Decreased 5-HT1A and increased 5-HT2A receptor binding after chronic corticosterone associated with a behavioural indication of depression but not anxiety

Pergamon

Psychoneuroendocrinology, Vol. 22, No. 7, pp. 477-491, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0306-4530/97 $17.00 + .00

PII: S0306-4530(97)00052-8

DECREASED 5-HT1A AND INCREASED 5-HT2A RECEPTOR BINDING AFTER CHRONIC CORTICOSTERONE ASSOCIATED WITH A BEHAVIOURAL INDICATION OF DEPRESSION BUT NOT ANXIETY Cathy Fernandes, ~ Christina R. McKittrick, 2 Sandra E. File 1 and Bruce S.

McEwen2 IPsychopharmacology Research Unit, UMDS Division of Pharmacology, Guy's Hospital, London SE1 9RT, UK; and 2Laboratory of Neuroendocrinology, Box 165, The Rockefeller University, New York, NY 10021, USA

(Received 7 March 1997; in final form 17 June 1997)

SUMMARY The effects of chronic corticosterone treatment (100 mg pellet implanted for l week) were assessed in animal tests of anxiety, exploration and motor activity, and changes in binding to 5-HT1A and 5-HTea receptors, and the 5-HT transporter, were measured. At the end of the week's treatment, the corticosterone concentration was significantly elevated and there were significant decreases in adrenal, thymus and body weights. However, there were no changes in the measures of anxiety in the social interaction test or on trials 1 and 2 of the elevated plus-maze. Also supporting a dissociation between anxiety and elevated corticosterone concentrations are previous findings that benzodiazepine withdrawal causes increased anxiety but no change in corticosteroid concentrations. Therefore these two situations provide a double dissociation between anxiety and elevated corticosteroids. Decreased 5-HTIA receptor binding in the dentate gyrus and increased 5-HTzA receptor binding in the parietal cortex was found following chronic corticosterone treatment. This reciprocal relationship between 5-HTIA and 5-HTzA receptors has been proposed to be important in mediating depression. The significant decreases in motor activity observed in all the test situations would be compatible with this proposal. Thus the constellation of behavioural and biochemical changes detected after chronic conicosterone treatment is more pertinent to depression than anxiety. One week after removal of the pellets, the behavioural and neurochemical changes had disappeared and the only differences to remain were decreased adrenal, thymus and body weights in the animals that had been treated chronically with corticosterone. © 1997 Elsevier Science Ltd Keywords--5-HT~A; 5-HT2A; 5-HT transporter; Corticosterone; Depression; Anxiety.

INTRODUCTION The hypothalamic-pituitary-adrenal (HPA) axis is one of the main systems controlling an organism's response to stressful stimuli and has been implicated in the pathophysiology of

Address correspondence and reprint requests to: Professor Sandra E. File, Psychopharmacology Research Unit, U M D S Division of Pharmacology, G u y ' s Hospital, London SE1 9RT, U K (Tel: 44171-955-4629; Fax: 44-171-955-4627; E-mail: [email protected]) 477

