St John's wort, hypericin, and imipramine: a comparative analysis of mRNA levels in brain areas involved in HPA axis control following short-term and long-term administration in normal and stressed rats


Clinical studies demonstrate that the antidepressant efficacy of St John's wort (Hypericum) is comparable to that of tricyclic antidepressants such as imipramine. Onset of efficacy of these drugs occurs after several weeks of treatment. Therefore, we used in situhybridization histochemistry to examine in rats the effects of short-term (2 weeks) and long-term (8 weeks) administration of imipramine, Hypericum extract, and hypericin (an active constituent of St John's wort) on the expression of genes that may be involved in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Imipramine (15 mg kg−1), Hypericum (500 mg kg−1), and hypericin (0.2 mg kg−1) given daily by gavage for 8 weeks but not for 2 weeks significantly decreased levels of corticotropin-releasing hormone (CRH) mRNA by 16–22% in the hypothalamic paraventricular nucleus (PVN) and serotonin 5-HT1A receptor mRNA by 11–17% in the hippocampus. Only imipramine decreased tyrosine hydroxylase (TH) mRNA levels in the locus coeruleus (by 23%), and only at 8 weeks. The similar delayed effects of the three compounds on gene transcription suggests a shared action on the centers that control HPA axis activity. A second study was performed to assess the effects of long-term imipramine and Hypericum administration on stress-induced changes in gene transcription in stress-responsive circuits. Repeated immobilization stress (2 h daily for 7 days) increased mRNA levels of CRH in the PVN, proopiomelanocortin (POMC) in the anterior pituitary, glutamic acid decarboxylase (GAD 65/67) in the bed nucleus of the stria terminalis (BST), cyclic AMP response element binding protein (CREB) in the hippocampus, and TH in the locus coeruleus. It decreased mRNA levels of 5-HT1A and brain-derived neurotrophic factor (BDNF) in the hippocampus. Long-term pre-treatment with either imipramine or Hypericum reduced to control levels the stress-induced increases in gene transcription of GAD in the BST, CREB in the hippocampus, and POMC in the pituitary. The stress-induced increases in mRNA levels of CRH in the PVN and TH in the locus coeruleus were reduced by imipramine but not by Hypericum. The stress-induced decreases in BDNF and 5-HT1AmRNA levels were not prevented by either drug. Taken together, these data show: (1) that Hypericum and hypericin have delayed effects on HPA axis control centers similar to those of imipramine; and (2) that select stress-induced changes in gene transcription in particular brain areas can be prevented by long-term treatment with either the prototypic tricyclic antidepressant imipramine or the herbiceutical St John's wort. However, imipramine appears to be more effective in blocking stress effects on the HPA axis than the plant extract.


St John's wort (Hypericum perforatum L, Hyperi- caceae) is a plant that has been used as a medicinal herb since ancient times. Today, Hypericum is used in many countries for the treatment of mild to moderate forms of depression, and its efficacy has been confirmed in several clinical studies.1 The therapeutic action of Hypericum is comparable to that of tricyclic antidepressants, whereas its side effects are less pronounced, resulting in a better compliance of the patients.2

Several behavioral studies support the antidepressant activity of Hypericum extracts. A methanolic extract significantly reduced immobility time in the forced swimming test.3 After 14 days of daily treatment in rats, the extract dose dependently reduced prolactin and corticosterone plasma levels.4 In in vitro studies, various extracts have been shown to inhibit synaptosomal uptake of norepinephrine, serotonin and dopamine5 and to inhibit ligand binding to the GABAA, the mu-, delta- and kappa-opioid, and the 5-HT6 and 5-HT7 receptors.6

St John's wort is a complex mixture of over two dozen constituents.7 The antidepressant activity of Hypericum extracts has been attributed to the phloroglucinol derivative hyperforin,8,9 to the napthodianthrones hypericin and pseudohypericin,10 and to several flavonoids.11,12 The activities of the different Hypericum compounds are still a matter of debate, but taking the previous findings together, it is likely that the different individual substances contribute to the antidepressant activity of the crude plant extract in a complicated manner. In the present study, we studied hypericin in addition to the full Hypericum extract because hypericin displayed high affinity for several aminergic receptor subtypes in vitro,13 and was active in the Porsolt forced swim test.10,14

