Regulation of Serotonin1A, Glucocorticoid, and Mineralocorticoid Receptor in Rat and Human Hippocampus: Implications for the Neurobiology of Depression

A.E. BENNETT RESEARCH AWARD Regulation of Serotonin1A, Glucocorticoid, and Mineralocorticoid Receptor in Rat and Human Hippocampus: Implications for the Neurobiology of Depression Juan F. Lo´pez, Derek T. Chalmers, Karley Y. Little, and Stanley J. Watson Background: Disturbances of the limbic– hypothalamic– pituitary–adrenal axis and the serotonin system are commonly found in depressive illness. Studying the effect of stress on these two neurobiological systems may give us important clues into the pathophysiology of affective illness and help us understand how stress and mood disorders are related. Methods: We studied the effect of chronic unpredictable stress and antidepressant treatment on serotonin 1A (5HT1A), glucocorticoid (GR), and mineralocorticoid (MR) receptor levels in rat hippocampus, using in situ hybridization and receptor autoradiography. We also used in situ hybridization to quantify hippocampal 5-HT1A, GR, and MR messenger (mRNA) levels in a small group of suicide victims with a history of depression, compared to matched controls (n 5 6). Results: We found that rats subjected to chronic unpredictable stress showed a significant elevation of basal plasma corticosterone compared to nonstressed rats. Chronic stress also caused a decrease in 5-HT1A mRNA and binding in the hippocampus. In addition, chronic stress produced alterations on the MR/GR mRNA ratio in this same region. The decreases in 5-HT1A mRNA and binding, as well as the MR/GR alterations, were prevented in animals that received imipramine or desipramine antidepressant treatment. Zimelidine was unable to reverse stress-induced increases in corticosterone, and was only partially successful in preventing the stress-induced receptor changes in the hippocampus. Suicide victims with a history of depression showed changes that were very similar to the changes found in chronic stress. Conclusions: Alterations in hippocampal 5-HT1A levels and in the MR/GR balance may be one of the mechanisms by which stress may trigger and/or maintain depressive episodes. Biol Psychiatry 1998;43:547–573 © 1998 Society of Biological Psychiatry

From the Department of Psychiatry, Mental Health Research Institute (JFL, SJW) and the Veterans Administration Hospital (KYL), University of Michigan Medical Center, Ann Arbor, Michigan; and Arena Pharmaceuticals, San Diego, California (DTC). Address reprint requests to Juan F. Lo´pez, MD, Mental Health Research Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109. Received April 22, 1997; revised September 25, 1997; accepted October 10, 1997.

© 1998 Society of Biological Psychiatry

Key Words: Serotonin1A receptors, hippocampus, stress, imipramine, corticosteroids

Introduction

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esearch studies have implicated disturbances in the serotonin (5-HT) system (Meltzer 1988, 1989) and the limbic– hypothalamic–pituitary–adrenal (LHPA) axis (Gold et al 1988; Kathol et al 1989) as the neurobiological “alterations” most consistently associated with affective illness. Although abnormalities in these two systems are usually studied individually, their interaction in the brain, as it relates to the pathophysiology of depression, has not been as extensively studied. In reviewing the clinical, psychological, and biological literature on depressive illness, one factor that emerges as being closely associated with depression is stress. Stress and depression have been linked in a variety of ways; for example, both physical and psychological stressors have been shown to be temporally (and perhaps causally) related to the onset of depressive episodes (Post 1992). Some studies have suggested that, at least for recurrent depression, stressful life events are more common in “nonendogenous depression” (Frank et al 1994). Other studies have found that stressful life events are significantly correlated even with the first episode of psychotic/ endogenous depression (Brown et al 1994). Another important link between depression and stress is the fact that both the LHPA and 5-HT systems, in addition to being involved in the pathophysiology of depression, are also critical contributors to the neurobiology of stress (McEwen 1987). Therefore, studying the neurobiology of stress by focusing on these two systems may give us important clues into the pathophysiology of affective illness, shed light on the actions of antidepressants, and begin to reveal how stress and mood disorders are related. The LHPA axis is a neuroendocrine system that is closely linked to stress in mammals. This system is geared to allow a swift response to stressful stimuli and ultimately a return to homeostasis, through complex feedback mech0006-3223/98/$19.00 PII S0006-3223(97)00484-8

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anisms. The parvocellular neurons of the paraventricular nucleus (PVN) in the hypothalamus represent the final common path for the integration of the stress response in the brain. They express corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), both of which are secretagogues of the LHPA. In response to physiological and psychological stressors, CRH and AVP are secreted into the hypophyseal portal circulation to reach the anterior pituitary, where they synergistically stimulate the proopiomelanocortin (POMC)-producing cells of the anterior lobe to release adrenocorticotropic hormone (ACTH), an end product of POMC precursor molecule processing. ACTH transported in the circulation interacts with the adrenal, causing elevation of plasma glucocorticoids. Repeated exposure to elevated glucocorticoid levels has been implicated in neuronal death, among other deleterious effects. Therefore, inhibition of steroid secretion is also an important component of this system. This inhibition is partly achieved by glucocorticoid binding to specific corticoid receptors in the pituitary and in limbic structures (Lopez et al 1991). Two types of corticoid receptors have been described in the brain based on biochemical and functional characteristics (McEwen 1991). Type I or mineralocorticoid receptor (MR) resembles the kidney mineralocorticoid receptor and has stringent specificity, binding selectively corticosterone, the main glucocorticoid of the rat. In the brain, MR is most densely localized in hippocampal and septal neurons and is described as a “high-affinity, low-capacity corticoid receptor system.” Conversely, type II or glucocorticoid receptor (GR) is known as a “low-affinity, high-capacity receptor system.” GR receptors are widely distributed in brain, including hippocampus, hypothalamus, and pituitary cells; however, they bind corticosterone with a lower affinity compared to MR. Their highest affinity is for potent synthetic glucocorticoids, such as dexamethasone (McEwen 1991). These receptor characteristics complement each other and put the MR and GR in a position to modulate LHPA responses. The MR receptors appear to be operative at low corticosterone concentrations and may offer tonic inhibition to the axis during the nadir of the circadian rhythm (De Kloet et al 1988; McEwen 1991). When high concentrations are present, MR receptors saturate, and the GR receptors appear to ensure the return of homeostasis. It is thus apparent that dysfunction on any of the complex pathways within the system could severely affect the organism’s adaptive capacity and expose the brain to high glucocorticoid concentrations. Hyperglucocorticoid states have been of interest to psychiatric research both as the consequence and the cause of depressive symptoms. Studies that investigate glucocor-

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ticoid hypersecretion as a consequence of depression have focused on the fact that a significant proportion of depressed patients show dysregulation of the LHPA axis. This dysregulation is manifested by cortisol hypersecretion, failure to suppress cortisol secretion after dexamethasone administration, and blunted ACTH response to CRH administration (Carroll et al 1976; Gold et al 1986; Kathol et al 1989; Sachar et al 1973). Although these findings are concerned with peripheral measures of LHPA activity, there is also good evidence of increased central drive, based on increased activity at the nadir of the circadian rhythm (Young et al 1995), impaired negative feedback (Young et al 1991), as well as more direct findings of elevated CRH in the cerebrospinal fluid (CSF) of depressed patients (Nemeroff et al 1984). These LHPA alterations are thought to reflect either a central neurotransmitter abnormality, the stress of the illness, or a combination of both. Studies that investigate hyperglucocorticoid states as a cause for depressive illness are less numerous, and have been stimulated by the fact that the final products of the LHPA axis (cortisol in humans, corticosterone in rats) have been shown to have profound effects on mood and behavior (McEwen 1987). For example, a high incidence of depression is linked to pathologies involving elevated corticosteroid levels, such as Cushing’s syndrome. This corticosteroid-induced depression usually disappears when corticosteroid levels return to normal (Kathol 1985; Murphy 1991). It is clear, from both animal and clinical studies (Kathol 1985; McEwen 1987; Murphy 1991), that circulating steroid levels provide important hormonal control of affect, which may be mediated by steroid-induced modulation of central limbic circuitry. The precise mechanism by which corticosteroids exert this influence on affect is not well understood; however, this mechanism is likely to involve interactions with brain neurotransmitters, since we know that central control of affect is intimately associated with the actions of the monoaminergic molecules 5-HT, norepinephrine, and dopamine. Effective clinical treatment of affective disorders such as depressive illness has been accomplished with drugs that act to potentiate the action of these monoaminergic neurotransmitter systems (Baker and Greenshaw 1988). For 5-HT in particular, a new generation of specific agents with strong antidepressant properties has been developed (Lucki 1991); consequently, alterations in serotonergic circuitry has been proposed as one of the central components of affective disease. Thus, it is likely that functional interactions between corticosteroids and the central serotonergic system may be significant, providing a biochemical basis for hormonally induced alterations in central nervous system (CNS) circuits related to emotional control.