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affective disorders. Elevated cortisol concentrations, changes in diurnal rhythm of cortisol secretion, and blunted response of the HPA axis have been reported in a variety of anxiety disorders and depression (Anisman and Zacharko, 1982; File, 1991a; Holsboer, 1989; Holsboer et al., 1995). Furthermore, the HPA axis has been shown to have an important role in animal models of anxiety and depression (File, 1991a). Recent research has focused on the relationship between mediators of the HPA axis and the serotonergic (5-HT) system, which also plays a central role in anxiety and depression (File, 1984; Graeff et al., 1996; Meltzer and Lowy, 1987; Van Praag, 1982, 1996). Manipulations of the HPA axis, such as adrenalectomy and administration of corticosterone, alter 5-HT turnover (Singh et al., 1990), and 5-HT1A (Mendelson and McEwen, 1992a,b) and 5-HT2A (Kuroda et al., 1992) receptor densities. Stressful stimuli have also been shown to regulate 5-HT1A (Mendelson and McEwen, 1991) and 5-HT2A (Torda et al., 1990) receptor binding. The HPA axis can in turn be modulated by central 5-HT systems (Lesch and Lerer, 1991). Animal experiments using central drug injections have provided evidence for the roles of particular 5-HT receptor sub-types in specific brain regions. Activation of the presynaptic 5-HT~A receptors, following administration of the 5-HTIA agonist, 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) into the raph6 nuclei, has been found to produce anxiolytic effects (Andrews et al., 1994; File and Gonzalez, 1996; File et al., 1996; Higgins et al., 1988; Hogg et al., 1994), whereas it was shown that activation of the post-synaptic 5-HT~A receptors in the amygdala and hippocampus resulted in anxiogenic effects (Andrews et al., 1994; File and Gonzalez, 1996; File et al., 1996; Gonzalez et al., 1996; Hodges et al., 1987). Similarly, the 5-HT2A/2C receptor has been implicated in the mediation of anxiety (Deakin, 1988), with administration of the 5-HTzA/2C receptor agonist, 1-(3-chlorophenyl) piperazine (mCPP), into the hippocampus producing anxiogenic effects (Whitton and Curzon, 1990). The aim of the present experiment was to investigate the effects of sub-chronic (i week) corticosterone treatment on animal tests of anxiety and on 5-HTIA and 5-HT2A receptors, and the 5-HT transporter. The reversibility of any such changes was assessed by testing the animals 1 week after removal of the corticosterone pellets. The social interaction and elevated plus-maze tests of anxiety were selected since these have both been extensively validated (File, 1980 and Pellow et al., 1985, respectively) and shown to be sensitive to anxiolytic and anxiogenic effects of the hormones from the HPA axis (File, 1991a). For example, corticotrophin-releasing-factor (CRF) and adrenocorticotrophic hormone (ACTH) have both been shown to increase anxiety (Dunn and File, 1987; File, 1979; File et al., 1988; File and Vellucci, 1978), whereas an acute injection of corticosterone itself appears to have anxiolytic effects (File et al., 1979). The animals were tested in the low light unfamiliar condition of the social interaction test as these conditions provide a suitable baseline for the detection of either anxiolytic or anxiogenic effects (File, 1980). We selected more than one test of anxiety because of the growing evidence that different animal tests are sensitive to different types of anxiety (File, 1991b, 1992, 1996). There is also strong evidence that the nature of the anxiety measured in trial 2 of the elevated plus-maze is quite distinct from that measured in trial 1 (Fernandes and File, 1996; File, 1990, 1993a; Rodgers and Shepherd, 1993) and thus the animals were tested in two trials of the elevated plus-maze. In addition, we assessed the effects of corticosterone treatment on exploration and motor activity, measured in the holeboard (File, 1991b; File and Wardill, 1975a,b). Following behavioural testing, binding to 5-HTlA and 5-HTzA receptors and the 5-HT transporter was determined in hippocampal, cortical and raph6 brain areas. The brain areas were selected for autoradiography based on previous studies demonstrating altered receptor

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binding following chronic corticosterone treatment (Kuroda et al., 1992; Mendelson and McEwen, 1992a,b). In addition, these areas are thought to play an important role in the mediation of anxiety (Gray, 1982).