Studies that investigate the molecular targets of St John's wort relevant to its antidepressant effects have not been done. It is not known how St John's wort affects key neurochemical systems in the brain in tests in which animals receive therapeutic dose regimens for protracted periods of time and under normal and stressed conditions. In this study, we employed two such tests in rats. The first test is designed to reveal neurochemical changes that are selectively associated with the delayed onset of therapeutic efficacy in humans. The second test uses an animal model of chronic stress that produces defined neurochemical changes against which the actions of long-term antidepressant drug administration can be evaluated. In both tests, the actions of St John's wort have been concurrently compared with those of the prototypic tricyclic antidepressant drug imipramine.

The first test is based on a characteristic feature common to both tricyclic antidepressants and St John's wort—the delay of 2 weeks or more before the therapeutic effect becomes evident. In an earlier animal study designed to examine the association between long-term antidepressant administration and the delayed alteration in hypothalamic-pituitary-adrenal axis (HPA) axis activity, corticotropin releasing hormone (CRH) mRNA levels in the hypothalamic paraventricular nucleus (PVN) of rats were found to be decreased following long-term (8 weeks) but not short-term (2 weeks) treatment with imipramine.15 The same results were found with several other antidepressant drugs selected for their distinctly different primary pharmacological actions.16 Thus in this study, we measured changes in CRH mRNA levels at these two time points, and we also measured mRNA expression in a number of CNS areas involved in and related to HPA axis control.

The second test of the antidepressant efficacy of St John's wort employed an animal model of chronic stress. In humans, prolonged stress is associated with certain mood disorders.17,18 An important link between depression and stress is established by the facts that the HPA axis and particular sets of 5-hydroxytryptamine (5-HT), norepinephrine, and glutamic acid decarboxylase (GABA) containing neuronal systems are involved both in the pathophysiology of depression and the neurobiology of stress.19,20 Several clinical studies give evidence that depression is associated with an activation of the HPA axis.20,21,22 In the course of chronic antidepressant drug treatment, the hyperactivity of the HPA axis normalizes with clinical recovery from depression.23

Chronic stress in animals results in elevated adrenocorticotropic hormone (ACTH) and glucocorticoid levels in plasma and elevated production of CRH in the PVN.24,25,26 In addition, tyrosine hydroxylase (TH) gene expression in the locus coeruleus is elevated,24,27 suggesting that stress activates the norepinephrine-containing neurons that play an important role in behavioral arousal. Recently, other work has extended these findings in animals to include observations of neurochemical changes in brain structures known to influence the HPA axis. These include GABA in the bed nucleus of the stria terminalis (BST), assessed by measures of GAD mRNA levels,28 and several neurochemical markers in the hippocampus.29

The direction of changes in mRNA levels following stress is generally opposite to that of the same genes in the same locations following long-term antidepressant treatment. However, the relationship between the neurochemical effects of chronic stress and those of antidepressant drug administration has received little study. Thus, in the second study we used an immobilization stress procedure to: (1) show stress-induced changes in gene expression in selected brain regions: (2) determine whether long-term pre-treatment with either imipramine or St John's wort blocks the stress-induced changes; and (3) find differences between the synthetic tricyclic antidepressant imipramine and the natural herbiceutical St John's wort in their ability to modify the effects of chronic stress.

Material and methods

Experiment 1—short-term/long-term treatment design

Male CD rats (150–180 g, Charles River WIGA, Sulzfeld, Germany) were single housed in a 12 h light/dark cycle, with lights off at 19.00 h, at a constant temperature of 25 ± 1°C and free access to food (Altromin 1324) and tap water. Rats were randomly assigned to the various experimental groups and weighed daily. The experimental procedures used in this work were officially approved by the Regierungspräsident, Münster (A 38/93).

