Physiology&Behavior.Vol. 51, pp. 1129-1133, 1992
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
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Adrenal Hormones in Rats Before and After Stress-Experience: Effects of Ipsapirone S. M. KORTE, 1 G. A. H. B O U W S A N D B. B O H U S
Department of Animal Physiology, University of Groningen, Center for Behavioral, Cognitive and Neuro-Sciences, P.O. Box 14, 9750 AA Haren, The Netherlands Received 9 September 1991 KORTE, S. M., G. A. H. BOUWS AND B. BOHUS. Adrenal hormones in rats before and after stress-experience:Effects of ipsapirone. PHYSIOL BEHAV51(6) 1129-1133, 1992.--The present study was designedto investigatethe effectsof the anxiolytic 5-HTjAreceptor agonist ipsapirone on the hormonal responses in rats under nonstress and stress conditions by means of repeated blood sampling through an intracardiac catheter. Ipsapirone was given in doses of 2.5, 5, 10, and 20 mg/kg (IP) under nonstress conditions in the home cages of the rats. Plasma corticosterone levels increased in a dose-dependent way in the dose range of 5 to 20 mg/kg, whereas the plasma catecholamines were only significantlyincreased with the highest dose of the drug. The effect of ipsapirone in control and in stressed rats was studied with the selected dose of 5 mg/kg. Conditioned fear of inescapableelectric footshock (0.6 mA, AC for 3 s) given one day earlier was used as stressor. Surprisingly,ipsapirone potentiated the magnitude of the neuroendocrine responses. Rats receiving an inescapable footshock 1 day earlier showed a further elevated corticosterone response to the 5-HTIAreceptor agonist ipsapirone even before exposing them to the conditioned stress situation. The present findingssuggest that if an animal has no possibilitiesto escape or avoid a noxious event, functional hypersensitivitywill develop in the serotonergic neuronal system, which is reflected in the increased responsivenessof the HPA axis to a 5-HT~Aagonist challenge. Norepinephrine
Epinephrine
Corticosterone
lpsapirone
THERE is a large body of evidence that serotonin (5-hydroxytryptamine, 5-HT) is involved in mechanisms of neuroendocrine activation and anxiety ( 17,24,51,57). The widespread projections of 5-HT neurons from raphe nuclei to the limbic system, the hypothalamus and the cortex (2,35,40,48), provide the anatomic substrate for a functional system that may be involved in anxiety and neuroendocrine regulation. Autoradiographic studies suggest that 5-HT~A receptors are located presynaptically on serotonergic cell bodies in the raphe nuclei and postsynaptically on neurons in the septo-hippocampal-amygdaloid system, cortex, and hypothalamus (20,39). Binding of ipsapirone to these receptors may exert complex effects on 5-HT neurotransmission. The anxiolytic effects are thought to be mediated by suppression ofserotonergic neuronal activity via a full agonistic action on presynaptic 5-HTIA autoreceptors ( 16,17,51) or via a partial agonist/antagonist action at postsynaptic 5-HTtA receptor rich sites (23,43). There is a large body of evidence that serotonergic (5-HT) 1A receptor mechanisms are involved in the neural activation of both the hypothalamic-pituitary-adrenocortical (HPA) axis (7,14,18,19,21,26,31,53,54) and the adrenomedullary system (4,5,9,10,14,30). Ipsapirone is a novel anxiolytic compound with a high degree of specific binding to 5-HTjA receptors (15,45,52). Therefore, ipsapirone can be used as a tool to study serotonergic mechanisms underlying anxiety and neuroendocrine activation. Requests for reprints should be addressed to S. M. Korte.
1129
5-HT~^receptor
Stress
It was hypothesized that absence of control in a social stress situation (defeat) may change the state of 5-HT receptors for subsequent stimulation and may lead to an elevation in circulating adrenal hormones (25,54). In contrast, ipsapirone appears to have anxiolytic-like effects in a nonsocial stress situation if behavior and heart rate are used as markers for stress (27,28). Therefore, the aim of the present study was to investigate the influence of ipsapirone on the plasma levels of catecholamines and corticosterone after the experience of absence of control in a nonsocial stress situation. METHOD
Animals Male Wistar rats weighing 290-340 g at the beginning of the experiments were used. They were housed individually in clear Plexiglass cages (25 × 25 × 30 cm) on a 12-h light-dark regime (0730 h-1930 h lights on) at a room temperature of 21 _+ 2°C. All animals had free access to standard food (Hope Farms rat chow) and water.
Surgery Under complete ether anaesthesia, the animals were provided with a silicon heart catheter (0.95 mm o.d., 0.50 mm i.d.) through
1130
the right jugular vein and externalized on the top of the skull according to a technique described earlier (47). This method allows frequent blood sampling in freely moving rats (56). After each sample an equal quantity ofcitrated donor blood was given to avoid diminution of the blood volume with related changes in hemodynamics (47). Donor blood was obtained from unstressed rats with permanent heart catheters. The rats were given 1-2 weeks to acclimatise and recover from surgeD'.