5-HT1A, GR, and MR mRNA Regulation

Animal studies have in fact shown that corticosteroids can alter several elements of serotonergic neurotransmission. Removal of circulating corticosteroids by adrenalectomy (ADX) results in anatomically specific decreases in indices of serotonin metabolism, while stressful procedures, which raise corticosteroid levels, produce corresponding increases in serotonin turnover (Curzon et al 1972; Van Loon et al 1981); however, corticosteroids may also act to directly modulate serotonergic neurotransmission by regulating 5-HT receptors. Autoradiographic studies (Biegon et al 1985) first identified increased 5-HT1 receptor binding in the rat hippocampal formation 1 week after bilateral adrenalectomy. Subsequent investigations confirmed the sensitivity of 5-HT1 receptors to circulating corticosteroid levels (De Kloet et al 1986) and indicate that specific hippocampal subfields are exquisitely sensitive to adrenal steroids. More recent electrophysiological studies have shown a suppression of 5-HT-induced hyperpolarization within Ammon’s horn 1 (CA1) pyramidal cells after brief application of steroids (Joels et al 1991), establishing a functional coupling for steroid–serotonin receptor interactions within the hippocampus. It, therefore, seems likely that the high concentration of corticosteroid receptors within the hippocampus underlies the sensitivity of 5-HT receptors to corticosteroid regulation in this region. It is also well established that the hippocampus is a central component of limbic circuitry and is fundamental in controlling aspects of cognitive and behavioral functions. The ascending serotonergic innervation of hippocampal neurons arising in midbrain raphe nuclei provides one means by which the serotonergic system may act to regulate limbic function. Thus, the hippocampus represents a key anatomical structure involved in the central control of LHPA axis function and limbic circuitry. As such, this area provides a unique anatomical environment in which to study the molecular interplay between serotonergic systems and corticosteroids. The actions of 5-HT are mediated by multiple receptors. Serotonin receptors were originally divided into two subtypes, 5-HT1 and 5-HT2, on the basis of pharmacologic profile (Peroutka and Snyder 1979); however, more recent pharmacologic and biochemical data indicate the presence of at least six major 5-HT receptor families or classes: 5-HT1 through 5-HT7, with some families containing multiple subtypes (Hoyer and Martin 1996). Thus, the 5-HT1 receptor family, defined as exhibiting high affinity for 5-HT, has been subdivided into five receptor subtypes, 5-HT1A–1F. (The 5-HT1c receptor was reclassified as 5-HT2c, due to its resemblance to members of the 5-HT2 family.) The 5-HT1A receptor in particular has been identified both as an inhibitory somatodendritic receptor in raphe

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serotonergic cells and a postsynaptic receptor in selective serotonergic terminal fields (Hall et al 1985). Its abundance in the limbic system, as well as evidence from animal and clinical studies, strongly suggest that the 5-HT1A receptor plays an important role in the pathophysiology of mood disorders. Some investigators have hypothesized that a “balance” between the 5-HT1A and the 5-HT2A receptors is essential to antidepressant action. This is based on the observation that, while the net effect of antidepressant administration seems to be functional down-regulation of 5-HT2A receptors in the neocortex, the opposite is often observed for 5-HT1A receptors in the hippocampus. For example, antidepressant drugs have been found to up-regulate hippocampal 5-HT1A receptor electrophysiological responsiveness (Blier et al 1988) and possibly increase receptor number (Welner et al 1989), although this last observation is not universal (Watanabe et al 1993). This suggests that this receptor subtype may be important in relation to the therapeutic actions of these compounds. In humans, selective 5-HT1A agonists, such as gepirone and buspirone, can effectively treat generalized anxiety disorder (Rickels 1990) as well as depression (Fabre 1990), including the melancholic subtype (Robinson et al 1990). Buspirone has also been reported to augment the antidepressant response in patients who are antidepressant nonresponders (Jacobsen 1991). 5-HT1A is the most abundant serotonin receptor in the hippocampus, where it is colocalized with glucocorticoid and mineralocorticoid receptors, making this structure an ideal anatomical substrate for studying steroid–5-HT1A receptor interactions. Several investigators have reported increases in 5-HT1A receptor binding and gene expression in response to ADX (Chalmers et al 1994; Mendelson and McEwen 1990), demonstrating that this receptor subtype is under tonic inhibition by corticosterone. Thus, it is possible that the mechanisms underlying corticosteroid regulation of 5-HT1A receptors may contribute to steroidmediated modulation of affective state and, as such, may represent an important linkage both to the pathophysiology of affective disorders and to psychotherapeutic drug action. The present paper reports previously unpublished series of preclinical experiments in the rodent, designed to further our understanding of the mechanisms involved in corticosteroid-mediated modulation of the 5-HT1A receptor at the molecular and neuroanatomical level under conditions related to chronic stress and pathophysiological levels of steroids. Specifically, we have used a chronic unpredictable stress model to ask: 1) How do hippocampal 5-HT1A receptors react to endogenously elevated corticosteroids? 2) Can antidepressant treatment alter the 5-HT1A receptor under conditions of chronic stress? 3) What is the

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effect of a milder stress (swim), which produces only modest elevations of corticosterone? 4) Can we modify the effects of chronic stress on hippocampal 5-HT1A receptors by preventing the increase in corticosterone, using adrenalectomy? 5) Are there differences between a specific serotonin reuptake inhibitor (SSRI) and a noradrenergic reuptake inhibitor in their ability to modify the effects of chronic stress? 6) What are the roles of MR and GR in mediating hippocampal 5-HT1A receptor regulation? We also report results from a postmortem study, investigating the regulation of 5-HT1A, MR, and GR in the hippocampus of suicide victims with a history of major depressive disorder. Here we ask: Will we find evidence of 5-HT1A, GR, and/or MR dysregulation that parallels the abnormalities found in models of chronic stress? In this way we have identified a molecular linkage between the LHPA axis and the 5-HT system and discussed the potential relevance of these to the mechanism of action of antidepressant drugs and the pathophysiology of affective disorders.

Methods for Animal Studies Animals In all studies male Sprague-Dawley rats were used (250 –300 g). Rats were housed 6 per cage in a 12-hour light;dark cycle and allowed free access to food and water. Animals are maintained in accordance with the NIH guidelines for the Care and Use of Laboratory Animals. All the protocols were approved by the University of Michigan Committee on Use and Care of Animals.

Treatment Protocols 1. CHRONIC UNPREDICTABLE STRESS AND IMIPRAMINE:

Rats were randomly assigned to one of the following experimental groups (n 5 6 per group): 1) daily intraperitoneal (IP) injections of saline (Saline); 2) daily IP injections of the tricyclic antidepressant imipramine, at a dose of 10 mg/kg (Imipramine); 3) daily stress and daily saline injections (Stress Saline); and 4) daily stress and daily injections of imipramine (10 mg/kg) (Stress Imipramine). The stress protocol used was a modified version of a chronic unpredictable stress paradigm (Chappell et al 1986; Katz and Sibel 1982). The daily schedule was as follows: Day 1, 9:00 AM: forced swim in a water tank at 24°C for 20 min, 1:00 PM: restraint stress for 2 hours; Day 2, 12:00 noon: restraint stress for 3 hours; Day 3, 10:00 AM: exposure to ether vapor until loss of consciousness, 2:00 PM: 4-hour exposure to a 4°C cold room; Day 4, 2:00 PM: forced swim for 20 min; Day 5, 8:00 AM: 4-hour exposure to a 4°C cold room, 3:00 PM: exposure to ether vapor; Day 6, 11:00 AM: restraint stress for 4 hours; Day 7, 10:00 AM: forced swim for 20 min, 2:00 PM: 3-hour exposure to a 4°C cold room; Day 8, 6:00 PM: exposure to ether vapor; Day 9, 8:00 AM: restraint stress for 4 hours, 1:00 PM: exposure to ether vapor; Day 10, 11:00 AM: 5-hour exposure to a 4°C cold room; Day 11, 9:00 AM: forced 14 DAYS TREATMENT.

swim for 20 min, 10:00 AM: exposure to ether vapor; Day 12, 2:00 PM: one electroconvulsive shock (80 mA, 0.2 sec, 60 Hz); Day 13, 11:00 AM: exposure to ether vapor, 12:00 noon: 5-hour exposure to a 4°C cold room; and Day 14, 8:00 AM: restraint stress for 5 hours. Animals in all treatment groups were sacrificed by decapitation on day 15, between 9:00 AM and 10:30 AM. Brains were rapidly removed, frozen in liquid isopentane (242°C), and stored at 280°C until ready for sectioning. At the time of sacrifice, trunk blood was collected in tubes containing edetic acid (EDTA) and spun at 1500 rpm for 10 min. Subsequently plasma samples were transferred to tubes containing 0.5 mL HCl and assayed for corticosterone using radioimmunoassay (Vazquez et al 1993). 2. DAILY SWIM STRESS STUDY. In this second experiment, rats (n 5 5 per group) underwent swim stress for 3, 7, 14, or 21 days. An unhandled group served as control. The stress consisted of daily 20-min sessions of swim at 24°C. All animals were sacrificed by rapid decapitation, 24 hours after the last swim session, between 9:00 AM and 10:30 AM. As in the previous experiment, trunk blood was collected for corticosterone measurement, and brains were rapidly removed, frozen in liquid isopentane, and stored at 280°C until sectioning. 3. CHRONIC STRESS–ADRENALECTOMY STUDIES: 14

In a third experiment, rats were divided into two major groups; half of the rats received chronic unpredictable stress as before, while the other half did not. Each of these groups was subdivided and underwent ADX, or sham surgery, with or without corticosterone replacement (50 mg/mL in their drinking water). The animals were therefore divided into six groups (n 5 6/group): 1) Sham Nonstress (control); 2) Sham Stress; 3) ADX Nonstress; 4) ADX Stress; 5) ADX– corticosterone replaced nonstress (Cort Nonstress); and 6) ADX– corticosterone replaced stress (Cort Stress). Chronic unpredictable stress (CUS) was administered for 14 days. Rats were either ADX or Sham-ADX bilaterally using a dorsal approach. All surgeries were performed between 9:00 AM and 10:30 AM. Postsurgery ADX animals received 0.9% saline as drinking water. All animals were sacrificed 24 hours after the last stress session; brains and plasma were handled as above. DAYS TREATMENT.