Imipramine hydrochloride was obtained from Sigma (Deisenhofen, Germany), and hypericin was obtained from Roth (Karlsruhe, Germany). A lyophilized methanolic native Hypericum extract (LI 160, drug/extract ratio: 4–7:1) was obtained from Lichtwer Pharma AG (Berlin, Germany). The extract LI 160 was subsequently characterized by HPLC using the method published by Butterweck et al.3 The main constituents were: chlorogenic acid 5.5 mg g−1, rutin 20.1 mg g−1, hyperoside 20.15 mg g−1, isoquercitrine 10.8 mg g−1, astilbin 2.4 mg g−1, quercitrine 5.0 mg g−1, quercetin 4.6 mg g−1, 13.118-biapigenine 3.8 mg g−1, hyperforin 53.1 mg g−1, and hypericin 2.21 mg g−1.

All substances were administered orally using the gavage technique. The oral administration route had to be chosen because: (1) both the Hypericum extract and hypericin are barely soluble in aqueous solvents and thus must be administered as a suspension; (2) the Hypericum extract suspension is pH 4–5 (for intraperitoneal injections, solutions should have a 7.0–7.5 pH); (3) for chronic application, intraperitoneal injections are contraindicated;30 and (4) gavage—if done properly—is less stressful for animals than intraperitoneal injection.30 For consistency of method, imipramine was also given by gavage.

Drug solutions were prepared fresh daily. Stock compounds were kept in light-tight containers. Control animals received deionized water with an ethanol content of 160 μl per 10 ml. Imipramine (15 mg) and Hypericum extract (500 mg) were first moisturized in 160 μl ethanol and then diluted with deionized water to a final volume of 10 ml. A homogeneous suspension of the Hypericum extract was obtained by sonication for 5 min. Hypericin is barely soluble in water, therefore 5 mg hypericin were nearly dissolved in 2.5 ml ethanol and further diluted with water to a final concentration of 0.2 mg per 10 ml. The ethanol concentration of the hypericin solution was 1%. The final application volume of each preparation was 10 ml kg−1 b.w. A dosage of 15 mg kg−1 imipramine was chosen because previous behavioral experiments (V Butterweck, unpublished data) indicated that oral dosages below 15 mg were only partially active; for chronic treatment, 15 mg kg−1 proved to be active without toxic effects, whereas higher doses administered chronically were toxic. The Hypericum extract (500 mg kg−1) and the hypericin (0.2 mg kg−1) dosages were chosen because of their demonstrated efficacy in the forced swimming test.3,10

Animals were killed by decapitation between 9.00 and 11.00 h; the last drug administration was the day before between 16.00 and 17.00 h. Brains were removed, frozen by immersion in 2-methyl butane at −30°C, and stored at −70°C prior to sectioning. Trunk blood was collected on ice-chilled EDTA-coated (10 ml) tubes containing 500 KIU aprotinin ml−1, centrifuged, and plasma was frozen at −70°C.

Experiment 2—stress plus drugs design

Male Sprague–Dawley rats (150–180 g. Taconic Farms, Germantown, NY, USA) were housed two per cage in a 12 h light/dark cycle, with lights off at 18.00 h, at a constant temperature of 25 ± 1°C and free access to food and tap water. Rats were randomly assigned to the various experimental groups and weighed daily. Drugs (imipramine and Hypericum extract) were prepared and administered as in the first experiment. Control animals received deionized water only. Administration was performed between 8.00 and 10.00 h every day.

The immobilization procedure was approved by the Animal Care and Use Committee of the National Institute of Mental Health Intramural Research Program. Rats were immobilized for 2 h in the morning during the nadir of their circadian rhythm. Animals were immobilized by placing the head through stainless steel loops and securing each limb with tape to a stainless steel platform as described previously.31 The immobilization schedule used in this study was the stress design from Mamalaki et al.24 A group of 48 animals was subdivided as follows: 16 animals were treated with imipramine (15 mg kg−1, p.o.), 16 were treated with Hypericum extract (500 mg kg−1, p.o.), and 16 animals received vehicle (deionized water) over a period of 7 weeks. In the seventh week, eight animals of each treatment group were immobilized for 2 h once daily for 7 days and killed immediately after the last stress session.