Drug Treatment lpsapirone (TVX Q 7821, Troponwerke GmbH and Co., Cologne, Germany) was dissolved in saline. Injections were given intraperitoneally (IP) 29 rain before the test session in a constant volume of 2.0 ml/kg. The dose of 5 mg/kg, IP, was used in the postshock test because this dose has shown to have anxiolytic-like effects in different test situations if behavior and heart rate were used as markers of stress (27,28,51).
KORTE. BOUWS AND BOHUS
Chemical Determinations Blood samples of 0.45 ml were withdrawn for determination of plasma E, NE, and CORT. The samples were transferred immediately into chilled (0°C) centrifuge tubes containing 0.01% EDTA as antioxidant and 10 #1 heparin solution (500 IU/ml) as anticoagulant. Blood was centrifuged at 4°C for 10 min at 5000 rpm, and 100 ul of the supernatant were stored at - 2 0 ° C for CORT and at - 8 0 ° C for the catecholamine (CA) measurements. Plasma CORT was measured by means of reversed phase high performance liquid chromatography (HPLC) (13). Determination of plasma CA concentrations was performed by HPLC in combination with electrochemical detection (44).
Statistics The two-tailed Dunnett's test (36) was used to compare the vehicle control group with the ipsapirone-treated groups under baseline conditions. The neuroendocrine data of drug-treated control and stressed animals were evaluated by means of t tests to determine significances. A probability level of p < 0.05 was taken as significant.
Procedures and Blood Sampling Home cage measurements. The catheters of the animals were connected with a polyethylene blood sampling tube (0.4 m length, 1.45 mm o.d. and 0.75 mm i.d.) in their home cages 40 minutes before the start of an experiment. Four doses of the drug (2.5, 5, 10, and 20 mg/kg) were used. Each rat received only one dose of the drug. Blood samples were taken in the home cages at T = 0 min and at T = 29 min after IP injection of the drug. Control measurements. This experiment was performed to control for the effects of handling, moving the animals, and exposing them to the apparatus. The rats were trained in a twocompartment apparatus in which the stressor would be applied (1). Briefly, the rats were placed in a waiting cage next to the apparatus after which they were trained to enter from a platform to a dark compartment where they were allowed to stay for 5 min. This procedure was repeated three times in order to allow habituation. Blood samples were taken in the home cage before treatment (T = 0 min) followed by injection of saline or ipsapirone. Subsequent samples were taken at T = 29 min in the home cage. They were then transferred to the waiting cage for 1 min and after this directly exposed to the dark compartment. Exploring the environment took place most of the 5-min observation period (27). Samples were taken at T = 32.5 and T = 36.5 rain (i.e.. 1st and 5th min of the exposure to the dark compartment). Following retransfer to the home cage, blood samples were taken at T - 45, 65, and 95 min. Postshock measurements. After a washout period of 6 days all animals received an additional training trial in the apparatus to enter the dark compartment, but an inescapable scrambled footshock (0.6 mA, AC for 3 s) was given immediately upon entering the dark from the lit platform and the sliding door was closed. The rats were removed from the dark 30-40 s after termination of the footshock. On the next day they were allocated to ipsapirone or saline treatment groups. Each group consisted of an equal number of rats with earlier placebo or drug administration in order to exclude that any possible effects of prior treatment influenced the results. The blood sampling procedure was identical to the one used in the control (nonshock) condition. No further footshock was administered during the reexposure to the dark compartment. Under the nonstress and stress conditions a single dose ofipsapirone (5 mg/kg) was used. This dose was selected on the basis of the baseline home cage studies.
RESULTS
Circulating Hormone Concentrations During Baseline Conditions: Effects of lpsapirone Table 1 shows the alterations in the level of E, NE, and CORT, 29 min after IP injection of ipsapirone in doses of 2.5, 5, 10, and 20 mg/kg (IP) as measured in the home cage of the rats. The actual zero time values are comparable with those in Fig. 1. Plasma CORT levels increased in a dose-dependent manner in the dose range of 5 to 20 mg/kg. Plasma E, NE levels remained unchanged unless 20 mg/kg dose of ipsapirone was used.
Home Cage Data of Circulating CAs and CORT in Control and Postshock Animals: Effects of lpsapirone Figure 1 shows that no significant changes occurred in drugtreated control rats during the measurements. However, the same dose of ipsapirone (5 mg/kg) increased plasma levels of CORT (p < 0.01), E and NE (both p < 0.05) in postshock rats in the home cage at T = 29 min even before reexposure to the twocompartment apparatus.
Plasma Levels of CAs and CORT in Control and Postshock Animals During Forced Reexposure to the Dark Compartment: Effects of lpsapirone Figure 1 shows that ipsapirone produced an increase in both NE and E levels relative to saline-treated controls during the
TABLE 1 CHANGES IN PLASMA E, NE, AND CORT CONCENTRATIONS 1N RATS
lpsapirone (IP)
AE (pg/ml)
ANE (pg/ml)
ACORT (ugldl)
0.0 mg]kg 2.5mg/kg 5.0 mg]kg 10.0 mg/kg 20.0 mg/kg
33 _+38 10_+ 8 23 _+ 16 80 +_ 15 412 _+ 196t
0 +_26 0_+33 70 +_27 6 _+ 11 383 +- 127J/
6 _+6 3_+3 21 _+6 35 +_2* 44 +_5*
The values represent the means + SEM of plasma concentrations (before-drug treatment minus after-drug treatment). *t$ Significantchanges: *p < 0.05, tp < 0.025, Sp < 0.01 compared to saline control (Dunnett's test).
