Introduction

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Corticotropin-releasing factor (CRF) is a 41 amino acid peptide that is synthesized and secreted in many regions of the brain, particularly in limbic-associated areas such as the hypothalamus, amygdala, and bed nucleus of the stria terminalis. There is extensive evidence that CRF functions as both a neurohormone and neurotransmitter in coordinating endocrine, autonomic, and behavioral aspects of the stress response (Owens and Nemeroff, 1991). Major depression is a condition that has been associated with a predisposing influence of major stressors, particularly early in life, and with neurochemical and neuroendocrine findings of CRF hypersecretion (Arborelius et al., 1999). On the basis of these observations, we hypothesized that antidepressant agents, whose ultimate therapeutic mechanism(s) of action are poorly understood, may act in part by reducing CRF synthesis or secretion, either tonically or in response to stress.

There is clinical evidence that antidepressants exert effects on CRF systems. Hypothalamic-pituitary-adrenal (HPA) axis abnormalities, hypothesized to result at least in part from CRF hypersecretion and elevated cerebrospinal fluid (CSF) CRF concentrations in depressed patients, have been reported to normalize after antidepressant treatment or electroconvulsive therapy. However, in human studies it is currently impossible to delineate the anatomical basis of antidepressant/CRF interactions nor the neurobiological step(s) (e.g., gene expression, peptide processing and release) at which these interactions take place.

In this series of experiments, we sought to determine regions of the rat brain in which CRFergic neurotransmission may be influenced by antidepressant administration. Chronic, rather than acute, drug effects were examined in view of the fact that therapeutic efficacy of these drugs requires a delay period of several weeks (Owens et al., 1996). There was no drug washout period, because we were interested in neuroregulation that may occur in patients during antidepressant treatment, rather than in the absence of the drug. CRF and CRF receptor gene expression, and CRF receptor density were measured in areas of peak expression, and CRF peptide concentration was determined in the median eminence, amygdala, and locus coeruleus. These brain regions were selected on the basis of previous experiments in which experimental manipulations altered CRF concentration, on functional associations with the HPA axis, and/or hypothesized roles in the mechanism of action of antidepressant drugs. Measurements of CRF receptor expression and density were included because changes in these parameters, in response to antidepressant treatment and/or synaptic CRF release (i.e., receptor up-regulation) could potentially modify CRFergic neurotransmission. Measures of HPA axis activity at the time of sacrifice were also obtained.

A major property of CRF-containing neurons, including but not limited to those in the hypothalamus, is the capacity to increase neurotransmitter synthesis and secretion in response to various forms of stress. Major depression occurs disproportionately in individuals with exposure to significant early and/or chronic life stressors and is accompanied by symptoms of anxiety. It is plausible that increased CRF and HPA axis activity noted in a substantial subset of these patients (Arborelius et al., 1999) represents a chronic hypersensitivity to stress. Therefore, in addition to measuring basal gene expression and peptide concentration, we determined whether the effects of acute or chronic stress on these indicators of CRF neuronal function were altered by prior antidepressant treatment. To this end, a probe directed against intronic, or "heteronuclear", CRF RNA was most sensitive in detecting the rapid transcriptional response to acute stressors, whereas a messenger RNA probe was used to measure total CRF gene expression under basal conditions or after a 2-week, intermittent stress paradigm. The purpose of these studies was not to construct an animal model of depression, but to test for antidepressant effects on CRF neurons that might not be detectable under resting conditions but that could be involved in the therapeutic effect of these agents.

	Materials and Methods

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Subjects. Adult, male Sprague-Dawley rats (Harlan Bioproducts for Science, Indianapolis, IN) weighing 250 to 300 g at the beginning of each experiment were handled daily. Except as otherwise noted, rats were housed three per cage in a humidified room with a 12-h light/dark cycle (lights on at 7:00 AM), and lab chow and water available ad libitum. Rats were killed by decapitation between the hours of 8:00 and 10:00 AM, within 5 min of removing the first rat from each cage. All procedures, including stressors, were approved by the Emory University Institutional Animal Care and Use Committee and were conducted in accordance with guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Antidepressant Administration. Three groups of antidepressant-treated rats were used in the experiments herein. Doses (at the time of sacrifice) for each experiment are provided with each result. Treatment duration was 27 days (venlafaxine/immobilization experiment) or 26 days (other two experiments). In each case, rats weighing 250 to 300 g were anesthetized with methoxyflurane and implanted in the subcutaneous, infrascapular region with osmotic minipumps (Alza, Palo Alto, CA) containing antidepressant drug or vehicle. Minipumps were manipulated each day to prevent adhesions, and were rotated 180° under anesthesia at least 1 week before sacrifice. There was no drug washout period before sacrifice. In the case of fluoxetine treatment, it was necessary to implant serial model 2 ML2 pumps rather than a single 2 ML4 minipump due to solubility limitations.