An acute paradigm was designed to investigate whether a single exposure of rats to immobilization stress could induce changes in mRNA expression in brain areas involved in HPA axis control, and in addition, whether acute pre-treatment with imipramine could antagonize possible changes in stress-induced gene expression. Thus, a second group of 32 animals was divided into an unstressed and a stressed group. Unstressed animals received either imipramine (15 mg kg−1 b.w., n = 8) or vehicle (deionized water, n = 8) 2 h prior to decapitation. Stressed animals (n = 8 per group) received the same treatment, respectively. Two h after oral administration of test substances, animals were exposed to a single immobilization stress for 2 h and were killed immediately after the stress session. Unstressed animals from the acute and chronic experiment were habituated by daily handling. All groups were killed between 12.00 and 14.00 h.

Measurement of corticosterone and adrenocorticotropic hormone (ACTH)

Radioimmunoassay (RIA) of corticosterone was performed using [125I]corticosterone, antiserum, and standard solution in a kit from ICN Biomedical (Costa Mesa, CA, USA). The assay was adapted to rat serum conditions. Precipitation was done using a second antibody solid phase. ACTH was measured using a DSL kit (Webster, Texas, USA). Plasma samples contained 500 KIU aprotinin ml−1. Both assays were performed according to manufacturer's instruction. The inter- and intraassay coefficients of variance for ACTH were 10.6% and 6.9%, respectively, with a detection limit of 10 pg ml−1. For corticosterone, the inter- and intraassay coefficients of variance were 7.2% and 4.4%, with a detection limit of 25 ng ml−1.

In situ hybridization histochemistry

Guided by Nissl-stained sections collected during the cutting and by the atlas of Paxinos and Watson,32 coronal frozen sections (15 μm-thick) were collected at the levels of the midportion of the parvocellular region of the PVN at the level where the magnocellular nucleus is largest (−1.8 mm), bed nucleus of the stria terminalis (BST) (−0.26 mm), dorsal hippocampus (−3.3 mm), pituitary, and locus coeruleus (−9.7 mm). Sections were thaw-mounted onto gelatin-coated slides, dried, and stored at −40°C prior to processing for in situ hybridization histochemistry.

The in situ hybridization histochemistry procedures were performed as described previously for ribonucleotide (cRNA) probes.15 First, tissue sections were processed by fixation with 4% formaldehyde solution, acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine-HCI, pH 8.0 solution, dehydration with ethanol, and delipidation with chloroform. Second, the antisense probes were transcribed from linearized plasmids using the Riboprobe System (Promega Biotech, Madison, WI, USA) with 35S-UTP (specific activity >1000 Ci mmol−1; New England Nuclear, Boston, MA, USA) and T7, T3, or SP6 RNA polymerase. The cDNA probes were: a 760-bp fragment of rat CRH (gift of Dr James Herman, University of Kentucky, Lexington, KY, USA), 923 bp of mouse POMC (gift of Dr James Douglass, Vollum Institute, Portland, OR, USA), 384 bp of rat TH (gift of Dr Barry Kosofsky, Harvard Medical School, Boston, MA, USA), 229 bp of rat arginine-vasopressin (AVP) (gift of Dr W Scott Young, NIMH, Bethesda, MD, USA), 2.3 kb and 3.2 kb full-length sequences of the rat GAD65 and GAD67 (gift of Drs A Tobin and N Tillakaratne, UCLA, Los Angeles, CA, USA), rat full-length BDNF33 (gift of Drs J Lauterhorn and C Gall, University of California Irvine, CA, USA), rat full-length CREB (gift of Dr Stephen Hyman, NIMH, Bethesda, MD, USA) and a 900-bp BAL I/PVU II fragment of the rat serotonin 5-HT1A receptor gene (gift of Dr Paul Albert, University of Ottawa, Ontario, Canada).

The radiolabeled probes were diluted in a riboprobe hybridization buffer and applied to brain sections (approximately 500 000 CPM per section). After overnight incubation at 55°C in a humidified chamber, slides containing brain sections were washed first in 20 μg ml−1 RNase solution and then 1 h each in 2× SSC (50°C) and 0.2× SSC (55°C and 60°C) solutions to reduce non-specific binding of the probe. The slides were then dehydrated with ethanol and air-dried for autoradiography.