5-HT~A RECEPTOR STIMULATION AND STRESS RESPONSES
DARK
DARK COMPARTMENT
COMPARTMENT HOME
CAGE
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rapidity of reaching high CORT levels after ipsapirone treatment in the animals in the home cage 1 day after experience of inescapable shock.
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CONTROL
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FIG. 1. The effectofipsapirone on the plasma levelsof E, NE and CORT measured in home cage, shock compartment and again in the home cage. Rats were tested before and 24 h after inescapablefootshock, t: IP injection; O-O: ipsapirone (5 mg/kg);(3-0: saline. Data represent means _+SEM of eight animals in each group. *p < 0.05, **p < 0.01: statistically significantdifference from corresponding control.
exposure to the dark compartment of the apparatus, whereas the CORT response remained unaffected. These elevations in CAs levels were significant for the control condition (nonshock) at T = 32.5 (p < 0.05) and T = 36.5 min (p < 0.01) during confinement in the dark compartment. Both E and NE levels remained elevated after return to the home cage at T = 45 min Co < 0.05). For the postshock condition significant elevations in E and CORT were seen for both sampling periods during confinement in the dark compartment (p < at least 0.05). In these rats the CORT levels remained significantly elevated after return to the home cage relative to controls at T = 45 min, T = 65 min and T = 95 min. Ipsapirone failed to affect NE levels in the stressed animals. Although the absolute CORT concentrations in ipsapironetreated control and stressed rats showed no large differences, it must be underlined that marked differences were seen in the
The present study suggests that ipsapirone, a 5-HTIA receptor agonist, has a complex dose- and stress-state-dependent effect on the circulating CORT and CAs concentrations in the rat. Under baseline, stress-free conditions very low basal plasma levels of the sympatho-adrenal hormones were present. Ipsapirone elevated CORT concentrations in a dose-dependent way in rats in their home cage. Elevations in plasma levels ofcatecholamines were only seen after 20 mg/kg of the drug. In contrast, the selected dose of the drug (5 mg/kg), led to a significant elevation of the plasma levels of E and NE in the control (nonshock) rats during exposure to the apparatus. The CORT levels were not significantly affected in this state. This dose range of the drug has shown to reduce anxiety in different test situations (27,28). However, in the home cage 24 h after the experience of shock, the animals showed after drug treatment a strong elevation of plasma CORT concentration, and a minor elevation of both plasma levels of E and NE even before reexposure to the twocompartment apparatus. Furthermore, ipsapirone elevated both the E and CORT but not the NE response to the conditioned emotional stressor. The actual response magnitude to the emotional stressor of fear for shock was not much altered. Serotonin (5-HT) uses different located 5-HT receptors to convey its messages. High densities somatodendritic 5-HT~Aautoreceptors are located on cell bodies and/or dendrites in the raphe nuclei (15-17,20,39). High densities of postsynaptic 5HTIA receptors have been found in the septum, hippocampus, amygdala, hypothalamus, and neocortex (17,20,39). These populations of 5-HT~A receptors may be differentially involved in anxiolytic and neuroendocrine effects of the 5-HT~A receptor agonist. Evidence now exists that the 8-OH-DPAT, ipsapirone, buspirone, etc. have anxiolytic effects by acting as agonists on the 5-HTIA autoreceptors on the raphe nuclei (16,22). The inhibition of neuronal firing and the decrease of 5-HT release in the various terminal regions are the consequence of autoreceptor stimulation and is suggested to underlie the anxiolysis caused by ipsapirone and related compounds (5,46). There is indirect evidence that 5-HT~Aagonists activate presynaptic autoreceptors at low doses, and pre- and postsynaptic receptors at high ones (17,33). This may be explained by the fact that these presynaptic 5-HT~A autoreceptors possess a large receptor reserve for the agonists (32). High doses (10 mg/kg, IP) ipsapirone may facilitate 5-HT~A postsynaptic neurotransmission and result in the release of ACTH (18,19) and CORT (26,31). This higher dose may still show anxiolytic effects (28). The 5-HT~A receptor agonists (e.g., 8-OH-DPAT, ipsapirone, buspirone) after either systemic administration (4,8,9,14,38,49) or central infusion into the hypothalamic paraventricular nucleus cause an elevation of both plasma levels of E and CORT in freely moving rats (21,30). These results suggest an activation of the both sympathoadrenomeduilary system and the hypothalamo-pituitary-adrenocortical (HPA) axis. The increase in plasma E and CORT concentrations is probably due to activation of 5-HT~Areceptors because the increases can be blocked by pretreatment with the mixed 5HT~A.B/fl-adrenoceptor antagonists (-)-pindolol and (-)-propanolol (9,26). It is suggested that the 5-HT~A receptors located on CRH-containing neurons in the hypothalamic paraventricular nucleus (PVN) are involved in activation of both the HPA-axis and the adrenal medulla (7,30,38). Furthermore, stimulation of 5-HT~A receptors results in the release of ~3-endorphin which