METHODS Animals Male hooded Lister rats (Harlan UK, Bicester) weighing approximately 180-200 g at the start of the study were singly housed in a room maintained at 22°C, with lights (30 scotopic lux) on from 0700-1900h. Food and water were freely available. The experimental procedures carried out in this study were in compliance with the UK Animals Scientific Procedures Act 1986 (Home Office Project Licence Number: 90/00656). Corticosterone Treatment Animals were randomly allocated to four treatment groups (n = 16/group); placebo (P), corticosterone (C), placebo recovery (Pr) and corticosterone recovery (Cr). Placebo or corticosterone (100 mg) pellets (Innovative Research of America, USA) were implanted subcutaneously under light halothane anaesthesia (Rhone-Poulenc, UK), as previously described by Meyer et al. (1979), at 1 week prior to behavioural testing. (The carrier-binder excipients of the matrix of the placebo and corticosterone pellets included cholesterol, lactose, celluloses, phosphates and stearates.) In the recovery groups, rats were again lightly anaesthetized 1 week after pellet implantation and their pellets removed. A week later, they were tested. Throughout both the treatment and recovery periods, rats were handled and weighed daily. Behavioural Tests The social interaction test arena was a wooden box 60 cm square x 35 cm deep. Infrared photocells were mounted in the walls of the box, 4.5 and 12.5 cm from the floor, and the interruption of these beams provided automated measures of locomotor activity and rearing, respectively. A closed-circuit camera was mounted vertically above the arena and the rats were observed from a monitor in an adjacent room. Photocell output and the scores of the observer, who was blind to the hormone treatment, were entered into a computer. The light level in the test arena was 33 scotopic lux. For further details of this test and the behavioural measures, see File (1980, 1993b). The elevatedplus-maze was made of wood and had two open arms (50 x 10 cm) and two enclosed arms of the same size with walls 40 cm high, elevated 50 cm above the ground. A camera was mounted vertically above each maze, and the behaviour was scored by an observer blind to the hormone treatment, from a monitor in an adjacent room. For further details of this test and the behavioural measures, see File (1992) and Pellow et al. (1985). The holeboard was a wooden box 60 x 60 x 35 cm with four holes, each 6.5 cm in diameter, equally spaced in the floor. The interruption of infrared beams from cells located immediately beneath the edges of the holes recorded the number of head-dips and the time spent head-dipping. Locomotor activity and rearing were measured by the interruption of infrared beams from cells located in the walls of the box, 4.5 and 12.5 cm, respectively, from the floor. For further details of this test and the behavioural measures, see File and Wardill (1975a,b).

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Procedure All testing took place under quiet conditions, in an order randomised for hormone treatment and the behaviour was scored by an observer who was blind to the hormone treatment, between 0800 and 1300h.

Social Interaction Test. Animals were allocated to test partners within their treatment group (n -- 8 pairs/group) on the basis of weight, such that members of a pair did not differ by more than 10 g. Pairs of rats were tested for 4.5 min, and the time each pair spent in active social interaction was recorded. The following behaviours were scored: sniffing, following, grooming, crawling under or over a partner, boxing, wrestling, kicking, biting, mounting and horizontal submission. At the end of each social interaction test, the arena was wiped with a damp cloth and faecal pellets removed. Elevated Plus-Maze Trial 1. Immediately after the end of the social interaction test, rats were tested for 5 min in the elevated plus-maze. The times (s) spent on the open and enclosed arms were recorded. Four paws into, and two paws out of, an arm defining an arm entry and exit, respectively. At the end of each trial, the maze was wiped clean with a damp cloth. After completion of the elevated plus-maze trial, animals were returned to their home cage. Elevated Plus-Maze Trial 2. The following day, rats were tested again in the elevated plus-maze for 5 min, as described above. Holeboard. Immediately after the second trial in the elevated plus-maze, the animals were tested in the holeboard for 7.5 min and then returned to their home cage. The holeboard was wiped clean with a damp cloth at the end of each trial. Corticosterone Concentration, Body and Organ Weights On the third day, between 0800 and 1300h, rats were decapitated, trunk blood collected for assay of plasma corticosterone concentration, and brains rapidly removed, frozen on dry ice and maintained at -70°C until use. The adrenals and the thymus were also dissected and weighed. Plasma corticosterone concentrations were measured in triplicate by radiostereoassay (competitive protein-binding assay) as described by Murphy (1967). The intraand inter-assay variations were less than 10%.

