assistance of Michael Valentino and Wei Ping Pu is greatly appreciated.
| |
Footnotes |
|---|
Accepted for publication January 22, 1999.
Received for publication November 5, 1998.
1 This work was supported by U.S. Public Health Service Grants MH42796, MH40008, and MH00840 (an RSDA award to R.J.V.) and an NARSAD Young Investigator Award (to L.A.P.).
Send reprint requests to: Dr. Rita J. Valentino, Department of Psychiatry, MS 403, Medical College of Pennsylvania and Hahnemann University, Broad and Vine St., Philadelphia, PA 19102-1192. E-mail: valentinor{at}auhs.edu
| |
Abbreviations |
|---|
CRF, corticotropin-releasing factor;
aCSF, artificial cerebrospinal fluid;
DPheCRF12-41, [D-Phe12,Nle21,38,CMeLeu37]r /hCRF(12-41);
LC, locus ceruleus;
PSB, pontamine sky blue;
VIP, vasoactive intestinal
peptide.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  B. A. S. Reyes, R. J. Valentino, and E. J. Van Bockstaele Stress-Induced Intracellular Trafficking of Corticotropin-Releasing Factor Receptors in Rat Locus Coeruleus Neurons Endocrinology, January 1, 2008; 149(1): 122 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Buckley and A. F. Schatzberg On the Interactions of the Hypothalamic-Pituitary-Adrenal (HPA) Axis and Sleep: Normal HPA Axis Activity and Circadian Rhythm, Exemplary Sleep Disorders J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3106 - 3114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. P. Jedema and A. A. Grace Corticotropin-Releasing Hormone Directly Activates Noradrenergic Neurons of the Locus Ceruleus Recorded In Vitro J. Neurosci., October 27, 2004; 24(43): 9703 - 9713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E A MAYER The neurobiology of stress and gastrointestinal disease Gut, December 1, 2000; 47(6): 861 - 869. [Full Text] [PDF] < |
, solid line) and nonswim control animals (
, dashed line) and
are monophasic for both groups. In contrast, the CRF dose-response
curve generated in swim stress rats (
, dashed line) is biphasic and
indicates sensitization to low doses of CRF and desensitization to
higher doses. Each point is the mean of 4 to 10 cells, and vertical
lines represent ± 1 S.E.M. See Table 
, dashed line, n = 6), in nonswim control animals (
),
K1 = 0.01K2
(
). Note that as
K1 and K2
diverge, the slope of the curve becomes more shallow and an inflection
becomes apparent at 50%. B, experimental (
) dose-response curves for CRF in control