K O R I E , BOUWS A N D BOHUS
1132
causes marked increases in plasma CA levels, especially of E (3,6,55). Neither can it be excluded that the circulating CAs promote A C T H secretion (10,50). An exaggerated plasma catecholamine response was tbund under both control and stress (postshock) conditions after ipsapirone treatment during exposure to the apparatus. It can not be excluded that the augmented catecholamine response in the stressful condition may be merely an epiphenomenon of the increased arousal and even anxiety-like reactions as induced by ipsapirone or its metabolite l-(2-pyrimidinyl)-piperazine via adrenergic receptors ( 11,37,41,42). Under baseline conditions the 5-HT~A agonist ipsapirone had a dose-dependent effect on C O R T levels and led to a significant elevation at a dose of 10 mg/kg and higher, whereas 24 h after inescapable footshock 5 mg/kg ipsapirone led to an exaggerated C O R T and CA response before the exposure to the stressful environment in the home cages of the animals. A similar phenomenon was observed 24 h following the social stress of defeat (29). We have hypothesized that absence of control of the stressor, which occurs during social defeat or inescapable footshock, causes a functional supersensitivity of 5-HT~A receptor system to subsequent agonist stimulation. Although the mechanisms of this supersensitivity are not understood yet, it is likely that generalization occurs to other 5-HT receptors (24), Other neuroendocfine evidence suggests that corticotrophin, corticosterone, renin, and prolactin release by 5-HT agonists is enhanced as the consequence of denervation supersensitivity of 5-HT receptors (54). Changes in receptor density, a shift from low to high affinity state of the receptor or an increased functional coupling of 5-
HT receptors to their second messengers are suggested as possible underlying mechanisms. In addition, daily repetition of restraint stress (up to I0 days) increases the behavioral sensitivity to induce postsynaptic effects by 5-HT releasers, selective and nonselective 5-ttT~ receptor agonists in the rat (25). There is evidence that restraint stress increases binding at 5-HT~A receptors in CA4 and the infrapyramidal dentate gyms, but no effects on 5-HT~ or 5-ttT2 receptors in rat hippocampus (34). Whether this serotonergic hypersensitivity is adaptive or maladaptive needs further investigation. Kennett and Curzon (12,25) suggest that an increased postsynaptic 5-HT function is adaptive to prevent behavioural depression. Accordingly, behavioral adaptation occurs after repeated exposure to stressors and this is associated with increased 5-HT~A postsynaptic receptor functions induced enhancement of 5-HT neurotransmission (12,25). Together these findings reinforce the view that 5-HTIA receptor-mediated mechanisms play an important role in the neuroendocrine control of the adrenal cortex and medulla. Furthermore, it is suggested that the functional state of these receptors and whether pre- or postsynaptic processes dominate is affected by environmental and/or conditioned thctors. The present findings suggest that if an animal has no possibilities to escape or avoid a noxious event, functional hypersensitivity will develop in the serotonergic neuronal system, which is reflected in the increased responsiveness of the HPA axis and sympatho-adrenomedullary system to a 5-Hl~,~ agonist challenge. It remains to be determined by which mechanisms supersensitivity in the serotonergic system develops or at least in some of its branches.
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56.
57.
1133 after intraventricular administration of [3H]5-hydroxytryptamine. Neuroscience 6:115-138; 1981. Rimele, T. J.; Henry, D. E.; Lee, D. K. H.; Geiger, G.; Heaslip, R. J.; Grimes, D. Tissue-dependent alpha adrenoceptor activity of buspirone and related compounds. J. Pharmacol. Exp. Ther. 241: 771-778; 1987. Sanghera, M. K.; Coke, J. A.; Williams, H. L.; McMillen, B. A. Ipsapirone and l-(2-pyrimidinyl)-piperazine increase rat locus coeruleus noradrenergic activity. Brain Res. Bull. 24:17-22; 1990. Sinton, C. M.; Fallon, S. L. Electrophysiological evidence fora functional differentiation between subtypes of the 5-HTI receptor. Eur. J. Pharmacol. 157:173-181; 1988. Smedes, F.; Kraak, J. C.; Poppe, H. Simple and fast solvent extraction system for selective and quantitative isolation of adrenaline, noradrenaline and dopamine from plasma and urine. J. Chromatogr. 231:25-39; 1982. Spencer, D. G., Jr.; Traber, J. The interoceptive discriminative stimuli induced by the novel putative anxiolytic TVX Q 7821: Behavioral evidence for the specific involvement ofserotonin 5-HT 1A receptors. Psychopharmacology (Berlin) 91:25-29; 1987. Sprouse, J. S.; Aghajanian, G. K. Responses of hippocampal pyramidal cells to putative serotonin 5-HT IA and 5-HT 1B agonists; a comparative study with dorsal raphe neurons. Neuropharmacology 27:707-715; 1988. Steffens, A. B. A method for frequent sampling of blood and continuous infusion of fluids in the rat without disturbing the animal. Physiol. Behav. 