4. CHRONIC STRESS AND ZIMELIDINE OR DESIPRAMINE:

In the fourth experiment, rats were divided in six groups (n 5 6/group): 1) saline-injected animals (Nonstress Saline) (controls); 2) saline-injected animals that were also stressed (Stress Saline); 3) desipramine-treated animals (Nonstress Desipramine); 4) desipramine-treated and stressed animals (Stress Desipramine); 5) zimelidine-treated animals (Nonstress Zimelidine); and 6) zimelidine-treated animals that were also stressed (Stress Zimelidine). The stress paradigm used was CUS, which was administered for 28 days, instead of 14 days. The first 2 weeks of CUS followed the schedule described under experiment 1; the second 2 weeks repeated the same stress schedule of the first 2 weeks. Desipramine (10 mg/kg), zimelidine (20 mg/kg), and saline were injected daily (IP) for the

28 DAYS TREATMENT.

5-HT1A, GR, and MR mRNA Regulation

duration of the stress. Animals were always injected at the same hour (between 12:00 and 13:00). All animals were sacrificed the morning after the last stress session. Brains and trunk blood were obtained and processed as described above. In a similar experiment, rats also underwent CUS for 28 days and received the antidepressant fluoxetine (10 mg/kg). Five groups of rats (n 5 6/group) were studied: 1) a nonstressed saline control group (Saline); 2) a nonstressed group treated with daily injections of fluoxetine (Fluoxetine); 3) a stressed group receiving saline injections (Stress Saline); 4) a group that was stressed and received daily fluoxetine injection (Stress Fluoxetine); and 5) a group that was not stressed and did not receive any saline injections, which served to “control” for the potential stressful effect of daily saline injections (Unhandled). For this experiment, only trunk blood for plasma corticosterone was collected. For all studies (except the fluoxetine experiment), brains were sectioned in coronal plane at 15 mm on a cryostat (Hacker Inst., Fairfield, NJ) at 220°C and thaw mounted onto polylysinecoated microscope slides. Sections were stored at 280°C until processed for in situ hybridization and receptor autoradiography.

Technical Methods RIBOPROBE DESIGN. 5-HT1A complementary RNA (cRNA) riboprobe was produced from a BalI–PvuII fragment of the rat 5-HT1A receptor gene (Albert et al 1990) ligated into HincII-cut pGEM blue (Promega™). This fragment is composed of a 910-bp insert covering the sequence from the beginning of the second putative transmembrane domain to the middle of the extracytoplasmic domain found between transmembrane domains VI and VII, encompassing the entire sequence of the third cytoplasmic loop. This represents the region of least homology for G-protein coupled receptors. 5-HT1A probe specificity was confirmed by absence of signal in both sections labeled with sense 5-HT1A probe and sections pretreated with ribonuclease (RNase) prior to hybridization with antisense (cRNA) 5-HT1A probe. GR messenger RNA (mRNA) was visualized using a cRNA probe synthesized from a 456-bp fragment of GR cDNA (provided by Keith Yamamoto) subcloned into the XbaI–EcoRI site of pGEM 4, while the MR cRNA probe was synthesized from a 347-bp PstI–EcoRI fragment of MR cDNA (Patel et al 1989) ligated into pGEM 3. The specificity of these GR and MR riboprobes has been previously confirmed (Herman et al 1989a). Riboprobes were produced using either SP6 or T7 transcription systems in a standard labeling reaction mixture consisting of: 1 mg linearized plasmid, 5X SP6 transcription buffer, 125 mCi 35 S-uridine triphosphate (UTP), 150 mmol/L nucleoside triphosphates, 12.5 mmol/L dithiothreitol, 20 U RNAse inhibitor, and 6 U of the appropriate polymerase. The reaction was incubated at 37°C for 90 min, labeled probe being separated from free nucleotides over a Sephadex G50-50 column. IN SITU HYBRIDIZATION. Sections were removed from storage at 280°C and placed directly into 4% buffered paraformaldehyde at room temperature. After 60 min, slides were rinsed in isotonic phosphate-buffered saline and treated with proteinase K (1 mg/mL in 100 mmol/L TRIS/HCl, pH 8.0) for 10 min at

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37°C. Subsequently, sections underwent successive washes in water (1 min), 0.1 mol/L triethanolamine (pH 8.0, plus 0.25% acetic anhydride) for 10 min and 2X SSC (0.3 mmol/L NaCl, 0.03 mmol/L sodium citrate, pH 7.2) for 5 min. Sections were then dehydrated through graded alcohols and air dried. Postfixed sections were hybridized with 1.0 3 106 dpm [35S]UTP-labeled riboprobes in hybridization buffer containing 50% formamide, 10% dextran sulphate, 3X SSC, 50 mmol/L sodium phosphate buffer (pH 7.4), 1X Denhardt’s solution, 0.1 mg/mL yeast transfer RNA (tRNA), and 10 mmol/L dithiothreitol in a total volume of 25 mL. The probe was applied to sections on a glass coverslip and hybridized overnight at 55°C. Next day the sections were washed in 2X SSC for 5 min and then treated with RNase A (200 mg/mL in 10 mmol/L TRIS/HCl, pH 8.0, containing 0.5 mol/L NaCl) for 60 min at 37°C. Subsequently, sections were washed in 2X SSC for 5 min, 1X SSC for 5 min, and 0.5X SSC for 60 min at hybridization temperature, and 0.5X SSC at room temperature for 5 min, and then dehydrated in graded alcohols and air dried. For signal detection, sections were placed on Kodak XAR-5 X-ray film and exposed for 2 days at room temperature. [3H]-8-Hydroxy-2-(N,N-di-N-propylamino-tetralin) [ H]-8-OH-DPAT) binding to detect 5-HT1A receptor sites was performed according to published methods (Palacios et al 1987). Slide-mounted tissue sections were preincubated in 0.17 mol/L TRIS/HCl, pH 7.6, containing 4 mmol/L CaCl2 and 0.1% ascorbic acid for 30 min at room temperature. Subsequently, sections were incubated with 2 nmol/L [3H]-8-OH-DPAT for 60 min at room temperature. Postincubation, slides were washed in incubation buffer (2 3 5 min) at 4°C and dried in a stream of cold air. Nonspecific binding was determined in the presence of 2 mmol/L 5-HT. Sections were apposed to tritium-sensitive Hyperfilm™ (Amersham) and exposed at room temperature for 10 days. As 2 nmol/L has been determined as a saturating concentration for 8-OH-DPAT (Palacios et al 1987), resulting autoradiograms are a measure of Bmax for 5-HT1A receptors. For detection of 5-HT transporter binding sites, slide-mounted tissue sections were brought to room temperature and incubated with 70 –150 pmol/L 3H-paroxetine in 50 mmol/L TRIS HCl containing 120 mmol/L NaCl and 5 mmol/L KCl (pH 7.7 at 23°C) at room temperature. Blanks were incubated in the same medium with the addition of 1 mmol/L citalopram. After incubation with 3H-paroxetine, tissue sections were washed in the same buffer at room temperature, dipped in deionized water, and dried rapidly under a stream of cold, dry air. In preliminary nonautoradiographic studies to determine the time courses of dissociation and association kinetics and pharmacology of 3H-paroxetine binding to slide-mounted tissue sections, the washed brain sections were wiped from the slides with Whatman GFR/B glass fiber filters. Filters were equilibrated with 15 mL of Formula 963 scintillation fluid and counted at 54% efficiency in a Beckman 3801 scintillation spectrometer. In all subsequent autoradiographic studies, the brain sections were incubated with 3H-paroxetine for 120 min at room temperature and washed for two 60-min periods in buffer at room temperature. The dry, labeled, slide-mounted brain sections were apIN VITRO RECEPTOR AUTORADIOGRAPHY. 3

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posed to 3H-Ultrofilm (LKB) (Palacios et al 1987), and the autoradiograms were developed after 6 – 8 weeks of exposure. MICRODENSITOMETRIC ANALYSIS. Autoradiograms generated from both in situ hybridization and in vitro receptor autoradiography were analyzed using an automated image analysis system (Dage camera, MAC II/IMAGE program). Anatomical regions of interest were interactively selected, and mean optical density measurements for each region were determined from at least six coronal sections. Hippocampal subfields were determined with reference to Nissl-stained sections and the anatomical atlas of Paxinos and Watson (1986). Nonspecific labeling of [35S]-riboprobes was determined from an area of section exhibiting apparent lack of hybridization signal. For in vitro receptor autoradiograms, nonspecific binding was determined from adjacent sections incubated with [3H]-8-OH-DPAT in the presence of 2 mmol/L 5-HT. Statistical differences were determined by analysis of variance (ANOVA) and posthoc Fisher test.

Methods for Human Postmortem Study Experimental Design TISSUE COLLECTION. The postmortem human brain specimens were collected at The University of North Carolina by one of us (KYL). Details of the collection and sectioning methods have been published elsewhere (Little et al 1993). Briefly, brains were obtained at autopsy from control subjects dying in accidents, by assault, or of cardiac causes. Similarly, specimens were obtained from subjects committing suicide by various methods. Many subjects received severe wounds that led to death within minutes. No subject survived long enough to receive treatment at a hospital. The brains were removed at autopsy, blocked, and rapidly frozen in dry ice. These brain blocks were stored at 280°C until studied. SUBJECTS CHARACTERISTICS. We selected sections of hippocampal blocks (6 suicides and 6 control subjects) from a larger group of subjects (Little et al 1993). These 12 subjects were selected after a careful examination of their Nissl stains by an experienced neuropathologist in our institution revealed that the hippocampal tissue was in good condition (i.e., no freezing artifact, no profound ischemic changes). The suicide victims all had a history of major depression, were not taking psychotropic medications chronically at the time of death, and had no history of substance abuse. Controls met the following criteria: 1) suitable method of death (rapid, not due to a chronic condition); and 2) appropriate age, sex, and postmortem interval to match suicides. Suicides all died of rapidly fatal injuries and were without evidence of chronic medical illness. A research psychiatrist (KYL) and psychologist interviewed at least one informant for each subject. The average number of informants was 3.6 for suicides and 2.1 for controls. All interviews focused on ascertaining if the subject met DSM-III-R criteria for mood disorders, psychoactive substance abuse disorders, anxiety disorders, or psychotic disorders. During the interview, the Family History– Research Diagnostic Criteria checklists of symptoms for mood

disorders, alcoholism and antisocial personality, anxiety disorders, and psychotic disorders were completed. Table 2 lists the clinical and demographic characteristics of each individual subject. There were no statistically significant differences in age (t 5 23.76, df 5 10, p 5 .71), or postmortem interval (t 5 20.91, df 5 10, p 5 .38) between the two groups.