Autoradiography of 5-HTIA and 5-HT2A Receptors and 5-HT Transporter Sites Binding to 5-HT1A and 5-HTzA receptors and the 5-HT transporter in hippocampal, cortical and raphd brain areas were measured in animals randomly selected from each group (n = 8/group). Coronal sections 16 ~m thick were cut on a cryostat microtome, thawmounted onto gelatin-coated slides, air dried for 15 rain and maintained at -70°C until use. Sections containing medial hypothalamus, parietal cortex, occipital cortex, cingulate gyms, dorsal hippocampus and amygdala were taken 3.0-4.0 mm posterior to bregma; ventral hippocampus was cut 5.2--6.0 mm posterior to bregma and sections containing dorsal and median raphd were taken 7.3 to 8.0 mm posterior to bregma, according to Paxinos and Watson (1986).

5-HT1A Receptors. Autoradiography of 5-HTIA receptors was performed as previously described (Mendelson and McEwen, 1991, 1992a). Sections were pre-incubated for 30 min at room temperature in buffer containing 50 mM Tris-HC1, 180 mM NaCI, 5 mM CaCI2,

Corticosterone,5-HT, Anxietyand Depression

481

and 1.2 mM MgC12 (pH 7.4). Sections were then incubated for 60 min at room temperature in the same buffer with the addition of 0.01% ascorbic acid and 1.5 nM [3H]-8-hydroxy-2(di-n-propylamino)tetralin ([3H]-8-OH-DPAT, specific activity = 135 Ci/mmol; NEN, Boston, MA). Non-specific binding was determined by incubation of adjacent sections in the presence of 1 #M serotonin (Sigma, St Louis, MO). Following incubation, sections were washed twice for 5 min in 4°C pre-incubation buffer then dipped for 5 s in 4°C distilled water and air dried.

5-HT2A Receptors. Autoradiography of 5-HT2A receptors was performed using the method of Mendelson and McEwen (1991), with minor modification. Sections were preincubated for 10 min at room temperature in buffer containing 50 mM Tris-HCl (pH 7.4). Sections were then incubated for 60 min at room temperature in the same buffer with the addition of 1/~M prazosin (Sigma) in order to block ~l-adrenergic receptors and 1/~M tetrabenazine (Fluka, Ronkonkoma NY) in order to block vesicular monoamine release sites. 5-HT2A binding sites were labelled with 2 nM [3H]-ketanserin (specific activity = 77 Ci/mmol; NEN). Non-specific binding was determined by incubation of adjacent sections in the presence of 500 nM spiperone (Research Biochemicals, Natick, MA). Following incubation, sections were washed twice for 10 min in 4°C pre-incubation buffer then dipped for 5 s in 4°C distilled water and air dried. 5-HT Transporter. Sections were processed for 5-HT transporter autoradiography as described in Mendelson et al. (1993). Slides were pre-incubated in 50 mM Tris-HCl (pH 7.7) for 15 min at room temperature. Sections were then incubated for 2 h at room temperature in the same buffer with the addition of 120 mM NaCI, 5 mM KCI and 0.4 nM [3H]-paroxetine (specific activity = 22 Ci/mmol; NEN). Non-specific binding was determined in the presence of 40/~M fiuoxetine (courtesy of Eli Lilly, Indianapolis IN). Following incubation, sections were washed twice for 45 min each in 50 mM Tris-HCl (pH 7.7), 120 mM NaC1 and 5 mM KC1 at room temperature, then dipped for 5 s in 4°C distilled water and air dried. Radiolabelled sections were apposed to tritium-sensitive 3Hyperfilm (Amersham, Arlington Heights IL) at room temperature. Autoradiograms for 5-HT1A and 5-HT2A receptors and the 5-HT transporter were developed after 14, 21 and 23 days, respectively. Films were developed in Kodak D-19 developer for 4 min, placed in a stop bath for 1 min, and fixed for 8 min in Kodak Rapid Fixer with Hardener (Rochester, NY). Autoradiograms were analysed by computer-assisted densitometry (Imaging Research, St Catharine's, Ontario) with optical densities converted into moles of radioligand bound per milligram tissue (wet weight) using curves generated from tritiated microscale standards (Amersham) co-exposed with the labelled sections. Statistics The effects of corticosterone treatment were assessed by two-way analyses of variance (ANOVA), with hormone (corticosterone or placebo) as one factor and pellet (present or removed) as the second. Comparisons between individual groups were made using Tukey's tests and it is these post-hoc significances that are cited in the figures and tables. We have included F-ratios for the pellet factor when this reached significance, suggesting that the presence of an implanted pellet (whether placebo or corticosterone) had a significant consequence.

