Abstract
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.
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
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 MΩ) 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.
For experiments involving intracerulear administration of a CRF antagonist, double-barrel glass micropipettes were used to record single-unit LC discharge and simultaneously microinfuse the peptide (Akaoka et al., 1992; 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 MΩ) 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,CαMeLeu37]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.
For experiments that examined LC responses to sciatic nerve stimulation, LC discharge was recorded for at least 9 min, and a trial of 60 sciatic nerve stimuli (1.3 mA, 0.5-ms duration, 0.5 Hz) was initiated. Stimuli were applied through a pair of 25-gauge hypodermic needles inserted into the medial aspect of the contralateral hindpaw using a Master 8 pulse stimulator and stimulus isolation unit (Isoflex; AMPI). LC discharge activity during these trials was recorded and stored as peristimulus time histograms. Synchronizing pulses initiated 2 s sweeps 500 ms before the stimulus; thus, discharge activity was recorded for 500 ms before the stimulus and for the next 1.5 s. After recording spontaneous and sensory-evoked discharge, the electrode was moved ventrally until another cell was isolated, and the protocol was repeated. In this way, sensory-evoked discharge was quantified from each of three cells in five animals (n= 15 total) of both swim stress and nonswim control animals.
In antagonist experiments that involved the i.c.v. administration of DPheCRF12–41, a cannula guide (22 gauge; Plastic Products) was inserted at the coordinates described above with its tip 4.6 mm ventral to skull surface. This was affixed to the skull using machine screws and cranioplastic cement. A cannula (26 gauge) connected by PE tubing to a 10-μl syringe and filled to the tip with DPheCRF12–41 (0.33 mg/ml) fit into the guide cannula such that the tip extended 1 mm past the guide cannula, placing it into the lateral ventricle. LC discharge was recorded for at least 9 min, and DPheCRF12–41 (3 μg in 3 μl of aCSF) was then microinfused into the LC. This dose of DPheCRF12–41 was determined to be selective for antagonism of CRF and approximately 20 times the IC50 value (Curtis et al., 1994). 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’st 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’st 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 DPheCRF12–41were 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
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.
Behaviors during the forced swim test. The bars indicate the incidence of different behaviors (as defined inMaterials and Methods) during a 5-min forced swim test 24 h after manipulation for swim stress (hatched bars,n = 11) or in nonswim control animals (filled bars,n = 11) or in naive rats (open bars,n = 11). A, active behaviors represent the sum of swimming and climbing. B, these active behaviors are broken down into the individual components. *p < .05, **p < .005.
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; andF4,36 = 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, andF2,26 = 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.
Prior swim stress markedly alters the CRF dose-response curve for LC activation. The abscissa indicates the dose of CRF (micrograms, log scale), and the ordinate indicates the maximum increase produced by a dose of CRF expressed as a percentage of the mean baseline rate. CRF dose-response curves are similar in naive rats (●, 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 1 for the mean spontaneous discharge rates before the administration of different doses of CRF.
Mean basal LC discharge rate before CRF administration
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 DPheCRF12–41prevented 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, ttest 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 a CRF antagonist on LC responses of swim stress rats to CRF. A, effects of i.c.v. administered DPheCRF12–41 (3.0 μg). The abscissa indicates CRF dose administered i.c.v. The ordinate indicates the maximum increase in LC discharge rate expressed as a percentage of the baseline rate. Bars indicate the mean effects of CRF in the absence (open bars) and presence (solid bars) of DPheCRF12–41. Each bar represents the mean of 5 to 10 cells, and vertical lines represent ±1 S.E.M. DPheCRF12–41 attenuated the effects of the lowest and highest doses of CRF but not that produced by 1.0 μg of CRF (*p < .05). B, effects of intracerulear administered DPheCRF12–41 (10 ng). The abscissa indicates the time (min) before and after injection of CRF (0.1 μg i.c.v.) that occurred at time = 0, and the ordinate indicates LC discharge rate expressed as a percentage of the mean baseline rate. Shown are the effects of CRF in swim stress rats not pretreated with the antagonist (▴, solid line, n = 7), swim stress rats pretreated with the antagonist (▵, dashed line, n= 6), in nonswim control animals (○, solid line,n = 7), and in naive rats (●, solid line,n = 10). Intracerulear administration of DPheCRF12–41 prevented LC activation by 0.1 μg of CRF in swim stress rats, suggesting that mechanisms for sensitization lie within the nucleus LC. Vertical lines represent ±1 S.E.M.