Stress Experiments. In two acute stress experiments, rats were subjected to immobilization in wire mesh or swim stress, immediately before sacrifice. The latter consisted of individual placement in transparent tanks (30 cm in diameter, 60 cm in height), half-filled with fresh 30 ± 2°C water for 15 min. Swim stress was used in the second experiment because it was found in an intermediate pilot study (data not shown) that PVN CRF hnRNA synthesis is stimulated to a greater and more reproducible degree after this stressor.

In Situ Hybridization Histochemistry. Unfixed rat brains were sectioned coronally at 15 to 20 µm, thaw-mounted onto Fisher Scientific (Pittsburgh, PA) Superfrost Plus slides at a sampling interval of 100 to 105 µm, and stored at 80°C. Slides were brought to room temperature, postfixed in 4% paraformaldehyde, pH 7.5, and rinsed in phosphate-buffered saline. The remaining steps, including proteinase K treatment, acetylation, dehydration, overnight hybridization in a humidified chamber (60-62°C), RNase A digestion and washes to a final stringency of 0.1× standard saline citrate, 0.1% dithiothreitol, 60°C for 30 min, were performed as previously described (Simmons et al., 1989). Between 1.4 and 2 × 10⁶ cpm of probe solution were used per slide.

CRF Radioimmunoassay. Brains were thawed on a chilled glass plate and dissected into brain regions according to anatomic landmarks. The locus coeruleus was obtained as a bilateral micropunch dissection (0.8 mm in diameter, 0.6-0.9 mm in thickness per side); two locus coeruleus samples from the same group were pooled in each tube. CRF was extracted from tissue dissections in 1 M HCl with protease inhibitors as previously described (Ladd et al., 1996). CRF content was determined in duplicate by radioimmunoassay, using a rat/human CRF antiserum obtained from Peninsula Laboratories (Belmont, CA) (final dilution 1:23,000). Synthetic peptides were used for the CRF standard (Bachem, Philadelphia, PA), and ¹²⁵I-⁰Tyr-r/hCRF tracer (PerkinElmer Life Sciences, Boston, MA). The limit of sensitivity was 2.5 pg/tube. Data were normalized with respect to protein content as determined by the Lowry method with bovine serum albumin as the standard (Lowry et al., 1951).

CRF Receptor Autoradiography. Brain sections (20 µm) were incubated with 0.1 nM [¹²⁵I-Tyr⁰]-sauvagine (PerkinElmer Life Sciences), a radioligand with similar affinity to both the rat CRF₁ and CRF₂ receptors (Primus et al., 1997); doubling this concentration did not yield any additional specific binding. Serial sections were processed with 0.1 µM CP-154,526 (Pfizer, Groton, CT), a specific CRF₁ receptor antagonist, with CP-154,526 plus 1 µM unlabeled sauvagine, or in the absence of competing ligand. Slides were apposed to X-ray Film (Biomax). Image analysis was conducted as described in the in situ hybridization section, by using ¹²⁵I (Amersham Biosciences, Inc.) rather than ¹⁴C standards. Subtraction was used to determine CRF₁ or CRF₂ receptor signal.

Corticotropin (ACTH) and Corticosterone Assays. Plasma concentrations of ACTH were determined using a two-site immunoradiometric assay (Nichols, San Juan Capistrano, CA). Serum concentrations of corticosterone were determined using a commercially available radioimmunoassay (ICN Biochemicals, Costa Mesa, CA). Intra- and interassay variability for these assays were less than 8%.

Monoamine Transporter Ligand Binding Assays. All ex vivo estimates of transporter occupancy used standard binding protocols modified from Owens et al. (1997).

Serum Drug Determinations. Drug extractions and high-performance liquid chromatography determination were adapted from previously described methods (Stowe et al., 1997). In brief, antidepressant drugs and internal standards (fluoxetine and paroxetine: citalopram; venlafaxine: Wy45818) were extracted over 1-ml C₁₈ columns (Lida, Nalge nunc, Pittsburgh, PA), desiccated under a nitrogen stream, and dissolved in 120 µl of mobile phase before injection onto a 3-µm C₁₈ column (Keystone, Bellefonte, PA), with ultraviolet detection at 215 nm.