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Table I. Mean ( ± S E M ) corticosterone concentration (ng/ml plasma) of adrenal, thymus and body weights of rats after 1 week implantation with either a placebo (P) or corticosterone (C) pellet, or 1 week after removal of the placebo (Pr) or corticosterone (Cr) pellet (n = 16/group) Corticosterone (ng/ml plasma) P C Pr Cr

59.3 i 4.2 72.1 ±4.3* 40.5 ± 6.2 45.7-/-5.7

Adrenal/body wt (mg/g × 100)

Thymus/body wt (mg/g × 100)

14.3 7.8 16.0 13.2

148.3 17.2 157.7 85.6

~ ± ± J:

0.5 0.6"* 0.8 0.7"*

± ± ± ±

5.8 1.4"* 5.2 7.0"*

Body wt (g) 293.8 ± 4.5 228.3 14.3"* 288.1 ± 4.3 242.0±5.1"*

*p < .05, **p < .01 for P compared with C and Pr compared with Cr; Tukey's tests after ANOVA.

RESULTS

Corticosterone Concentration, Body and Organ Weights Plasma corticosterone concentration was significantly elevated in the corticosteronetreated rats compared with the rats implanted with a placebo pellet, see Table I. The Table II. Mean ( + S E M ) time spent in social interaction, percent number of entries onto, and time spent on, the open arms of the elevated plus-maze during trials 1 and 2, and number of head dips and time spent head-dipping in the holeboard after 1 week implantation with either a placebo (P) or corticosterone (C) pellet, or 1 week after removal of the placebo (Pr) or corticosterone (Cr) pellet

Placebo (e) Social interaction (n = 8 pairs of rats/group) Time spent in social 93.15 + 11.0 interaction (s) Elevated plus-maze Trial 1 (n = 16/group) % Number of open arm 21.1 ± 2.9 entries % Time spent on the open 14.1 5:3.0

Corticosterone (C)

Placebo recovery (el-)

Corticosterone recovery (Cr)

76.5 ± 9.2

100.3 ± 9.1

104.1 ± 10.9

24.4 ± 2.6

22.4 ± 3.2

29.6 ± 2.6

17.0 ± 3.3

14.4 5= 2.9

19.2 ± 3.5

15.9 ± 3.8

13.9 ± 2.6

18.1 ± 3.7

9.5 ± 3.0

10.0 ± 2.5

12.7 ± 3.4

23.6 ± 0.9 28.2 ± 1.3

25.8 ± 1.2 25.7 ± 1.2

25.6 ± 1.0 29.7 ± 2.4

arms

Trial 2 (n = 16/group) % Number of open arm 11.0 ± 2.5 entries % Time spent on the open 5.3 ± 1.6 at'ins

Holeboard (n = 16/group) Number of head dips Time spent head-dipping (s)

26.9 ± 1.1 27.2 ± 2.3

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483

corticosterone treatment significantly decreased body, adrenal and thymus weights, see Table I. One week after pellet removal, there were no significant differences in the corticosterone concentrations; however, body, adrenal and thymus weights were still significantly reduced in those that had received the corticosterone pellet, see Table I. The groups with the pellet implants had significantly higher corticosterone concentrations than the groups tested 1 week after removal of the pellets (pellet factor--F(1,60) = 19.2, p < .001), suggesting that the presence of an implanted pellet was sufficient to elevate endogenous corticosterone, even if this was a placebo pellet, see Table I.