Technical Methods Fresh frozen hippocampal blocks from these brains were thawed to 220°C and cryostat sectioned at a thickness of 20 mm. Sections were thaw-mounted onto poly-L-lysine subbed microscope slides, dried, and stored at 280°C until processing. For this study, five slides per probe, per subject were prepared for in situ hybridization, as we have previously described (Lopez et al 1992). In situ hybridization was performed for GR mRNA, MR mRNA, and 5-HT1A mRNA. To control for mRNA loss due to postmortem factors, we also performed in situ hybridization with P1B15, a complementary DNA (cDNA) encoding for the nonregulatable protein cyclophilin (Danielson et al 1988). Therefore, the GR, MR, and 5-HT1A mRNA values were “corrected” for P1B15. We quantified the molecules in the following hippocampal subfields: CA1, CA2, CA3/4, and dentate gyrus (DG). Quantification was done blind to the clinical status of the subject. The 5-HT1A cRNA riboprobe was produced from a BalI–PvuII fragment of the rat 5-HT1A receptor gene ligated into HincII-cut pGEM blue (Promega™). This fragment is composed of a 910-bp insert covering the sequence from the beginning of the second transmembrane domain to the middle of the extracytoplasmic domain between transmembrane domains VI and VII, encompassing the entire sequence of the third cytoplasmic loop. This represents the region of least homology for other G-protein coupled receptors and is highly homologous to the human 5-HT1A. Human GR mRNA was visualized using a cRNA probe synthesized from a 456-bp fragment of GR cDNA subcloned into the XbaI–EcoRI site of pGEM 4, while the human MR cRNA probe was synthesized from a 347-bp PstI–EcoRI fragment of MR cDNA ligated into pGEM 3. For both of these cRNAs the sequence corresponds to the steroid binding domain, the region of least homology between these two receptors, to avoid potential cross-hybridization. The p1B15 cRNA was transcribed from a 680-bp insert subcloned into a pSP64 plasmid. This insert has the sequences from position 5 to 685 of the rat cyclophillin cDNA (Danielson et al 1988). The plasmid was linearized with Hind III and the riboprobe synthesized with SP6 polymerase. The mean value of the five slides per probe was taken as the individual value for each subject. For each probe, comparisons between suicides and controls were done using two-factor analysis of variance ANOVA (diagnosis 3 hippocampal subfield). When ANOVA indicated a significant difference between the groups, unpaired t tests were performed for each hippocampal subfield, between the suicides and controls. Significance was determined at p , .05. To compare the distribution of 5-HT1A mRNA with 5-HT1A receptor binding, hippocampal sections from 2 control subjects were processed for receptor binding autoradiography using [3H]-8-OH-DPAT. Binding was performed according to pub-

5-HT1A, GR, and MR mRNA Regulation

Figure 1. Photomicrographs of 5-HT1A receptor mRNA expression (A) and [3H]-8-OH-DPAT binding (B) in adjacent hippocampal sections. CA1, Ammon’s horn 1; CA2, Ammon’s horn 2; CA3-4, Ammon’s horn 3 and 4; DG, dentate gyrus. Note the decreased hybridization signal in the CA2 relative to the other subfields. lished methods (Pazos et al 1987), as detailed under the animals methods above.

Results Chronic Unpredictable Stress and Imipramine Study Figure 1 illustrates the distribution of 5-HT1A receptor mRNA and binding in the hippocampus, as well as the particular subfields analyzed. As previously described (Chalmers and Watson 1991), 5-HT1A receptor mRNA is very abundant within the pyramidal cell layer of CA1 and CA3 subfields and dentate gyrus granule cells with lower levels of expression within the CA2 subfield. This mRNA distribution is closely matched by the 5-HT1A receptor binding, although the 5-HT1A binding signal is not limited to the pyramidal cell layer, since it is also detected in stratum oriens, radiatum, and moleculare. To investigate the effects of endogenous corticosteroid secretion on 5-HT1A receptor regulation, we subjected rats to 14 days of CUS. This paradigm was chosen because it

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produces profound changes in the LHPA axis (Chappell et al 1986), and it has been proposed by some investigators as an animal model of depression (Armario et al 1988; Katz and Sibel 1982). We also investigated the effect of concomitant imipramine administration, since imipramine has been reported to affect aspects of the LHPA axis (Lopez et al 1994b). Analysis of variance followed by posthoc comparisons revealed that rats submitted to chronic unpredictable stress showed a significant elevation of basal plasma corticosterone compared to the nonstressed rats (F 5 4, df 5 3/20, p , .05); however, imipramine significantly inhibited the stress-induced corticosterone increase. There were no differences in corticosterone levels between nonstressed rats injected with saline and those injected with imipramine (Figure 2A). A two-factor ANOVA also revealed a significant drug by stress interaction (F 5 7, p 5 .01). The corticosterone values for the treatment groups were: Saline 5 3 6 1.3 mg/dL, Imipramine 5 4 6 1.5 mg/dL, Stress 1 Saline 5 6.9 6 1.2 mg/dL, Stress 1 Imipramine 5 1.6 6 0.3 mg/dL. The 2-week chronic stress paradigm caused a decrease in 5-HT1A mRNA levels across all hippocampal subfields relative to saline-treated animals (Figure 2B). Although 5-HT1A expression was reduced to a similar extent across all subfields (;22%), statistically significant differences between Saline and Stress groups were only detected within CA1 and CA3/4 subfields. These stress-induced decreases in 5-HT1A gene expression were prevented in animals in which imipramine was administered concomitantly with stress. Posthoc analysis indicated significant differences between Imipramine and Stress Saline groups in all subfields except CA2. No statistically significant differences were found for imipramine administration alone. In a analogous fashion to 5-HT1A mRNA expression, mean 5-HT1A binding levels were reduced in all hippocampal subfields of Stress Imipramine animals relative to Saline control (Figure 2C). Statistically significant differences between Saline and Stress Saline groups were evident in CA1, CA3/4, and dentate gyrus. Changes 5-HT1A binding were proportionally smaller than changes in 5-HT1A mRNA (;15%). In all subfields, imipramine prevented 5-HT1A down-regulation in response to stress. No differences were found between saline and imipramine administration in the nonstressed animals. A two-factor ANOVA revealed both a drug (F 5 12.36, p , .005) and a stress effect (F 5 6.32, p 5 .02) across the subfields. The 5-HT1A receptor is located postsynaptically in the hippocampus. To investigate if CUS was affecting any presynaptic elements in this same anatomical region, we quantified, in the same animals, the serotonin transporter (5-HTt) binding sites, using 3H-paroxetine binding

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Figure 2. Effect of chronic unpredictable stress performed for 2 weeks on: plasma corticosterone levels (A), hippocampal 5-HT1A receptor mRNA expression (B), and hippocampal [3H]-8OH-DPAT binding (C) obtained from animals treated with: Saline (n 5 6), Imipramine (n 5 6), Stress Saline (n 5 6), and Stress Imipramine (n 5 6). Overall one-way ANOVA indicated significant effects of treatment on plasma corticosterone, 5-HT1A mRNA expression, and [3H]-8-OH-DPAT binding in each hippocampal subfield (see text for details of analysis). Post hoc analysis using Fisher protected least significant difference test (Fisher PLSD test) confirmed significant differences when Saline was compared to Stress Saline (*p , .05) and when Stress Saline was compared to Stress Imipramine (†p , .05). In addition, several hippocampal subfields exhibited significant differences when compared to the Imipramine group only (zp , .05).

(Figure 3A). The 5-HTt is a key component of the serotonergic synapse, since it controls the amount of 5HTt in the synaptic cleft and is believed to mediate the therapeutic action of antidepressants (Lopez et al 1994a). As can be observed in Figure 3B, no significant

differences in 5-HTt sites were observed across all four groups in any of the hippocampal regions. A two-factor ANOVA revealed no drug effect (F 5 0.38, p 5 .76) or stress effect (F 5 0.143, p 5 .93) in any of the subfields analyzed.

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Figure 3. Effect of chronic unpredictable stress performed for 2 weeks on hippocampal 5-HT transporter binding sites of animals treated with: Saline (n 5 6), Imipramine (n 5 6), Stress Saline (n 5 6), and Stress Imipramine (n 5 6). Panel A is a representative photomicrograph of hippocampal paroxetine receptor autoradiography performed to quantify 5-HT transporter binding capacity. In this study, one-way ANOVA did not indicate significant effects of treatment on any of the hippocampal subfields when compared to the saline group (B).

Daily Swim Stress Study Chronic unpredictable stress is a paradigm that is associated with persistent elevations of corticosterone (Armario et al 1988; Chappell et al 1986). It would be important to

determine whether the changes in 5-HT1A receptor observed after CUS can be found in other stress paradigms; in particular, situations where modest increases in corticosterone are observed. Swim stress is a mild stressor that

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causes an acute release of ACTH and corticosterone (Young 1990); however, this ACTH and corticosterone release is of a lesser magnitude than those observed with other stressors, such as footshock (Lopez et al 1994b; Young 1990). In addition, because the same stress is applied every day, rats may habituate to the stressor more readily (Terrazzino et al 1995). We therefore investigated the effect of daily swim stress, given at various durations (3, 7, 14, and 21 days) on 5-HT1A receptor mRNA and binding levels in the hippocampus. Animals treated with daily swim stress showed corticosterone levels that were lower than the levels observed after nonhabituating stress (Figure 4A). Plasma corticosterone on days 3 and 21 of swim were significantly higher than the control animals (F 5 3.21, df 5 4/20, p , .05). Nevertheless, no significant differences in 5-HT1A mRNA (Figure 4B) or 5-HT1A binding (Figure 4C) were detected between the unhandled (control) group and any of the other groups of rats that received daily swim sessions of several days duration.