4:833-836; 1969. Steinbusch, H. W. M. Distribution of serotonin-immunoreactivity in the central nervous system of the rat-cell bodies and terminals. Neuroscience 6:557-618; 1981. Taylor, J.; Hams, N.; Krieman, M.; Vogel, W. H. Effects ofbuspirone on plasma catecholamines, heart rate, and blood pressure in stressed and nonstressed rats. Pharmacol. Biochem. Behav. 34:349-353; 1989. Tilders, F. J. H.; Berkenbosch, F.; Vermes, I.; Linton, E. A.; Smelik, P. G. Role of epinephrine and vasopressin in the control of the pituitary-adrenal response to stress. Fed. Proc. 44:155-160; 1985. Traber, J.; Glaser, T. 5-HT IA receptor-related anxiolytics. Trends Pharmacol. Sci. 8:432-437; 1987. Traber, J.; Davies, M. A.; Dompert, W. U.; Glaser, T.; Schuurman, T.; Seidel, P.-R. Brain serotonin receptors as target for the putative anxiolytic TVX Q 7821. Brain Res. Bull. 12:741-744; 1984. Van de Kar, L. D. Neuroendocrine aspects of the serotonergic hypothesis of depression. Neurosci. Biobehav. Rev. 13:237-246; 1989. Van de Kar, I. D.; Carnes, M.; Maslowski, R. J.; Bonadonna, A. M.; Rittenhouse, P. A.; Kunimoto, K.; Piechowski, R. A.; Bethea, C. L. Neuroendocrine evidence for denervation super-sensitivity of serotonin receptors: Effects of the 5-HT agonist RU 24969 on corticotropin, corticosterone, prolactin and renin secretion. J. Pharmacol. Exp. Ther. 251:428-434; 1989. Van Loon, G. R.; Appel, N. M.; Ho, D. Beta-endorphin-induced stimulation of central sympathetic outflow: Beta-endorphin increases plasma concentrations of epinephrine, norepinephrine and dopamine in rats. Endocrinology 109:46-53; 1981. Wiersma, J.; Kastelijn, J. A chronic technique for high frequency blood sampling/transfusion in the freely behaving rat which does not affect prolactine and corticosterone secretion. J. Endocrinol. 107: 285-292; 1985. Wise, C. D.; Berger, B. D.; Stein, L. Benzodiazepines: Anxiety-reducing action by reduction of serotonergic turnover in the brain. Science 177:180-183; 1972.
0031-9384/92 $5.00 + .00 Copyright© 1992PergamonPressLtd.
Printed in the USA.
Adrenal Hormones in Rats Before and After Stress-Experience: Effects of Ipsapirone S. M. KORTE, 1 G. A. H. B O U W S A N D B. B O H U S
Department of Animal Physiology, University of Groningen, Center for Behavioral, Cognitive and Neuro-Sciences, P.O. Box 14, 9750 AA Haren, The Netherlands Received 9 September 1991 KORTE, S. M., G. A. H. BOUWS AND B. BOHUS. Adrenal hormones in rats before and after stress-experience:Effects of ipsapirone. PHYSIOL BEHAV51(6) 1129-1133, 1992.--The present study was designedto investigatethe effectsof the anxiolytic 5-HTjAreceptor agonist ipsapirone on the hormonal responses in rats under nonstress and stress conditions by means of repeated blood sampling through an intracardiac catheter. Ipsapirone was given in doses of 2.5, 5, 10, and 20 mg/kg (IP) under nonstress conditions in the home cages of the rats. Plasma corticosterone levels increased in a dose-dependent way in the dose range of 5 to 20 mg/kg, whereas the plasma catecholamines were only significantlyincreased with the highest dose of the drug. The effect of ipsapirone in control and in stressed rats was studied with the selected dose of 5 mg/kg. Conditioned fear of inescapableelectric footshock (0.6 mA, AC for 3 s) given one day earlier was used as stressor. Surprisingly,ipsapirone potentiated the magnitude of the neuroendocrine responses. Rats receiving an inescapable footshock 1 day earlier showed a further elevated corticosterone response to the 5-HTIAreceptor agonist ipsapirone even before exposing them to the conditioned stress situation. The present findingssuggest that if an animal has no possibilitiesto escape or avoid a noxious event, functional hypersensitivitywill develop in the serotonergic neuronal system, which is reflected in the increased responsivenessof the HPA axis to a 5-HT~Aagonist challenge. Norepinephrine
Epinephrine
Corticosterone
lpsapirone
THERE is a large body of evidence that serotonin (5-hydroxytryptamine, 5-HT) is involved in mechanisms of neuroendocrine activation and anxiety ( 17,24,51,57). The widespread projections of 5-HT neurons from raphe nuclei to the limbic system, the hypothalamus and the cortex (2,35,40,48), provide the anatomic substrate for a functional system that may be involved in anxiety and neuroendocrine regulation. Autoradiographic studies suggest that 5-HT~A receptors are located presynaptically on serotonergic cell bodies in the raphe nuclei and postsynaptically on neurons in the septo-hippocampal-amygdaloid system, cortex, and hypothalamus (20,39). Binding of ipsapirone to these receptors may exert complex effects on 5-HT neurotransmission. The anxiolytic effects are thought to be mediated by suppression ofserotonergic neuronal activity via a full agonistic action on presynaptic 5-HTIA autoreceptors ( 16,17,51) or via a partial agonist/antagonist action at postsynaptic 5-HTtA receptor rich sites (23,43). There is a large body of evidence that serotonergic (5-HT) 1A receptor mechanisms are involved in the neural activation of both the hypothalamic-pituitary-adrenocortical (HPA) axis (7,14,18,19,21,26,31,53,54) and the adrenomedullary system (4,5,9,10,14,30). Ipsapirone is a novel anxiolytic compound with a high degree of specific binding to 5-HTjA receptors (15,45,52). Therefore, ipsapirone can be used as a tool to study serotonergic mechanisms underlying anxiety and neuroendocrine activation. Requests for reprints should be addressed to S. M. Korte.