Slides and 14C plastic standards containing known amounts of radioactivity (American Radiochemicals, St Louis, MO, USA) were placed in x-ray cassettes, apposed to film (BioMax MR, Kodak, Rochester, NY, USA) for periods ranging from 1 to 72 h, and developed in an automatic processor (X-OMAT, Kodak). To determine anatomical localization of hybridized AVP cRNA at the cellular level, sections were dipped in nuclear track emulsion (NTB-2, Kodak), exposed for 18 h, developed (D-19, Kodak), and lightly counterstained with cresyl violet.

Data analysis and presentation

Autoradiographic images were digitized with a solid-state camera (CCD-72, Dage-MTI) and a Macintosh computer using NIH Image software (Wayne Rasband, NIMH). Transmittance measurements were converted to dpm mg−1 plastic using the calibration curve (Rodbard equation) generated from the standards. Brain structures were identified according to the atlas of Paxinos and Watson.32 Light transmittance through the film at PVN (CRH and AVP in the parvocellular division), BST (GAD65/67), hippocampus (5-HT1A, BDNF, CREB), anterior and intermediate lobes of pituitary (POMC), and locus coeruleus (TH) was measured by outlining the structure with the mouse cursor. Mouse cursor control was used to outline the selected structure. The average value for each animal in experimental or control groups (based on four measurements per animal) was used to calculate group means (n = 5–8 per group).

For analysis of induction of AVP mRNA in cells within the parvocellular portion of the PVN that projects to the median eminence (the CRH-containing zone of the PVNpc), emulsion-coated slides were analyzed with a Leica DMR microscope. The silver grains in the emulsion layer were visualized with epi-illumination passing through a POL cube filter (which exclusively selects for polarized light reflected off the silver grains) and captured as a ‘darkfield’ image with the Dage-MTI camera using NIH Image. The captured POL image was inverted and background flattened using the 2D rolling ball function. Density slice was then used to select only labeled cells, and the median eminence projecting zone (determined on the basis of the corresponding CRH mRNA image from the same animal) was outlined using the mouse cursor. A calibrated area measurement was then made; this number was directly proportional to the number of labeled cells in the PVNpc.

Two-way ANOVAs (time and drug treatment or stress and drug treatment) were used to compare specific mRNA levels in control vs treated groups. The Student–Newman–Keuls test was used for post-hoc comparisons of mRNA levels in each region. A criterion level of P < 0.05 was used to determine significance.


Experiment 1—short-term/long-term treatment study

Examples of the expression patterns of each of the mRNA probes are shown in Figure 1. Densitometry showed that CRH mRNA levels in the PVN were not significantly changed at 2 weeks (short-term administration) but were significantly decreased at 8 weeks (long-term administration) in all three drug groups examined (Table 1). CRH mRNA levels were reduced 20–22% by imipramine and hypericin (P < 0.001) and 16% by Hypericum extract (P < 0.01) at 8 weeks. No significant changes were observed for mRNA expression of AVP, measured by both densitometry (data not shown) and cell counting (Table 1), in the parvocellular portion of the PVN at either time point.

Figure 1

Examples of autoradiographic images, obtained in control animals, for the ribonucleotide probes hybridized in experiment 1. A darkfield microphotograph of an emulsion-coated section hybridized for arginine vasopressin (AVP) mRNA illustrates the discrete cellular labeling in the magnocellular (condensed cluster of labeled cells) and parvocellular (scattered labeled cells) divisions of the hypothalamic paraventricular nucleus (PVN). A photograph of the film exposed to a corticotropin-releasing hormone (CRH) mRNA-hybridized section is shown at the same magnification. The zone of CRH mRNA labeling, in the parvocellular PVN, was used to determine the portion of the PVN that was outlined for AVP mRNA quantification. The remaining film images, captured at lower magnification, are examples of proopiomelanocortin (POMC) mRNA hybridization in the pituitary (long exposure is shown, used for measurement of the expression in the anterior lobe; a shorter exposure was used for measurement of the darkly labeled intermediate lobe); tyrosine hydroxylase (TH) mRNA in the locus coeruleus, and serotonin receptor1A (5-HT1A) mRNA in the hippocampus. Bars measure 0.5 mm.