Chronic Stress and Adrenalectomy Study In this study, we investigated if this CUS-induced 5-HT1A down-regulation is secondary to the elevated plasma corticosteroid levels observed in this paradigm, or if it is mediated by “central” activating stress mechanisms. For this purpose, we studied the effect of CUS in intact rats and in rats after ADX with and without corticosterone (Cort) replacement. By removing the adrenals, we are able to determine if increases in ACTH and/or CRH are responsible for the observed 5-HT1A changes. Replacing corticosterone in the ADX rats allows the animal to respond to stress while corticosterone is present in the circulation, but the corticosterone response to the stressor is eliminated. The results of this experiment are seen in Figure 5. We found that plasma corticosterone significantly increased in the Sham Stress animals compared to the other groups (F 5 3.62, df 5 5/30, p 5 .01). The corticosterone levels in the ADX animals were practically undetectable. In the Sham Stress animals, chronic stress caused a consistent decrease in 5-HT1A mRNA in all hippocampal subfields, although the decrease achieved statistical significance only in CA3 (F 5 6.386, df 5 5/30, p , .001) and DG (F 5 9.314, df 5 5/30, p , .001) by ANOVA. No 5-HT1A mRNA down-regulation was observed in the ADX stress or Cort-replaced stress groups compared to their respective unstressed controls. Therefore, elimination of the corticosterone rise after stress prevented the decrease in hippocampal 5-HT1A. These results indicate that the 5-HT1A down-regulation observed after chronic stress is mostly mediated by increases in plasma corticosterone

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levels. Interestingly, chronically stressed rats that were adrenalectomized and received Cort replacement also had lower 5-HT1A levels than the sham controls. Whether this represents a chance event or the fact that there is indeed a small effect of stress independent of corticosteroid levels remains to be investigated. The possibility exists that under certain conditions, corticosterone may have a permissive effect, such that “central” mechanisms may also be operant, but only in the presence of corticosterone.

Chronic Stress and Treatment with Desipramine or Zimelidine Imipramine is a tricyclic antidepressant, and as such has effects on several neurotransmitter systems. To test the effect of a more selective antidepressant, such as a specific serotonin reuptake inhibitor, we studied the effect of zimelidine on the LHPA and 5-HT1A response to CUS. We contrasted the effect of zimelidine with a potent norepinephrine reuptake inhibitor (desipramine). In this way, we could examine the relative contributions of the two principal neurotransmitter systems commonly thought to mediate the therapeutic action of antidepressants. To allow the antidepressants more time to exert their therapeutic action, we extended the treatment protocol to 4 weeks. The results are shown in Figure 6. Animals subjected to 4 weeks of CUS and injected with saline showed a significant increase in baseline corticosterone levels, compared to controls (F 5 2.72, df 5 5/30, p , .05). This corticosterone increase was prevented if stressed animals were simultaneously treated with the tricyclic antidepressant desipramine (Figure 6A). Surprisingly, zimelidine did not prevent the increase in basal corticosterone levels observed after chronic stress. In fact, the corticosterone levels in the CUS animals treated with zimelidine where somewhat higher that the Stress Saline animals, although the difference was not statistically significant. No differences in corticosterone levels were evident in the antidepressant treatments alone. Animals receiving CUS and saline injections again showed a decrease in 5-HT1A mRNA levels across all hippocampal subfields (Figure 6B), although these decreases only achieved statistical significance in CA1 (F 5 2.62, df 5 5/30, p 5 .04) and CA3 (F 5 2.5, p 5 .05), using one-factor ANOVA. A two-factor repeated-measures ANOVA revealed a significant group effect (p , .05). The 5-HT1A mRNA percent decreases in the stressed group (relative to the control group) ranged from 48% in CA1, to 22% in the DG. The stressed group treated with zimelidine had 5-HT1A mRNA levels that were higher than the stressed group receiving no antidepressant, but were consistently lower than the control group. The

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Figure 4. Effect of daily sessions of swim stress performed for 3, 7, 14, and 21 days on: plasma corticosterone levels (A), hippocampal 5-HT1A receptor mRNA expression (B), and hippocampal [3H]-8-OHDPAT binding (C). Notice the lower corticosterone levels compared to the chronic unpredictable stress. One-way ANOVA with post hoc Fisher PLSD test indicated that plasma corticosterone levels were significantly higher than controls after rats received swim stress for 3 and 21 days (*p , .05). ANOVA indicated no significant effects of treatment on 5-HT1A mRNA expression or [3H]-8-OH-DPAT binding in each hippocampal subfield, when compared to the unhandled group.

decreases in gene expression in the Stress Zimelidine group were 30% in CA1, 20% in CA2, 14% in CA3, and only 10% in the DG. Therefore, zimelidine treatment only partially reversed the stress-induced 5-HT1A down-regulation, even though the difference was not statistically significant (Figure 6B). Stressed animals treated with

desipramine showed no evidence of 5-HT1A down-regulation in the hippocampus. Because of the role of hippocampal glucocorticoid (GR) and mineralocorticoid (MR) receptors in modulating LHPA activity, and the fact that these receptors are the target of circulating corticosteroids, we performed in situ

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Figure 5. Effect of intermittent sessions of chronic unpredictable stress performed for 2 weeks on: resting plasma corticosterone levels (A), and hippocampal 5-HT1A receptor mRNA expression (B) obtained from animals that were sham operated or adrenalectomized (ADX). The following groups were studied: Sham Nonstress (n 5 6), Sham Stress (n 5 6), ADX Nonstress (n 5 6), ADX Stress, ADX Corticosterone Replaced Nonstress (Cort Non Stress) (n 5 6), and ADX Corticosterone Replaced Stress (Cort Stress) (n 5 6). One-way ANOVA indicated significant effects of ADX treatment on plasma corticosterone and 5-HT1A mRNA expression (see text for details of analysis). ADX animals had significantly decreased circulating corticosterone levels when compared to all groups. Chronic stress significantly decreased 5-HT1A mRNA expression in the CA3-4 and dentate gyrus regions. Removal of circulating steroids by ADX prevented the decrease in 5-HT1A mRNA expression after chronic stress. Adrenalectomy with corticosterone replacement had a similar effect. *p , .05 compared to Sham Nonstress; zp , .05 compared to Sham Stress.

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Figure 6. Effect of chronic unpredictable stress performed for 4 weeks on resting plasma corticosterone levels (A), and hippocampal 5-HT1A receptor mRNA expression (B) obtained from animals that were treated with saline, desipramine, or zimelidine. The following six groups were studied: 1) Nonstress Saline (n 5 6), 2) Stress Saline (n 5 6), 3) Nonstress Desipramine (n 5 6), 4) Stress Desipramine (n 5 6), 5) Nonstress Zimelidine (n 5 6), and 6) Stress Zimelidine (n 5 6). One-way ANOVA indicated significantly elevated basal corticosterone levels in the Stress Saline and Stress Zimelidine groups after 4 weeks of chronic stress (F 5 2.72, df 5 5/30, p , .05). Four weeks of stress significantly decreased 5-HT1A receptor mRNA expression in the CA1 region only. Both desipramine and zimelidine prevented this effect; However, 5-HT1A mRNA levels tended to be lower in the Stress Zimelidine animals compared to the unstressed animals. *p , .05 compared to Nonstress Saline.

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Figure 7. Photomicrographs of glucocorticoid receptor (GR) mRNA (A) and mineralocorticoid receptor mRNA (B) in adjacent rat hippocampal sections. CA1, Ammon’s horn 1; CA2, Ammon’s horn 2; CA3-4, Ammon’s horn 3 and 4; DG, dentate gyrus. Note the relatively lower hybridization signal in the CA2 and CA3-4 subfields for the glucocorticoid receptor.

hybridization to quantify these two receptors in these same animals. As can be seen in Figure 7, both MR and GR mRNA are present throughout the hippocampus, although each displays a distinctive pattern of abundance. GR mRNA was most abundant within CA1 cells, while MR mRNA was most densely labeled across CA2 and the CA2/CA3 border. Thus, although differentially distributed, steroid receptor mRNAs are expressed in all subfields in which 5-HT1A mRNA is evident. Quantification of GR mRNA revealed no significant differences in any of the treatment groups (Figure 8A); however, for MR mRNA (Figure 8B) a two-factor ANOVA revealed a significant effect for stress (p , .05). Rats that were subjected to chronic stress had lower MR

mRNA levels across all subfields: 18% decrease in CA1 (F 5 1.28, df 5 5/30, p 5 .3), 28% decrease in CA2 (F 5 4.39, df 5 5/30, p , .005), 20% decrease in CA3 (F 5 2.62, df 5 5/30, p 5 .05), and 10% decrease in DG (F 5 2.08, df 5 5/30, p 5 .09). Similar to what happened with 5-HT1A, the stressed animals receiving zimelidine showed MR mRNA levels that were consistently lower than the control group (see Figure 8B). Therefore, zimelidine was unable to fully reverse the stress-induced MR mRNA down-regulation in the hippocampus. Desipramine treatment prevented the MR decrease in the stressed animals. When the ratio of MR to GR (MR/GR) was calculated for all groups (Table 1), animals that received CUS had significantly lower MR/GR ratios than all other

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Figure 8. Effect of chronic unpredictable stress performed for 4 weeks on hippocampal glucocorticoid receptor (GR) mRNA levels (A) and mineralocorticoid receptor (MR) mRNA levels (B) obtained from animals that were treated with saline, desipramine, or zimelidine (n 5 6 for each group). Two-factor ANOVA indicated no significant effect of stress or drug treatment on GR gene expression. Mineralocorticoid receptor mRNA levels were significantly decreased by chronic stress in the CA2 and CA3-4 hippocampal regions (see text for details). This effect was prevented with coadministration of desipramine but not zimelidine. *p , .05 compared to Nonstress Saline.

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Table 1. Ratio of Hippocampal Mineralocorticoid Receptors to Glucocorticoid Receptors in the Animals Subjected to Four Weeks of Nonhabituating Chronic Stress While Receiving Antidepressant Treatment

Treatment group Nonstress Saline (24) Stress Saline (24) Nonstress Desipramine (24) Stress Desipramine (32) Nonstress Zimelidine (24) Stress Zimelidine (32)

MR;GR ratio (all hippocampal subfields) (mean 6 SE) 1.22 6 0.13 0.86 6 0.07a 1.27 6 0.14 1.24 6 0.15 1.25 6 0.15 1.13 6 0.13

Number in parentheses denotes number of subfields analyzed in the group. a Significantly different from all groups except Stress Zimiledine group, p , .05.

groups, except the Stress Zimelidine. In other words, CUS caused an alteration in the MR/GR ratio in the hippocampus, which was only partially reversed by zimelidine treatment. We wanted to investigate whether the inability of zimelidine to block the corticosterone increase after CUS was an idiosyncratic effect of this medication, or whether other SSRIs would share this characteristic. Therefore, we performed a small study in which we measured plasma corticosterone in chronically stressed animals injected with fluoxetine (10 mg/kg). An unhandled group was added in this experiment to control for the possible effects of daily saline injections. Fluoxetine, administered concomitantly with CUS, was also unable to prevent the increase in corticosterone levels, and the levels were in fact slightly higher than the Stress Saline group (Figure 9). No differences were observed between the unhandled and saline-injected groups, suggesting that chronic saline injection is not a particularly stressful procedure.