1129
5-HT~^receptor
Stress
It was hypothesized that absence of control in a social stress situation (defeat) may change the state of 5-HT receptors for subsequent stimulation and may lead to an elevation in circulating adrenal hormones (25,54). In contrast, ipsapirone appears to have anxiolytic-like effects in a nonsocial stress situation if behavior and heart rate are used as markers for stress (27,28). Therefore, the aim of the present study was to investigate the influence of ipsapirone on the plasma levels of catecholamines and corticosterone after the experience of absence of control in a nonsocial stress situation. METHOD
Animals Male Wistar rats weighing 290-340 g at the beginning of the experiments were used. They were housed individually in clear Plexiglass cages (25 × 25 × 30 cm) on a 12-h light-dark regime (0730 h-1930 h lights on) at a room temperature of 21 _+ 2°C. All animals had free access to standard food (Hope Farms rat chow) and water.
Surgery Under complete ether anaesthesia, the animals were provided with a silicon heart catheter (0.95 mm o.d., 0.50 mm i.d.) through
1130
the right jugular vein and externalized on the top of the skull according to a technique described earlier (47). This method allows frequent blood sampling in freely moving rats (56). After each sample an equal quantity ofcitrated donor blood was given to avoid diminution of the blood volume with related changes in hemodynamics (47). Donor blood was obtained from unstressed rats with permanent heart catheters. The rats were given 1-2 weeks to acclimatise and recover from surgeD'.
Drug Treatment lpsapirone (TVX Q 7821, Troponwerke GmbH and Co., Cologne, Germany) was dissolved in saline. Injections were given intraperitoneally (IP) 29 rain before the test session in a constant volume of 2.0 ml/kg. The dose of 5 mg/kg, IP, was used in the postshock test because this dose has shown to have anxiolytic-like effects in different test situations if behavior and heart rate were used as markers of stress (27,28,51).
KORTE. BOUWS AND BOHUS
Chemical Determinations Blood samples of 0.45 ml were withdrawn for determination of plasma E, NE, and CORT. The samples were transferred immediately into chilled (0°C) centrifuge tubes containing 0.01% EDTA as antioxidant and 10 #1 heparin solution (500 IU/ml) as anticoagulant. Blood was centrifuged at 4°C for 10 min at 5000 rpm, and 100 ul of the supernatant were stored at - 2 0 ° C for CORT and at - 8 0 ° C for the catecholamine (CA) measurements. Plasma CORT was measured by means of reversed phase high performance liquid chromatography (HPLC) (13). Determination of plasma CA concentrations was performed by HPLC in combination with electrochemical detection (44).
Statistics The two-tailed Dunnett's test (36) was used to compare the vehicle control group with the ipsapirone-treated groups under baseline conditions. The neuroendocrine data of drug-treated control and stressed animals were evaluated by means of t tests to determine significances. A probability level of p < 0.05 was taken as significant.
Procedures and Blood Sampling Home cage measurements. The catheters of the animals were connected with a polyethylene blood sampling tube (0.4 m length, 1.45 mm o.d. and 0.75 mm i.d.) in their home cages 40 minutes before the start of an experiment. Four doses of the drug (2.5, 5, 10, and 20 mg/kg) were used. Each rat received only one dose of the drug. Blood samples were taken in the home cages at T = 0 min and at T = 29 min after IP injection of the drug. Control measurements. This experiment was performed to control for the effects of handling, moving the animals, and exposing them to the apparatus. The rats were trained in a twocompartment apparatus in which the stressor would be applied (1). Briefly, the rats were placed in a waiting cage next to the apparatus after which they were trained to enter from a platform to a dark compartment where they were allowed to stay for 5 min. This procedure was repeated three times in order to allow habituation. Blood samples were taken in the home cage before treatment (T = 0 min) followed by injection of saline or ipsapirone. Subsequent samples were taken at T = 29 min in the home cage. They were then transferred to the waiting cage for 1 min and after this directly exposed to the dark compartment. Exploring the environment took place most of the 5-min observation period (27). Samples were taken at T = 32.5 and T = 36.5 rain (i.e.. 1st and 5th min of the exposure to the dark compartment). Following retransfer to the home cage, blood samples were taken at T - 45, 65, and 95 min. Postshock measurements. After a washout period of 6 days all animals received an additional training trial in the apparatus to enter the dark compartment, but an inescapable scrambled footshock (0.6 mA, AC for 3 s) was given immediately upon entering the dark from the lit platform and the sliding door was closed. The rats were removed from the dark 30-40 s after termination of the footshock. On the next day they were allocated to ipsapirone or saline treatment groups. Each group consisted of an equal number of rats with earlier placebo or drug administration in order to exclude that any possible effects of prior treatment influenced the results. The blood sampling procedure was identical to the one used in the control (nonshock) condition. No further footshock was administered during the reexposure to the dark compartment. Under the nonstress and stress conditions a single dose ofipsapirone (5 mg/kg) was used. This dose was selected on the basis of the baseline home cage studies.