Statistical Analysis. Data were analyzed by Student's t test or one-way or two-way analysis of variance (ANOVA) where appropriate (P = 0.05). In the second, acute stress/antidepressant/CRF hnRNA experiment (Table 1), two-way ANOVA was not performed due to violation of the equal variance assumption. HPA axis hormone concentrations were transformed to common logarithms before statistical analysis due to nonhomogeneity of variance. Several outliers >2.5 S.D. from the mean (raw values) were discarded from the HPA axis data; doing so did not change any "nonsignificant" results to "significant" or vice versa.

	Results

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Antidepressant Effects on CRF hnRNA Response to Acute Stress. Pilot experiments established reliable increases in CRF hnRNA expression in the PVN after 15- or 30-min immobilization in wire mesh, whereas increases in exonic, CRF mRNA at later time points were less consistent. CRF hnRNA expression in the central nucleus of the CeA was undetectable.

View larger version (182K): [in this window] [in a new window]   Fig. 1.   Effect of antidepressant pretreatment and/or immobilization on PVN CRF hnRNA. Rats received saline (A and B) or venlafaxine (21 mg/kg/day; C and D) for 27 days. Immediately before sacrifice, rats were left undisturbed (A and C) or were immobilized for 30 min (B and D). PVN CRF hnRNA was determined by in situ hybridization histochemistry; representative samples closest to the mean within each group are shown.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of antidepressant pretreatment and/or immobilization on PVN CRF hnRNA. Rats received saline or venlafaxine (21 mg/kg/day) for 27 days. Immediately before sacrifice, rats were left undisturbed or were stressed by immobilization for 30 min. CRF hnRNA expression was measured in every fifth 20-µm section through the PVN by in situ hybridization histochemistry (three nuclei averaged per brain). **, significant effect of immobilization (P < 0.001); *, stress-venlafaxine interaction (P < 0.05).

Antidepressant Effects on CRF mRNA Response to Chronic Stress. In saline-pretreated rats, chronic stress resulted in a 29% increase in mean PVN CRF mRNA expression, but no such increase occurred in venlafaxine (30 mg/kg/day)-treated rats (Table 2; P < 0.05, interaction, two-way ANOVA). No drug or stress effects were noted in the CeA. In the dorsolateral but not the ventral portion of the BNST, CRF mRNA expression was higher in venlafaxine-treated animals relative to the saline group (P < 0.05, two-way ANOVA); there was no effect of stress or a drug/stress interaction.

Antidepressant and Stress Effects on CRF Peptide Concentration. Chronic, variable stress resulted in reduced CRF content (24%) in both the median eminence and locus coeruleus (Table 3; P < 0.05, two-way ANOVA). There was no statistically significant effect of venlafaxine treatment (30 mg/kg/day) or any drug/stress interaction. Unexpectedly, a 19% increase in amygdaloid CRF concentration was noted in venlafaxine-treated rats, without any stress effect or drug-stress interaction.

Antidepressant and Stress Effects on CRF Receptor Binding and CRF Receptor mRNA Expression. Neither chronic, variable stress, nor venlafaxine treatment (30 mg/kg/day) had any detectable effect on CRF₁ mRNA expression in the paraventricular nucleus, frontal cortex, or basolateral amygdala, nor on CRF₂ mRNA expression in the ventromedial nucleus of the hypothalamus or the lateral septum (Table 4). Chronic administration of venlafaxine, fluoxetine, reboxetine, or tranylcypromine did not alter type 1 CRF receptor binding in the frontal or parietal cortex, basolateral or centromedial amygdala, or type 2 CRF receptor binding in the ventromedial nucleus, lateral septum, or centromedial amygdala (Table 5).

Antidepressant and Stress Effects on HPA Axis Activity. The effect of chronic antidepressant administration on HPA axis activity response to an acute stressor was tested in two experiments, by using various antidepressant agents (venlafaxine, fluoxetine, reboxetine, tranylcypromine) (Table 6). In each case, immobilization or swim stress resulted in greater than 10-fold, mean increases in plasma ACTH and corticosterone concentrations, 15 to 30 min after initiation of the stressor. There were no antidepressant effects on basal or poststress values.