Localization of 5-HT1A, GR, and MR mRNA in Human Hippocampus Hybridization with the 5-HT1A cRNA probe resulted in a strong and detectable signal in human hippocampus (see Figure 10). The highest concentration of 5-HT1A mRNA expression was observed in the granular layer of the dentate gyrus. A positive signal was also detected in the pyramidal cell layer of CA1, CA2, and CA3 subfields, although the mRNA levels were lower than in the DG. Consistent with a report by Burnet et al (1995), CA1 had higher levels of mRNA than CA2 and CA3. To compare 5-HT1A mRNA distribution with 5-HT1A binding, we performed receptor binding autoradiography in hippocampal slides from 2 controls, with the selective ligand [3H]-8-OH-DPAT (Figure 10). In agreement with previous binding studies (Dillon et al 1991), the highest levels of 5-HT1A binding in the human hippocampus were observed in the CA subfields and in the subiculum. The highest levels of binding were present in CA1 and CA2; intermediate levels were present in the pyramidal and molecular layers of CA2 and CA3 subfields. Lower levels of binding were observed in the granular and molecular layers of the dentate gyrus. Therefore, there is a general concordance between the areas expressing 5-HT1A mRNA with the areas showing specific 5-HT1A binding in human hippocampus; however, there are differences in distribution and concentration between mRNA and receptor sites within specific anatomical subfields. When adjacent slides for MR and GR mRNA were examined, it was clear that for the most part the same regions that are positive for 5-HT1A receptors are also positive for MR and GR mRNA, although the relative abundance of mRNA varied depending on the subfield (Figure 11). Analysis of the areas using computerized image analysis revealed that the areas positive for 5-HT1A receptors overlap with 95% of the areas positive for MR

Figure 9. Effect of chronic unpredictable stress performed for 4 weeks on resting plasma corticosterone levels obtained from Unhandled (n 5 6) animals or animals treated with: Saline (n 5 6), Fluoxetine (n 5 6), Stress Saline (n 5 6), and Stress Fluoxetine (n 5 6). One-way ANOVA indicated significantly elevated plasma corticosterone levels in the Stress Fluoxetine group (F 5 3.28, df 5 4/25, p , .05). *Significantly different from all other groups using Fisher PLSD post hoc test.

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Figure 10. Distribution of 5-HT1A mRNA (A) and [3H]-8-OH-DPAT binding (B) in adjacent human hippocampal sections. The highest concentration of 5-HT1A mRNA expression was observed in the granular layer of the dentate gyrus (DG). A positive signal was also detected in the pyramidal cell layers of CA1, CA2, and CA3, although the mRNA levels were lower than in the dentate gyrus. High levels of 5-HT1A binding in the human hippocampus are observed in the CA1 and in the CA2 subfields. Intermediate levels were present in the pyramidal and molecular layers of the CA3 subfield. Moderate levels of binding were also observed in the granular and molecular layers of the dentate gyrus.

mRNA. High concentrations of MR mRNA are observed in the granule cell layer of the dentate gyrus, with moderate concentrations in CA3, and lower concentrations in CA1 and CA2 (pyramidal layer). GR mRNA was mostly detected in the DG (granule cells) and to a lesser degree in CA3, but very low levels were apparent in the other CA subfields. Therefore, quantification of GR mRNA levels was only done in CA3 and DG.

Comparison between Suicides and Controls Table 2 shows the demographic characteristics of the suicide and control subjects. After correcting with P1B15, suicide victims showed lower 5-HT1A mRNA levels than controls (Figure 12A) across all hippocampal subfields, although these decreases only reached significance in the DG by unpaired t test (CA1: 37% decrease, p 5 .06; CA2: 31% decrease, p 5 .06; CA3:

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Figure 11. Distribution of mineralocorticoid receptor (MR) mRNA (A) and glucocorticoid receptor (GR) mRNA (B) in the human hippocampal formation. High concentrations of MR mRNA were observed in the granular cell layer of the dentate gyrus (DG), with lower concentrations in the CA1, CA2, and CA3 pyramidal cell layer. GR mRNA was also detected in the dentate gyrus and to a lesser extent in the CA3 subfield.

49% decrease, p 5 .08; DG: 43% decrease, p 5 .03; df 5 10 for each one). No significant correlations with age (R 5 .1, p 5 .77) or postmortem time (R 5 .3, p 5 .56) were found. Suicide subjects showed a significant (37%) decrease in MR mRNA in CA3 (Figure 12C), compared to controls (t 5 3.04, df 5 10, p 5 .01 by two-tailed t test). The other CA subfields showed a nonsignificant decrease in MR mRNA in the suicides, but no differences were apparent in the dentate gyrus (CA1: 221%, p 5 .1; CA2: 227%, p 5

.08; DG: 23%, p 5 .39); however, for GR mRNA (Figure 12B), no statistically significant differences were detected between the two groups across the subfields measured (CA3: 17% decrease, p 5 .19; DG: 12% decrease, p 5 .33). As was the case for 5-HT1A, no significant correlations with age or postmortem time were detected for either GR or MR mRNA. In control subjects, 5-HT1A and MR mRNA levels were significantly and positively correlated in each subfield (Table 3). GR and MR mRNA levels were also positively

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Table 2. Subjects’ Demographic Characteristics, Postmortem Interval (PMI) in Hours, and Cause of Death Subject 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Age (years)

Sex

PMI

Cause of death

54 44 47 61 22 43 57 43 69 61 22 39

Female Female Female Male Female Male Female Male Male Male Female Male

13 21 16 14 14 5 15 20 22 20 12 10

Drowning g.s.w., chest Hanging Suffocation g.s.w., chest g.s.w., head Bacterial sepsis m.i. g.s.w., neck Vehicular trauma g.s.w., chest m.i.

Suicide Suicide Suicide Suicide Suicide Suicide Control Control Control Control Control Control

g.s.w., gunshot wound; m.i., myocardial infarction.

correlated in the subfields where GR mRNA was detectable. In contrast, there was no correlation between any of the receptor mRNAs in the suicide group. We also found that suicide victims had an MR/GR ratio that was significantly lower than the control group’s, (2.8 6 0.2 vs. 3.5 6 0.3; t 5 21.82, df 5 22, p , .05 by one-tailed t test).

Discussion This series of studies demonstrates that corticosteroids can regulate the serotonin 1A receptor at the molecular level and in an anatomically specific manner. Although we have focused our efforts on a specific serotonergic receptor, it is likely that these studies pertain to other neurotransmitter systems. In fact, the present data illustrate the general principle that circulating hormones play a key role in modulating the complex neurobiological interactions that occur within central circuitry. The main findings of the present studies can be summarized as follows: 1) both in rodent and in human hippocampus, the 5-HT1A receptor is expressed in all subfields that express GR and MR mRNA; 2) chronic stress can down-regulate 5-HT1A receptor binding and gene expression in the hippocampus; 3) this down-regulation of 5-HT1A gene expression is mediated, for the most part, by the elevation in peripheral corticosteroids, since adrenalectomy prevents it from occurring; 4) chronic stress has no effect on the presynaptic 5-HT transporter sites, suggesting that corticosteroids are acting postsynaptically to modify 5-HT1A gene expression; 5) both imipramine and desipramine prevent stress-related 5-HT1A down-regulation, possibly by decreasing the stress-induced corticosteroid elevation; 6) specific serotonin reuptake inhibitors (zimelidine and fluoxetine) are unable to prevent the stress-induced rise in plasma corticosteroids; this failure to block the rise in corticosterone is associated

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with an inability to fully prevent down-regulation of 5-HT1A receptor gene expression; 7) chronic stress is associated with an alteration in the MR/GR ratio in the hippocampus; the changes in MR/GR ratio are prevented if chronically stressed rats are treated with antidepressants; and 8) suicide victims, with a history of major depressive disorder, show 5-HT1A, MR, and GR gene expression changes in the hippocampus similar to those found in animals subjected to chronic unpredictable stress.

Anatomical Localization The first and most obvious indication that steroids and serotonin receptors may interact within the brain is their anatomical colocalization in key brain structures. Using in situ hybridization, to visualize specific mRNA species in an anatomical context, we were able to identify 5-HT1A receptor producing neurons in specific hippocampal subfields that also contain very high concentrations of glucocorticoid and mineralocorticoid receptor mRNAs. The close association between 5-HT1A, GR, and MR mRNAs in these densely packed neuronal groups, although highly suggestive, does not absolutely confirm the colocalization of these species in the same neurons; however, peripheral electrophysiological studies indicate that these receptors are colocalized, at least, in pyramidal cells of the CA1 subfield (Joels et al 1991). Direct confirmation of this cellular colocalization can be done utilizing combined radioactive and nonradioactive (colorimetric) in situ hybridization to simultaneously visualize two mRNA species in the same cell. What is the importance of the anatomical localization of these molecules? The hippocampal formation is a key limbic region that participates in the modulation of cognition, mood, and behavior (Isaacson 1974). In addition, the presence of GR and MR receptors, as well as a multitude of physiological studies in animals, implicate the hippocampus as an area where corticosteroids act to modulate the activity of the limbic– hypothalamic–pituitary–adrenal axis, thereby controlling their own release (Herman et al 1989b; Sapolsky et al 1984). Thus, the hippocampus represents a logical anatomical substrate in which to study steroid–serotonin receptor interactions and their possible relevance to disorders of mood and behavior.