Behaviour Anxiolytic activity would be reflected in an increased time spent in social interaction and in increased percentage of entries onto, and time spent on, the open arms of the elevated plus-maze. Despite the clear corticosterone elevation that resulted from the implanted corticosterone pellet, there were no changes in the measures of anxiety in any of the tests of anxiety, see Table II. The measures of exploration (number of head-dips and time spent head-dipping) were also not changed, see Table II. However, the corticosterone treatment did significantly change motor activity in all the test situations. Locomotor activity in the social interaction and the holeboard tests and the number of closed arm entries in the elevated plus-maze (measures of horizontal activity) were all significantly lower in the corticosterone-treated rats compared with their placebo controls, see Fig. 1. Rearing was also reduced by chronic corticosterone treatment, but this reduction only reached significance in the holeboard. One week after pellet removal, there were no significant differences between the groups, see Fig. 1.

Autoradiography The only significant effect of corticosterone treatment on 5-HT1A binding was a decrease in rostral and caudal (dorsal) sections of the dentate gyms, see Fig. 2. In the median raph6 nucleus (MRN), there was increased 5-HT1A binding in the groups of animals tested with a pellet implanted, compared with those animals from which the pellets had been removed (pellet factor--F(1,28)= 9.9, p < .01), suggesting that the presence of a pellet (whether corticosterone or placebo) increased 5-HT1A receptor binding in this area. 5-HT2A binding in areas 1, 3 and 4 of the parietal cortex was significantly increased following corticosterone treatment, see Fig. 3. The chronic corticosterone treatment also increased 5-HT2A binding in area 2 of the parietal cortex, but this increase failed to reach significance, see Fig. 3. Paroxetine binding in the infrapyramidal blade of the dentate gyms was increased in the corticosterone-treated rats compared with those implanted with a placebo pellet, see Table III. There was also a non-significant increase in paroxetine binding to the 5-HT transporter sites in the dorsomedial nucleus of the hypothalamus and the lateral hypothalamic area following corticosterone treatment, see Table III. One week after pellet removal, there were no significant differences in 5-HT1A, 5-HT2A or 5-HT transporter binding between the groups, see Figs 2 and 3 and Table III.

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C. Fernandes et al.

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Fig. 1. Mean (±SEM) locomotor activity and number of rears made by rats in the social interaction test (n = 8 pairs of rats/group, top panel) and the holeboard (n = 16/group, middle panel), and the number of closed arm entries made by rats during trial 1 and 2 of the elevated plus-maze (n = 16/ group, bottom panel) after 1 week implantation with either a placebo (P) or corticosterone (C) pellet, or 1 week after removal of the placebo (Pr) or corticosterone (Cr) pellet. *p < .05, **p < .01 for P compared with C; Tukey's tests after ANOVA.

Corticosterone, 5-HT, Anxiety and Depression

ROSTRAL

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Fig. 2. Specific binding of [3H]-8-OH-DPAT to 5-HTaA receptors in hippocampal areas and raph6 nuclei after 1 week implantation with either a placebo ([]) or corticosterone (11) pellet, or 1 week after removal of the placebo (shaded) or corticosterone (cross-hatched) pellet (n = 6---8/group). Regions examined were rostral sections (upper panel) of the hippocampus (CA1, CA2, CA3 and CA4) and the suprapyramidal (DG-supra) and infrapyramidal (DG-infra) blades of the dentate gyrus; caudal sections (middle panel) of the dorsal (dCA1, dCA3 and dCA4), ventral (vCA1, vCA3) and mixed dorsal/ventral (d/vCA2) hippocampus and dorsal (dDG-supra and dDG-infra) and ventral (vDG) blades of the dentate gyrus; dorsal (DRN) and median (MRN) raph6 nuclei (lower panel). *p < .05, **p < .01 for placebo compared with corticosterone implanted groups; Tukey's tests after ANOVA. + + p < .01 for pellet implanted groups compared with groups in which the pellets had been removed; two-way ANOVA (pellet factor).