	Discussion

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Previous studies have reported that basal PVN CRF mRNA is decreased by 30 to 67% (Brady et al., 1991, 1992; Aubry et al., 1999) or is unchanged (Jensen et al., 1999) after chronic antidepressant treatment. Consistent with the latter study, we found that administration of a variety of antidepressant agents produced no effect on basal CRF hnRNA or mRNA expression in the PVN. Although there were differences in drug doses and durations of treatment among investigators, these are unlikely to account for the discrepancies. The doses in the present experiments were validated by several approaches, including measurement of monoamine transporter occupancy and serum drug concentrations. The approximately 4-week treatment period was comparable to the duration in one of the studies that demonstrated a drug effect in nonstressed rats (Aubry et al., 1999), and is well beyond the 10- to 14-day period required for virtually all known neurochemical effects of antidepressant administration (Owens et al., 1996). Methodological differences related to animal handling may have produced the disparate results. For example, in some of the studies (Brady et al., 1991, 1992), rats may have been chronically stressed due to twice-daily injections for 8 weeks. If so, a protective effect of antidepressant drugs on stress-induced CRF transcription may actually have been observed in these previous studies, consistent with the current data set.

Whereas antidepressant administration was without effect on basal CRF gene expression, stress-induced CRF gene expression in the PVN was attenuated by chronic antidepressant treatment. The increase in CRF hnRNA production after either immobilization or swim stress was diminished after antidepressant pretreatment (Fig. 2; Table 1). In the second study, statistical significance was reached in the venlafaxine and tranylcypromine groups, but not within the fluoxetine or reboxetine groups, although the same trend was present. A similar, statistically significant interaction also existed between venlafaxine administration and chronic stress with respect to PVN CRF mRNA density (Table 2). This same interaction was also observed in a study using the serotonin uptake inhibitor paroxetine (data not shown). In both cases, the effect of chronic stress in vehicle-treated rats was quite modest (29 and 11%, respectively); two-way ANOVAs were significant for interactions but not overall stress effects. More robust effects of chronic stress on CRF mRNA expression, as well as HPA axis activity have been documented after a longer-term stress regimen (Herman et al., 1995). Nevertheless, the current results support the argument that antidepressant treatment desensitizes PVN neurons to stress-induced CRF gene expression.

We hypothesized that an antidepressant effect on CRF neurons in the PVN could be associated with effects on CRF concentration in the median eminence and HPA axis hormone concentrations in plasma. However, in these studies there was no effect of antidepressant administration on basal, median eminence CRF concentration, on depletion of the peptide after chronic stress, on adrenal gland hypertrophy caused by chronic stress, or on plasma ACTH or corticosterone concentration in nonstressed or acutely stressed rats. In comparison, previous investigators have reported either a decrease (Fadda et al., 1995) or no change (Heilig and Ekman, 1995) in CRF concentration in the entire hypothalamus after chronic antidepressant treatment. We also observed no significant effect of chronic stress on plasma ACTH and corticosterone concentrations at the time of sacrifice, although adrenal gland hypertrophy was evident in the stressed rats (Table 7). Previous reports on the capacity of chronic, variable stress to affect these measures have varied (Chappell et al., 1986; Garcia-Marquez and Armario, 1987; Herman et al., 1995); varying intensities of the stressors as well as the stress level at the time of sacrifice may have accounted for these differences. In summary, a functional consequence of the antidepressant-stress interaction in the PVN noted in this study has yet to be demonstrated. It is plausible that stress activation of CRF and the HPA axis is more frequent and chronic in depressed patients than in the stressed rats, and a blunted effect of stress on CRF gene expression may contribute toward antidepressant normalization of HPA axis activity observed in these patients (Arborelius et al., 1999). Evidence for such a functional interaction in a rat model may require a longer-term or more intensive chronic stress regimen, or measurement of median eminence CRF concentration at various time points after an acute stressor. The latter experiment may prove difficult, because either increases or decreases in median eminence CRF concentration have been reported (Chappell et al., 1986; Moldow et al., 1987; Haas and George, 1988) at various time points after acute stress, and in some cases blockade of axonal transport or protein synthesis was necessary to demonstrate CRF depletion after acute manipulations (Haas and George, 1988; Owens and Nemeroff, 1991). Characterization of acute stress-induced changes in CRF peptide concentration in antidepressant-treated rats was not attempted in this study.