Effect of Chronic Stress on 5-HT1A A commonly used strategy to investigate the effect of steroids on hippocampal GR and MR consists of removing the adrenal glands and therefore eliminating circulating corticosteroids. GR and MR respond to this manipulation by up-regulating their mRNA levels in the hippocampus (Vazquez et al 1993), thereby suggesting that steroids

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Figure 12. Densitometric analyses of 5-HT1A receptor (A), glucocorticoid receptor (B) and mineralocorticoid receptor (C) in the hippocampus obtained from suicide victims and matched controls. In addition to the analysis for the genes of interest, an in situ hybridization was performed in adjacent sections using cyclophyllin (P1B15) mRNA. Thus, each bar graph presents 5-HT1A, GR and MR mRNA analyses after correction with P1B15. The numbers beneath each hippocampal subfield label indicate the percent reduction in each region, compared to control levels. A two factor ANOVA, followed by t test (see text for details) showed a significant decrease in 5-HT1A mRNA levels in the dentate gyrus of suicide victims. A similar decrease was observed for MR in the CA3 pyramidal cell region. *p , 0.05 by student’s t test.

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Table 3. Results of Simple Regression Analysis between the Different Receptor mRNAs in Controls and Suicides Using All Hippocampal Subfields Controls Y-5-HT1A mRNA

X-MR mRNA R: .536 R : .287

F test: 8.861 p 5 .007

X-GR mRNA

Y-5-HT1A mRNA

2

R: .150 R : .023

F test: 0.230 p 5 .642

X-GR mRNA

Y-MR mRNA

R: .829 R2: .688

F test: 22.034 p 5 .001

2

Suicides X-MR mRNA R: .041 R2: .002

Y-5-HT1A mRNA F test: 0.037 p 5 .850

X-GR mRNA

Y-5-HT1A mRNA

R: .045 R2: .002

F test: 0.020 p 5 .891

X-GR mRNA

Y-MR mRNA

2

R: .395 R : .156

F test: 1.851 p 5 .204

modulate these receptors and exert a tonic inhibition on their gene expression. By using the same strategy to study 5-HT1A regulation in the hippocampus, several investigators have shown that this receptor is also sensitive to circulating corticosteroids. Administration of low doses of corticosterone after adrenalectomy decreases 5-HT1A binding and mRNA up-regulation to baseline levels in all hippocampal subfields (Chalmers et al 1992). At low doses, corticosterone occupies MR selectively. Dexamethasone, which preferentially binds to GR, is less effective in decreasing 5-HT1A mRNA levels after adrenalectomy, and only in the CA2 and CA3 subfields (Chalmers et al 1994). The predominant role of MR in modulating hippocampal 5-HT1A under basal conditions has been confirmed by Meijer and De Kloet (1994) and by Kuroda et al (1994), using specific pharmacologic agents. Steroid modulation of 5-HT1A gene expression in the hippocampus also exhibits some degree of receptor specificity, since 5-HT2C and D1 receptor mRNAs are not significantly regulated by adrenalectomy. Corticosteroid regulation of 5-HT1A gene expression in the hippocampus is also anatomically specific, since no changes in presynaptic 5-HT1A mRNA expression are evident in the dorsal raphe (Chalmers et al 1992). Adrenalectomy studies indicate that 5-HT1A hippocampal receptors are under tonic inhibition by corticosteroids, and this effect is predominantly mediated by MR (Meijer and De Kloet 1994). We wanted to investigate what would be the effect of pathologically elevated corticosteroid levels on 5-HT1A. We chose the chronic unpredictable

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stress paradigm as a more physiological preclinical model to test 5-HT1A regulation, because it produces a persistent activation of the LHPA axis similar to that found in depressed patients (Armario et al 1988; Katz and Sibel 1982). We found in the present study that chronic unpredictable stress down-regulates both 5-HT1A gene expression and receptor binding in the hippocampus. The decrease in 5-HT1A gene expression and binding is small but significant, and consistent across various experiments. Watanabe et al (1993) have demonstrated a decrease in hippocampal 5-HT1A binding following chronic restraint stress. Our results, using a different type of stress, are consistent with Watanabe’s findings and suggest that the decrease in binding may be the consequence of a reduction in 5-HT1A gene expression. Although the direction of the effect is in agreement with what will be expected based on the adrenalectomy data, these results are somewhat remarkable in view of the fact that hippocampal 5-HT1A receptors, as assessed by ligand binding, have proven to be remarkably resistant to homologous regulation, even after complete loss of serotonin stimulation as a result of neurotoxic lesion (Verge et al 1986). Similarly, significant depletion of hippocampal serotonin appears to produce only relatively small, or no changes in 5-HT1A receptor sites within hippocampal subfields (Pranzatelli 1994). It is, therefore, most likely that changes in both 5-HT1A mRNA expression and 5-HT1A binding observed in response to stress do not result from stress-induced changes in serotonergic activity, but rather reflect a direct effect on hippocampal neurons. An important observation is that while chronic unpredictable stress can decrease 5-HT1A receptor binding and gene expression below baseline, administration of low doses of corticosterone and dexamethasone alone can only maintain these parameters at control levels. This suggests that in chronic stress, both MR and GR are occupied by the high steroid levels, thereby leading to a synergistic effect impossible to achieve by occupation of either receptor alone (Meijer and De Kloet 1994). This possibility is supported by the fact that swim, a milder form of stress that causes lower corticosterone levels, did not affect 5-HT1A receptors in the hippocampus, either acutely or under chronic conditions. Chronic swim stress does not result in an induction of pituitary POMC mRNA and peptide content (Lopez et al 1994b), which is a compensatory biochemical event indicating prolonged and persistent ACTH secretion (Lopez et al 1991). Therefore, it is likely that the LHPA activation during swim stress is either not persistent enough or not severe enough to modulate 5-HT1A mRNA. The severity or persistence of the stress may be a very important factor, since we have shown that significant increases in POMC mRNA and POMC peptide are present in the pituitaries of suicide

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victims (Lopez et al 1992), consistent with a history of prolonged LHPA activation in these subjects. It is also possible that stress-activated brain circuits may contribute to this 5-HT1A down-regulation, independently of corticosteroid levels (i.e., a “central” effect); however, our results with chronically stressed animals that were adrenalectomized indicate that peripheral corticosteroids are the principal mediator of this phenomenon. Thus, preventing the corticosteroid response to stress abolishes the effect on 5-HT1A receptor. This of course does not rule out the likely possibility that stress may be simultaneously affecting other neural systems directly (such as for example CRH) without the need of corticosteroid mediation.

Effect of Antidepressant Medications on the LHPA Axis and 5-HT1A Receptor Interestingly, and of relevance to clinical psychiatry, when imipramine is administered concomitantly with chronic unpredictable stress, down-regulation of 5-HT1A receptors is prevented. The fact that imipramine is also profoundly decreasing corticosterone levels in chronically stressed rats provides indirect evidence that this effect may be steroid mediated. Furthermore, administration of imipramine alone does not cause a quantifiable up-regulation of 5-HT1A receptors, at least when administered for 2 weeks. The mechanisms responsible for imipramine-induced changes in corticosteroid secretion are unknown. It is possible that alterations in central catecholaminergic, serotonergic, and/or cholinergic systems is involved; however, it may be relevant that hippocampal MR and GR receptors have been reported by some investigators to up-regulate in response to long-term imipramine treatment (Brady et al 1991; Peiffer et al 1991; Seckl and Fink 1992). Such a response may enhance negative feedback control and consequently lower circulating corticosteroid levels. Intriguingly, we found that zimelidine and fluoxetine, two specific serotonin reuptake inhibitors, are unable to prevent the stress-induced elevation in corticosterone levels. At least for zimelidine, this is associated with an impairment in the ability of this antidepressant to completely restore 5-HT1A gene expression to baseline levels. Desipramine, a potent norepinephrine reuptake inhibitor and a tricyclic, is as effective as imipramine in blocking the corticosterone rise and in preventing the associated 5-HT1A down-regulation. There may be several reasons for this phenomenon. It is possible that the LHPA activation in our particular stress paradigm is more dependent on catecholaminergic than on serotonergic mechanisms; therefore modulation of norepinephrine-related circuits by imipramine or desipramine can block the stress response in our model. It is also possible that tricyclics are more

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effective in this chronic unpredictable paradigm because they have a “broader” biochemical action (e.g., cholinergic, noradrenergic, serotonergic). The LHPA response to a severe, more chronic, or unpredictable stressor is likely to involve several neurotransmitter systems. Therefore, a compound that modulates a single neurotransmitter may not be able to prevent, or may even potentiate, the LHPA axis activation in response to a chronic stress. The failure of an antidepressant to restore peripheral corticosteroids to baseline levels may therefore impair its ability to correct a “central” monoaminergic deficit. It is possible that higher doses of SSRIs are necessary to achieve this effect in our animal model; however, the antidepressant doses used in our studies were selected from the doses shown in animal models to correlate with antidepressant activity. Because the stress-induced 5-HT1A reductions in the zimelidinetreated animals seem to be of a lesser magnitude than in the saline-treated animals, it will be interesting to examine whether longer treatment regimes and/or higher doses could prevent the 5-HT1A down-regulation, even in the presence of high corticosteroid concentrations. These preclinical observations may have important clinical correlates and may shed light into the mechanisms of treatment resistance. For example, persistence of LHPA overactivity after antidepressant administration in depressed patients has been associated with relapse and poorer treatment outcome (Greden et al 1983). In addition, some clinical studies have found that tricyclics are more effective than specific serotonin reuptake inhibitors in the treatment of melancholia (Roose et al 1994), and that venlafaxine, an antidepressant with both norepinephrine and 5-HT reuptake activity, was more effective than fluoxetine in treating melancholic depression (Clerc et al 1994). Since melancholia and severity of depression are associated with a higher incidence of hypercortisolemia (Kathol et al 1989), it is tempting to speculate that the presence of LHPA overactivity in this population may be contributing to the relative resistance to SSRI treatment. Interestingly, many “augmentation” strategies in treatment-resistant patients are in effect attempts to “broaden” the biochemical profile of the pharmacologic treatment, which may be more effective in reversing LHPA overactivity than a treatment whose main impact is in a single neurotransmitter system.