Like i.c.v. administration, intracerulear administration of DPheCRF12–41 (10 ng in 30 nl) to swim stress rats (24 h after the stress) did not alter the mean LC discharge rates, which were 1.7 ± 0.3 Hz versus 1.8 ± 0.3 Hz (n = 5) before and 9 min after DPheCRF12–41 administration, respectively. However, intracerulear administration of DPheCRF12–41 significantly prevented LC activation by 0.1 μg of CRF (F5,35 = 1.1,p > .1, one-way ANOVA, repeated measures) (Fig. 3B). The response to 0.1 μg of CRF in swim stress rats pretreated with the CRF antagonist was similar to that observed in nonswim control animals or in naive rats (Fig. 3B). The mean LC discharge rates before CRF administration were not different between different groups of rats (F3,29 = 1.8, p > .1, one-way factorial ANOVA): 1.8 ± 0.2 Hz (n = 6 swim stress, antagonist-pretreated rats), 1.8 ± 0.3 Hz (n = 7 swim stress, not pretreated), 1.2 ± 0.2 Hz (n = 7 nonswim control animals), and 2.0 ± 0.3 Hz (n = 10 naive rats).
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).
LC responses to a dose of the muscarinic agonist carbachol, which was previously demonstrated to be submaximal for activating LC neurons (Valentino and Aulisi, 1987) 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].
Prior swim stress does not alter LC responses to VIP or to a muscarinic agonist (carbachol). A, bars represent the mean maximum increase in LC discharge expressed as a percentage above the baseline rate produced by VIP (0.02–0.2 μg i.c.v.) in naive control animals (open bars, n = 5) versus swim stress rats (filled bars, n = 5). B, bars represent the mean maximum increase in LC discharge expressed as a percentage above the baseline rate produced by carbachol (0.1 μg i.c.v.) in nonswim control animals (open bar, n = 6) versus swim stress rats (filled bar, n = 6). Vertical lines represent ±1 S.E.M.
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:
Experimental and hypothetical CRF dose-response curves. A, hypothetical dose-response curves for an agonist binding at two independent sites that are in equal proportions, as described by the equation shown in Results. The series of curves are generated by varying K1 such thatK1 = K2(●), K1 = 0.1K2 (▪),K1 = 0.01K2(▴), and K1 = 0.001K2 (▾). Note that asK1 and K2diverge, the slope of the curve becomes more shallow and an inflection becomes apparent at 50%. B, experimental (●, ▪) and hypothetical (○, ■) dose-response curves for CRF in control ●, ○) and swim stress (▪, ■) rats. The hypothetical curve in control rats (○) was generated by substituting 100 for Emaxand 0.9 μg for ED50 in the equationE/Emax = [CRF]/[CRF] + ED50. ■, Hypothetical curve for CRF binding to two sites in a 40:60 ratio and with ED50 values of 0.06 and 10 μg. Note that three of five points on the CRF dose-response curve generated in swim stress rats overlap this hypothetical curve. C, experimental (▴, ▪) and hypothetical (▵, ■) CRF dose-response curves in swim stress rats in the absence (▴, ▵) and presence (▪, ■) of the competitive antagonist DPheCRF12–41. The hypothetical dose-response curve in the absence of the antagonist (▵) was generated as described in B. The hypothetical curve predicting the effects of a competitive antagonist (■) was generated using the equation described in Results. Note that this curve closely models the experimental results.
Figure 5B shows experimental and hypothetical CRF dose-response curves in naive (circles) and swim stress (squares) rats. Assuming a 100% increase in LC discharge rate as the maximum effect produced by CRF (Curtis et al., 1997), 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 andK2, 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:
Discussion
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.
Changes in LC sensitivity to CRF could arise if swim stress facilitated CRF activation of neurons outside of the LC that are sources of LC afferents. However, the finding that intracerulear administration of a CRF antagonist prevented the effects of a low dose of CRF (to which the LC was sensitized to) suggested that the mechanism for sensitization was at the level of the LC, although a direct action on LC neurons versus afferent terminals within the LC cannot be discriminated by this study.
Possible changes in the CRF-binding protein could not account for the altered LC sensitivity to CRF observed in this study. Either an increase or a decrease in the binding protein would be predicted to decrease or increase, respectively, the effective concentration of agonist and shift the curve in a parallel manner, as opposed to producing the complex shift observed in this study. Additionally, oCRF, which was used as the agonist in this study, is a poor ligand for the CRF-binding protein (Chalmers et al., 1996).
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.
Of the two effects produced by swim stress and repeated footshock (i.e., sensitization and desensitization), sensitization is likely to be the physiologically relevant consequence because it occurs in a low dose range. Sensitization of LC neurons to CRF would allow subthreshold stimuli to activate the LC-noradrenergic system and exert an impact on its targets. Because CRF release in the LC results in arousal, as indicated by forebrain electroencephalographic activation (Page et al., 1993; 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
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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
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↵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.).
- Abbreviations:
- CRF
- corticotropin-releasing factor
- aCSF
- artificial cerebrospinal fluid
- DPheCRF12–41
- [d-Phe12,Nle21,38,CαMeLeu37]r /hCRF(12–41)
- LC
- locus ceruleus
- PSB
- pontamine sky blue
- VIP
- vasoactive intestinal peptide
- Received November 5, 1998.
- Accepted January 22, 1999.
- The American Society for Pharmacology and Experimental Therapeutics