A variety of mechanisms may underlie the effect of antidepressant treatment on CRF transcription in the PVN. One possibility is that antidepressant treatment leads to negative regulation of CRF gene transcription by increasing glucocorticoid feedback sensitivity. Antidepressant administration has been shown to increase the expression of type I and II glucocorticoid receptors in the hippocampus (Brady et al., 1991; Seckl and Fink, 1992; Reul et al., 1993); it is not known whether glucocorticoid receptor expression in the PVN is modified by antidepressant administration. Alternatively, antidepressant treatment may influence the intracellular cascade regulating CRF transcription after stress; this pathway likely involves cyclic AMP-responsive element-binding protein (Seasholtz et al., 1988; Kovács and Sawchenko, 1996). In addition, antidepressant treatment may render PVN neurons less susceptible to depolarization and calcium entry, as demonstrated in non-neuronal tumor cells (Schwaninger et al., 1995). Other potential mechanisms for antidepressant regulation of CRF transcription include -adrenergic receptor down-regulation, which occurs in the PVN as in most other brain regions (Duncan et al., 1993) in response to some antidepressant agents, or altered strength of neuronal inputs to the PVN such as projections from noradrenergic locus coeruleus neurons (Palkovits et al., 1980; Nestler et al., 1990).

We also tested the hypothesis that antidepressant administration would reduce CRF synthesis or secretion in extrahypothalamic regions of rat brain. Such an effect could explain the normalization of elevated CSF CRF concentrations after antidepressant treatment of depression (Arborelius et al., 1999). However, we found that CRF gene expression was unchanged in the CeA, ventral BNST (Table 2), or Barrington's nucleus (data not shown) after venlafaxine treatment, and unexpectedly was increased 17% in the dorsolateral BNST (Table 2). Antidepressant treatment did not alter CRF peptide concentration in the locus coeruleus, whereas CRF concentration was slightly increased in the amygdala after venlafaxine administration (Table 3). Previous investigators have found an absence of antidepressant effects on CRF peptide concentration in a variety of brain regions (Tizabi et al., 1985; Heilig and Ekman, 1995). The only two extrahypothalamic effects noted in this series were opposite to the hypothesized direction; their significance and whether they can be ascribed to chance are uncertain.

It should be noted that measurements of peptide content do not distinguish between changes in synthesis versus secretion, and therefore are not definitive indicators of neuronal activity or extracellular fluid peptide concentration. It is possible that balanced changes in CRF synthesis and secretion occurred, and consequently escaped detection in the radioimmunoassays. Despite technical challenges resulting from the size and hydrophobicity of CRF, two groups have reported successful CRF microdialysis in brain regions containing high concentrations of the peptide (Pich et al., 1995; Merali et al., 1998); it remains to be established whether lower synaptic concentrations of CRF can be measured using this method. Ideally, subsequent studies will more precisely elucidate the effects of antidepressant treatment on CRF secretion under resting and perturbed conditions in the same animal. The new MicroPET method that allows for visualization of gene expression in vivo is one such technique.

Another means by which antidepressant drugs could potentially alter CRF neurotransmission is via an effect on CRF receptor function. We and others have found no effect of antidepressant treatment on brain CRF receptor density, by using homogenate binding methods (Grigoriadis et al., 1989; Stout et al., 1996). The current findings extend these results by demonstrating no effect of antidepressant treatment on subtype-specific binding by using receptor autoradiography. Moreover, antidepressant administration was without effect on CRF₁ or CRF_2A receptor gene expression in peak forebrain regions. We did not observe a reduction in amgydaloid CRF₁ receptor mRNA expression, as was previously reported after amitriptyline administration (Aubry et al., 1999).

In summary, chronic antidepressant drug treatment in these studies did not affect basal CRF gene expression in the PVN but rather reduced the sensitivity of these neurons to various forms of stress. Further experiments are required to determine the time course of antidepressant treatment required for these effects and to characterize, if any, the functional consequences of these interactions. It is plausible, but not as yet demonstrated, that reduced sensitivity of PVN CRF gene expression to stress may contribute to normalization of HPA axis activity in patients undergoing psychopharmacological treatment. Evidence collected to date has failed to demonstrate that antidepressant administration modulates CRF synthesis, secretion, or receptor sensitivity in extrahypothalamic brain regions, although such an interaction has been hypothesized to underlie reduction of CSF CRF concentrations in treated patients.