486

C. Fernandes et al. DISCUSSION

The corticosterone concentrations measured at the end of the week are likely to represent the levels achieved throughout the period of treatment, following an initial peak increase after implantation (Meyer et al., 1979). This chronic corticosterone treatment elevated corticosterone concentrations and reduced adrenal, thymus and body weights. However, no changes were detected in the measures of anxiety in any of the behavioural test situations. This dissociation between elevated corticosteroid concentration and anxiety has also been found following chronic benzodiazepine treatment. Patients chronically treated with benzodiazepines and challenged with the benzodiazepine receptor antagonist, flumazenil, displayed a precipitated withdrawal response with marked increases in anxiety, without any accompanying changes in cortisol concentrations (Harrison-Read et al., 1996). Also, when rats were withdrawn from 21 days of diazepam treatment there were significant increases in anxiety, but no rise in corticosterone concentrations (File et al., 1997). Thus, the chronic corticosterone and chronic benzodiazepine treatments provide a double-dissociation between raised corticosteroid concentrations and anxiety. Although the concentrations of corticosterone detected in this study are not those usually associated with high levels of stress, failure to find changes in anxiety are unlikely to be due to the corticosterone levels being below the necessary range. Manipulations resulting in similar increases in corticosterone (e.g. exposure to cat odour and to the open arms of the elevated plus-maze) do cause an increase in anxiety (File et al., 1993, 1994). However, the crucial difference between these manipulations and the present study is likely to be due to the production of an acute versus a chronic increase in corticosterone concentration. The presence of an implanted pellet (whether corticosterone or placebo) was in itself sufficient to elevate corticosterone and to produce increased 5-HT~A binding in the MRN. The elevations in corticosterone found in the animals with an implanted pellet cannot be explained by the procedures used for blood sampling. All animals were killed following minimal and equivalent disturbance, in an order randomised for treatment. However, the implanted pellets did result in some local tissue inflammation, which was probably the source of the mild stress detected in these animals. As previously reported, the chronic corticosterone treatment significantly decreased binding at 5-HTIA receptors in the dentate gyrus (Meijer and de Kloet, 1994; Mendelson and McEwen, 1992a,b) and increased 5-HT2A receptor binding in the parietal cortex (Kuroda et al., 1992). However, these combined biochemical changes clearly did not result in changes in anxiety. A pattern of decreased 5-HTIA and increased 5-HT2A receptor binding as a result of chronic corticosterone was observed in this study. A reciprocal relationship between 5-HT1A and 5-HT2A/2C receptors has previously been demonstrated, with 5-HT2A/2c receptors opposing the action of 5-HT1A receptors (Backus et al., 1990; Graeff et al., 1996). Furthermore, it has been suggested that this relationship between 5-HT receptor sub-types is important in the mediation of depression (Deakin, 1989; Marek et al., 1989). The changes observed in 5-HTIA, 5-HT2A and 5-HT transporter binding, corticosterone concentrations and motor activity following chronic corticosterone treatment had disappeared 1 week after pellet removal. The only persisting effects at this time point were decreased body, adrenal and thymus weights. The biochemical profile that resulted from the sustained elevations of corticosterone did not lead to altered anxiety, but did result in marked hypoactivity, a change which is more related to depression than anxiety. Indeed, stress-induced hypoactivity is used as an animal

Corticosterone, 5-HT, Anxiety and Depression

487

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Fig. 3. Specific binding of [3H]-ketanserin to 5-HT2A receptors in the cortex and hippocampal areas after 1 week implantation with either a placebo ([]) or corticosterone (11) pellet, or 1 week after removal of the placebo (shaded) or corticosterone (cross-hatched) pellet. Regions examined were parietal cortex (upper panel-PC1, PC2, PC3, PC4, PC5 and PC6); occipital cortex (middle panel-OC1, OC2, OC3, OC4, OC5 and OC6); hippocampal areas (CA1, CA3, CA4 and dentate gyrus---DG), lower panel. *p < .05 for placebo compared with corticosterone implanted groups; Tukey's tests after ANOVA.