RESULTS
Circulating Hormone Concentrations During Baseline Conditions: Effects of lpsapirone Table 1 shows the alterations in the level of E, NE, and CORT, 29 min after IP injection of ipsapirone in doses of 2.5, 5, 10, and 20 mg/kg (IP) as measured in the home cage of the rats. The actual zero time values are comparable with those in Fig. 1. Plasma CORT levels increased in a dose-dependent manner in the dose range of 5 to 20 mg/kg. Plasma E, NE levels remained unchanged unless 20 mg/kg dose of ipsapirone was used.
Home Cage Data of Circulating CAs and CORT in Control and Postshock Animals: Effects of lpsapirone Figure 1 shows that no significant changes occurred in drugtreated control rats during the measurements. However, the same dose of ipsapirone (5 mg/kg) increased plasma levels of CORT (p < 0.01), E and NE (both p < 0.05) in postshock rats in the home cage at T = 29 min even before reexposure to the twocompartment apparatus.
Plasma Levels of CAs and CORT in Control and Postshock Animals During Forced Reexposure to the Dark Compartment: Effects of lpsapirone Figure 1 shows that ipsapirone produced an increase in both NE and E levels relative to saline-treated controls during the
TABLE 1 CHANGES IN PLASMA E, NE, AND CORT CONCENTRATIONS 1N RATS
lpsapirone (IP)
AE (pg/ml)
ANE (pg/ml)
ACORT (ugldl)
0.0 mg]kg 2.5mg/kg 5.0 mg]kg 10.0 mg/kg 20.0 mg/kg
33 _+38 10_+ 8 23 _+ 16 80 +_ 15 412 _+ 196t
0 +_26 0_+33 70 +_27 6 _+ 11 383 +- 127J/
6 _+6 3_+3 21 _+6 35 +_2* 44 +_5*
The values represent the means + SEM of plasma concentrations (before-drug treatment minus after-drug treatment). *t$ Significantchanges: *p < 0.05, tp < 0.025, Sp < 0.01 compared to saline control (Dunnett's test).
5-HT~A RECEPTOR STIMULATION AND STRESS RESPONSES
DARK
DARK COMPARTMENT
COMPARTMENT HOME
CAGE
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rapidity of reaching high CORT levels after ipsapirone treatment in the animals in the home cage 1 day after experience of inescapable shock.
POST-SHOCK
CONTROL
HOME
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FIG. 1. The effectofipsapirone on the plasma levelsof E, NE and CORT measured in home cage, shock compartment and again in the home cage. Rats were tested before and 24 h after inescapablefootshock, t: IP injection; O-O: ipsapirone (5 mg/kg);(3-0: saline. Data represent means _+SEM of eight animals in each group. *p < 0.05, **p < 0.01: statistically significantdifference from corresponding control.
exposure to the dark compartment of the apparatus, whereas the CORT response remained unaffected. These elevations in CAs levels were significant for the control condition (nonshock) at T = 32.5 (p < 0.05) and T = 36.5 min (p < 0.01) during confinement in the dark compartment. Both E and NE levels remained elevated after return to the home cage at T = 45 min Co < 0.05). For the postshock condition significant elevations in E and CORT were seen for both sampling periods during confinement in the dark compartment (p < at least 0.05). In these rats the CORT levels remained significantly elevated after return to the home cage relative to controls at T = 45 min, T = 65 min and T = 95 min. Ipsapirone failed to affect NE levels in the stressed animals. Although the absolute CORT concentrations in ipsapironetreated control and stressed rats showed no large differences, it must be underlined that marked differences were seen in the
The present study suggests that ipsapirone, a 5-HTIA receptor agonist, has a complex dose- and stress-state-dependent effect on the circulating CORT and CAs concentrations in the rat. Under baseline, stress-free conditions very low basal plasma levels of the sympatho-adrenal hormones were present. Ipsapirone elevated CORT concentrations in a dose-dependent way in rats in their home cage. Elevations in plasma levels ofcatecholamines were only seen after 20 mg/kg of the drug. In contrast, the selected dose of the drug (5 mg/kg), led to a significant elevation of the plasma levels of E and NE in the control (nonshock) rats during exposure to the apparatus. The CORT levels were not significantly affected in this state. This dose range of the drug has shown to reduce anxiety in different test situations (27,28). However, in the home cage 24 h after the experience of shock, the animals showed after drug treatment a strong elevation of plasma CORT concentration, and a minor elevation of both plasma levels of E and NE even before reexposure to the twocompartment apparatus. Furthermore, ipsapirone elevated both the E and CORT but not the NE response to the conditioned emotional stressor. The actual response magnitude to the emotional stressor of fear for shock was not much altered. Serotonin (5-HT) uses different located 5-HT receptors to convey its messages. High densities somatodendritic 5-HT~Aautoreceptors are located on cell bodies and/or dendrites in the raphe nuclei (15-17,20,39). High densities of postsynaptic 5HTIA receptors have been found in the septum, hippocampus, amygdala, hypothalamus, and neocortex (17,20,39). These populations of 5-HT~A receptors may be differentially involved in anxiolytic and neuroendocrine effects of the 5-HT~A receptor agonist. Evidence now exists that the 8-OH-DPAT, ipsapirone, buspirone, etc. have anxiolytic effects by acting as agonists on the 5-HTIA