Effect of Stress and Antidepressant Medications on Mineralocorticoid and Glucocorticoid Receptors The hippocampus is exquisitely sensitive to circulating corticosteroids. Appropriate levels of corticosterone appear to be necessary for the normal morphology and ultimate survival of adult rat hippocampus pyramidal neurons. Chronic administration of glucocorticoids alters

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the morphology of CA3 pyramidal cells, as evidenced by atrophy of apical dendrites, and decreased number of branch points (McEwen 1991). Interestingly, repeated stress may also produce these changes (McEwen 1991; Sapolsky et al 1986). In aged animals, loss of hippocampal cells has been associated with a failure to terminate the corticosteroid response to stress (Sapolsky et al 1986). This impaired feedback mechanism has therefore been postulated to be the consequence of the loss of corticosteroid receptors in the hippocampus. In our hands, chronic unpredictable stress caused modest decreases in MR mRNA levels in the hippocampus, and no changes in GR mRNA concentration; however, and perhaps more important from a functional standpoint, chronic stress was associated with a decrease in the MR/GR ratio, compared to control animals. These changes in MR/GR ratio may reflect disturbances in MR/GR homeostasis and could have important consequences to the control of LHPA secretion and to hippocampal excitability. For example, decreases in MR/GR ratio have also been found in the hippocampus of neonatal rats subjected to maternal separation (Vazquez et al 1996). This altered MR/GR ratio in the developing rat is associated with an exaggerated LHPA response to stress and a failure to adequately turn off the corticosteroid response (Vazquez et al 1996). Therefore, it is possible that an alteration of the MR/GR ratio due to chronic unpredictable stress will also result in an impairment of glucocorticoid feedback, chronically elevated glucocorticoid levels, and a subsequent impairment in 5-HT1A receptor number. Some studies have found increases in MR and/or GR mRNA after 2 weeks of antidepressant treatment (Peiffer et al 1991; Seckl and Fink 1992), but others have not found changes in this short time frame (Brady et al 1991; Lopez et al 1994b). In the experiments reported here, we found no changes in GR and MR mRNA in the hippocampus after 4 weeks of desipramine treatment alone; however, we found that desipramine administration restores the abnormal MR/GR ratio in stressed animals to controls levels. This may represent one of the multiple mechanisms by which antidepressants may enhance feedback and maintain low corticosterone levels, even in the presence of stress. Zimelidine administered to chronically stressed animals also had some effect in the MR/GR ratio, but apparently not enough to offset the “drive” of the LHPA axis, at least within the time frame of our study. It is important to note that most animal studies investigating antidepressant effects are performed under baseline conditions; however, in humans with depression, antidepressants are given to an organism that is under an “altered” (one could argue “stressed”) condition. As it is clear from our chronic stress study, the effect of the antidepressant on hippocampal 5-HT1A levels and MR/GR

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ratio was only evident during chronic stress; it prevented 5-HT1A down-regulation and prevented changes in MR/GR ratio. Little or no effect was observed during the baseline condition. Therefore, investigating the effect of antidepressant medications on relevant monoamine receptors under conditions of chronic stress may be closer to the clinical situation, as a significant number of patients with depression show evidence of LHPA overactivity.

5-HT1A, MR, and GR Gene Expression in Suicide Victims Our mapping study in humans shows that there is a general concordance between the areas expressing 5-HT1A mRNA with the areas showing specific 5-HT1A binding in human hippocampus. The hippocampal areas rich in 5-HT1A also have high concentrations of MR and, to a lesser extent, GR. This suggests that in humans, as in rats, stress hormones are capable of directly affecting 5-HT receptors in the limbic system. We have also shown that in a small group of medication-free suicide victims with a history of major depression, 5-HT1A receptor mRNA levels are decreased in the hippocampus, compared to an age- and postmortem interval-matched control group. These mRNA changes seem to be receptor specific, since no differences were detected in GR mRNA levels. In general, MR mRNA levels tended to change in the same direction as 5-HT1A, but failure to find a significant difference may be due to the small number of subjects. Interestingly, MR and 5-HT1A mRNA levels are significantly correlated in controls, but not in suicides. As discussed above, under baseline conditions, hippocampal 5-HT mRNA levels are under tonic inhibition by MR (Chalmers et al 1992; Kuroda et al 1994; Meijer and De Kloet 1994). It is possible that this relationship is altered in pathological conditions, such as depression and/or suicide. The fact that we also find in suicide victims a decrease in the MR/GR ratio, similar to the chronically stressed animals, is further evidence of an alteration in hippocampal homeostasis. Because of the small number of subjects, these results should be interpreted cautiously and should be replicated with a larger sample; however, these results are consistent with our animal studies using chronic stress. These observations add a very interesting dimension to the interaction between serotonin and the LHPA axis; if hippocampal 5-HT1A receptors are partially mediating mood changes and/or predisposing psychiatric patients to suicide, it is possible that the presence of LHPA overactivity in this population may be contributing to, or exacerbating, a “dysfunction” of brain 5-HT receptors. It is also possible that an altered MR/GR ratio may be

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connected to the impairment in LHPA feedback mechanisms, which has been reported (Young et al 1991). Chronically elevated plasma steroids could also be affecting other 5-HT receptors believed to be involved in the control of mood. For example, 5-HT2A receptors are increased in the cortex of rats exposed to chronic social stress (McKittrick et al 1995). These 5-HT2A changes are in the same direction as the 5-HT2A changes found in the cerebral cortex of suicide victims (Arango et al 1990; Mann et al 1989). Arango et al (1995) have reported an increase in 5-HT1A binding in the prefrontal cortex of suicide victims. Although this is in apparent contradiction with our findings, the results of these two studies are not necessarily inconsistent. Regulation of 5-HT1A receptors may be different in different brain regions. For example, hippocampal 5-HT1A receptors, due to their colocalization with MR and GR (Biegon 1990; De Kloet et al 1986; Joels et al 1991; Kuroda et al 1994), may be more sensitive to circulating steroids, while receptors in the prefrontal cortex may be less responsive to steroids and more responsive to changes in local 5-HT levels. In addition, the population in Arango’s study are suicide victims (not necessarily with affective illness); our subjects are a subpopulation of suicides with a history of depression. Serotonin 1A receptor binding has in fact been found to be decreased in the pars opercularis and temporal lobe of elderly depressives (Bowen et al 1989). This last observation is of interest regarding the potential relationship between hypercortisolemia and 5-HT1A receptors, since aging is associated with worse impairment of LHPA axis function (Sapolsky et al 1986).

Conclusions Our animal studies show that stress, through corticosteroid secretion, negatively modulates 5-HT1A receptors in the limbic system and is associated with alterations of corticoid receptor balance. The fact that we find similar changes in the hippocampus of suicide victims with a history of depression suggests that the same mechanisms may be operant in humans. These observations may have important implications for the pathophysiology and treatment of affective disorders. Corticoid-mediated 5-HT receptor down-regulation may be one of the mechanisms by which stressful events can precipitate depressive episodes in some (genetically) vulnerable individuals (Post 1992). Another implication is that altered 5-HT levels or metabolism do not necessarily have to be present for 5-HT receptor abnormalities to occur. Based on our data and that of others (Kuroda et al 1994; Meijer and De Kloet 1994), it is apparent that specific 5-HT receptors may be directly regulated in response to alterations of corticosteroid levels,

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which can result from repeated stress. Thus, in depressed patients normal levels of serotonin and its metabolites may not necessarily reflect normal central 5-HT activity. Finally, as has also been suggested by others (Holsboer and Barden 1996), the present study demonstrates that the efficacy of antidepressant treatment may be dictated, in part, by its ability to decrease the activity of the LHPA axis. Keeping in mind these observations, it is possible to construct a model of the relationships between stress hormones, 5-HT receptors, and mood. If hypersecretion of endogenous corticosteroids can affect the 5-HT1A receptor; is it possible that the hypercortisolemia present in some depressed patients may be contributing to the affective disturbance? This possibility has been raised by some investigators (Kathol 1985; Wolkowitz et al 1992) based on the parallelisms between Cushing’s syndrome and depression. Interestingly, patients with major depression who are resistant to antidepressant treatment experienced a dramatic improvement when they received steroid suppression agents (Murphy et al 1991; Wolkowitz et al 1993). Additionally, patients who respond to antidepressant treatment, but who continue to show cortisol nonsuppression after dexamethasone administration, have a much greater risk of relapsing than patients who show dexamethasone suppression (Greden et al 1983). Therefore, antidepressant agents, in addition to having a direct effect by correcting the central neurotransmitter “disturbance,” may also be improving depression indirectly by decreasing cortisol hypersecretion. Although it is clear that corticosteroids can regulate 5-HT receptors, it is also important to remember that regulation can exist in the other direction. Acute administration of 5-HT1A and 5-HT2 agonists causes release of ACTH and corticosteroids, and destruction of central 5-HT neurons decreases hippocampal GR and MR gene expression (Seckl et al 1990). Therefore, the relationship between corticosteroids and 5-HT in the brain is complex and tightly controlled. We have shown that stress-induced corticosteroid release affects 5-HT receptor function. We cannot rule out, however, that stress can also affect other receptors through non– corticosteroid-mediated pathways (Post 1992). The interplay of these factors may lead to the emergence, or maintenance, of affective symptoms. Similarly, antidepressants can counteract this phenomenon by affecting 5-HT receptor function directly (Blier and de Montigny 1994; Welner et al 1989) and by simultaneously regulating stress-induced corticosteroid secretion. Failure to prevent corticosteroid hypersecretion may be associated with an inability to correct a central 5-HT abnormality and with treatment resistance. This of course does not exclude the possibility that steroids can be simultaneously acting through other systems (e.g., noradrenergic), thereby syn-

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ergistically affecting mood and behavior. The use of modern biochemical and molecular neuroanatomical techniques in postmortem human brain should now allow us to test these hypotheses directly in psychiatric illness. This work was supported by a MIRA/NARSAD Young Investigator Award, a grant from the American Suicide Foundation, and a Scientist Development Award (MH 01164) to JFL, DA09491 to KYL and MH42251 to SJW. The help and intellectual advice of Delia M. Va´zquez, Elizabeth A. Young, John F. Greden, and Huda Akil are gratefully acknowledged.

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