488

C. Femandes et al.

Table Ill. Specific binding of [3H]-paroxetine to 5-HT transporter sites in the hypothalamus, cortex, amygdala, hippocampal areas and raph6 nuclei after 1 week implantation with either a placebo (P) or corticosterone (C) pellet, or 1 week after removal of the placebo (Pr) or corticosterone (Cr) pellet (n = 6--8/group).

Placebo (P) Hypothalamus Ventromedial nucleus Dorsomedial nucleus Lateral hypothalamic area Cingulate gyrus Occipital cortex (area 2) Parietal cortex Amygdala Basolateral nucleus Medial nucleus Hippocampal areas CA1 CA2 CA3 CA4 Dentate gyrus Superpyramidal blade Infrapyramidal blade Raph(~ nuclei Dorsal raph~ nucleus Median raph6 nucleus

Corticosterone (C)

Placebo recovery (er)

Corticosterone recovery (Cr)

44.8 4- 1.2 44.5 4- 2.5 41.7 4- 1.6 26.0 4- 1.1 32.4 4- 1.6 47.9 ± 2.9

49.6 i 1.8 51.9 + 2.4 46.5 4- 2.0 32.0 4- 1.1 37.7 4- 0.8 49.9 4- 2.6

46.7 ± 2.4 48.3 4- 2.3 44.3 4- 2.2 31.6 4- 2.8 34.0 4- 2.8 50.3 4- 2.8

49.2 ± 2.2 53.3 4- 2.3 48.8 4- 1.8 32.9 4- 2.7 34.7 4- 2.6 51.1 4- 1.5

69.7 4- 1.1 35.0 ± 2.1

75.0 4- 2.9 35.1 4- 2.1

75.6 4- 3.3 39.0 4- 2.6

76.9 4- 2.4 35.3 4- 1.6

28.2 zk 1.9 37.7 4- 2.3 36.3 4- 1.9 31.1 + 1.4

33.0 4- 1.5 44.5 + 2.7 41.2 4- 2.2 36.1 4- 1.8

32.8 4- 2.6 43.3 4- 2.2 42.8 ± 2.5 36.2 4- 2.5

33.3 4- 1.9 45.6 4- 2.0 42.7 ± 2.5 36.7 4- 1.8

27.2 4- 1.5 24.7 4- 1.4

30.0 4- 1.3 30.6 4- 1.1"

29.9 4- 2.4 30.0 4- 2.0

31.5 4- 2.1 29.3 4- 1.3

49.3 4- 3.4 33.8 4- 2.6

51.3 4- 3.0 28.7 4- 2.4

49.3 4- 2.7 34.3 i 2.6

56.3 ± 2.3 33.0 4- 1.9

*p < .05 for P compared with C; Tukey's tests after ANOVA

model of depression (Curzon et al., 1992; Kennett et al., 1985; Willner, 1990). Additionally, elevated cortisol concentrations are typically seen in depressed patients (Rubin, 1989). In conclusion, our results suggest that the constellation of changes observed following chronic corticosterone treatment are more pertinent to the neurobiology of depression than anxiety. Previous work has highlighted the importance of 5-HT2A/2C receptors in depression (Deakin, 1988). For example, several 5-HT2A/2C receptor antagonists are effective antidepressants (Koek et al., 1992) and many of the established antidepressants have been shown to decrease 5-HTzA/2C receptor binding following chronic administration (see Cowen, 1990). Furthermore, an increase in 5-HT2A/2C receptor binding in the frontal cortex has been found in post-mortem studies of patients with major depression (Arango et al., 1992; D'Haenen et al., 1992; Hrdina et al., 1993). The results of the present study indicate that the 5-HT2A receptors in the parietal cortex might be of particular importance in depression.

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