autoreceptors on the raphe nuclei (16,22). The inhibition of neuronal firing and the decrease of 5-HT release in the various terminal regions are the consequence of autoreceptor stimulation and is suggested to underlie the anxiolysis caused by ipsapirone and related compounds (5,46). There is indirect evidence that 5-HT~Aagonists activate presynaptic autoreceptors at low doses, and pre- and postsynaptic receptors at high ones (17,33). This may be explained by the fact that these presynaptic 5-HT~A autoreceptors possess a large receptor reserve for the agonists (32). High doses (10 mg/kg, IP) ipsapirone may facilitate 5-HT~A postsynaptic neurotransmission and result in the release of ACTH (18,19) and CORT (26,31). This higher dose may still show anxiolytic effects (28). The 5-HT~A receptor agonists (e.g., 8-OH-DPAT, ipsapirone, buspirone) after either systemic administration (4,8,9,14,38,49) or central infusion into the hypothalamic paraventricular nucleus cause an elevation of both plasma levels of E and CORT in freely moving rats (21,30). These results suggest an activation of the both sympathoadrenomeduilary system and the hypothalamo-pituitary-adrenocortical (HPA) axis. The increase in plasma E and CORT concentrations is probably due to activation of 5-HT~Areceptors because the increases can be blocked by pretreatment with the mixed 5HT~A.B/fl-adrenoceptor antagonists (-)-pindolol and (-)-propanolol (9,26). It is suggested that the 5-HT~A receptors located on CRH-containing neurons in the hypothalamic paraventricular nucleus (PVN) are involved in activation of both the HPA-axis and the adrenal medulla (7,30,38). Furthermore, stimulation of 5-HT~A receptors results in the release of ~3-endorphin which
K O R I E , BOUWS A N D BOHUS
1132
causes marked increases in plasma CA levels, especially of E (3,6,55). Neither can it be excluded that the circulating CAs promote A C T H secretion (10,50). An exaggerated plasma catecholamine response was tbund under both control and stress (postshock) conditions after ipsapirone treatment during exposure to the apparatus. It can not be excluded that the augmented catecholamine response in the stressful condition may be merely an epiphenomenon of the increased arousal and even anxiety-like reactions as induced by ipsapirone or its metabolite l-(2-pyrimidinyl)-piperazine via adrenergic receptors ( 11,37,41,42). Under baseline conditions the 5-HT~A agonist ipsapirone had a dose-dependent effect on C O R T levels and led to a significant elevation at a dose of 10 mg/kg and higher, whereas 24 h after inescapable footshock 5 mg/kg ipsapirone led to an exaggerated C O R T and CA response before the exposure to the stressful environment in the home cages of the animals. A similar phenomenon was observed 24 h following the social stress of defeat (29). We have hypothesized that absence of control of the stressor, which occurs during social defeat or inescapable footshock, causes a functional supersensitivity of 5-HT~A receptor system to subsequent agonist stimulation. Although the mechanisms of this supersensitivity are not understood yet, it is likely that generalization occurs to other 5-HT receptors (24), Other neuroendocfine evidence suggests that corticotrophin, corticosterone, renin, and prolactin release by 5-HT agonists is enhanced as the consequence of denervation supersensitivity of 5-HT receptors (54). Changes in receptor density, a shift from low to high affinity state of the receptor or an increased functional coupling of 5-
HT receptors to their second messengers are suggested as possible underlying mechanisms. In addition, daily repetition of restraint stress (up to I0 days) increases the behavioral sensitivity to induce postsynaptic effects by 5-HT releasers, selective and nonselective 5-ttT~ receptor agonists in the rat (25). There is evidence that restraint stress increases binding at 5-HT~A receptors in CA4 and the infrapyramidal dentate gyms, but no effects on 5-HT~ or 5-ttT2 receptors in rat hippocampus (34). Whether this serotonergic hypersensitivity is adaptive or maladaptive needs further investigation. Kennett and Curzon (12,25) suggest that an increased postsynaptic 5-HT function is adaptive to prevent behavioural depression. Accordingly, behavioral adaptation occurs after repeated exposure to stressors and this is associated with increased 5-HT~A postsynaptic receptor functions induced enhancement of 5-HT neurotransmission (12,25). Together these findings reinforce the view that 5-HTIA receptor-mediated mechanisms play an important role in the neuroendocrine control of the adrenal cortex and medulla. Furthermore, it is suggested that the functional state of these receptors and whether pre- or postsynaptic processes dominate is affected by environmental and/or conditioned thctors. The present findings suggest that if an animal has no possibilities to escape or avoid a noxious event, functional hypersensitivity will develop in the serotonergic neuronal system, which is reflected in the increased responsiveness of the HPA axis and sympatho-adrenomedullary system to a 5-Hl~,~ agonist challenge. It remains to be determined by which mechanisms supersensitivity in the serotonergic system develops or at least in some of its branches.
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