Table 1 Expression of mRNAs in rat brain after short-term and long-term antidepressant treatment

After 2 weeks of oral treatment, imipramine and hypericin but not Hypericum extract significantly decreased POMC mRNA expression in the anterior lobe of the pituitary (30–32%; P < 0.001) (Table 1). After 8 weeks of chronic drug treatment, POMC mRNA levels were reduced 18% by imipramine (P < 0.05), 25% by Hypericum extract (P < 0.05), and 22% by hypericin (P < 0.05). No treatment effects were observed in the intermediate lobe of the pituitary.

After 2 weeks of daily treatment, no significant changes in 5-HT1A receptor mRNA levels were found for any of the drugs (Table 1). Long-term treatment (8 weeks) with imipramine, Hypericum extract, and hypericin significantly decreased 5-HT1A receptor mRNA expression by 11–17% (P < 0.001; P < 0.01) in hippocampal field CA1 relative to control (Table 1). Similar decreases were observed in CA3 and DG but were not significant in these areas.

No changes in TH mRNA levels were evident after 2 weeks for any treatment (Table 1). After 8 weeks of daily imipramine administration, TH gene expression levels were decreased by 23% (P < 0.001). Hypericum extract produced a slight (11%) but not significant decrease in TH gene expression after 8 weeks, and hypericin did not alter TH mRNA levels.

The decrease in POMC mRNA levels in the anterior pituitary of animals treated with imipramine, Hypericum extract, and hypericin for 2 weeks was associated with a significant reduction of plasma ACTH (25–39%, P < 0.001) and corticosterone (51–57%, P < 0.001) levels (Table 2). No changes in hormone levels were observed after 8 weeks.

Table 2 Plasma hormone levels and adrenal and body weights in short-term and long-term treatment groups

Adrenal gland weights were not significantly altered by 2 weeks or 8 weeks of chronic antidepressant treatment. Body weight was significantly decreased by imipramine (13%, P < 0.05) and Hypericum extract (12%, P < 0.05) after 2 weeks of daily treatment but was not affected after 8 weeks. Hypericin slightly increased body weight (8%, P < 0.05) after 8 weeks (Table 2).

Experiment 2—stress plus antidepressant drug administration

CRH and AVP mRNA in the PVNpc

Neither acute imipramine pre-treatment, acute immobilization stress, nor acute imipramine plus stress had a significant effect on gene expression of CRH and AVP in the parvocellular PVN (Table 3). A 28% non-significant increase in AVP mRNA level was seen by densitometry of the PVNpc in the acute stress condition relative to control (data not shown). In contrast, long-term administration (7 weeks) of imipramine (15 mg kg−1 p.o.) or Hypericum extract (500 mg kg−1 p.o.) significantly decreased CRH mRNA levels in the PVN (Figures 2 and 3), replicating the results of the first experiment. CRH mRNA levels were reduced 15–17% (P < 0.05) by imipramine and by Hypericum extract (P < 0.05). In untreated animals, chronic immobilization stress (2 h per day for 7 days) significantly increased CRH mRNA expression (26%, P < 0.01) in the PVN (Figures 2b, 3), replicating earlier work.24 The stress-induced elevation of CRH mRNA expression was blocked by chronic imipramine administration (16%, P < 0.01) but not by the extract (Figure 3). No changes were observed for either expression density or numbers of cells expressing AVP mRNA in the parvocellular PVN in any of the chronic conditions.

Table 3 Expression of mRNA in acute conditions
Figure 2

Autoradiographic images of mRNA expression of CRH (a–d) in the hypothalamic PVN and POMC (e–h) in the pituitary. The CRH and POMC film images were selected from cases whose individual densitometric transmittance values represented the mean for that group. The images are the same as were used for densitometry; they were cropped and assembled into the composite without any image alteration. The cases shown are from chronic control (a and e), chronic stress (b and f), chronic imipramine (c and g), and chronic imipramine + stress (d and h). Bar measures 0.5 mm.

Figure 3

CRH and AVP mRNA expression in the parvocellular paraventricular nucleus (PVN) in the chronic conditions. Groups are: 1, chronic no stress; 2, chronic stress; 3, chronic imipramine; 4, chronic imipramine + stress; 5, chronic Hypericum extract; and 6, chronic Hypericum extract + stress. *P < 0.05; †P < 0.01.

POMC mRNA levels in the pituitary

Neither acute imipramine pre-treatment, acute immobilization stress, nor acute imipramine plus stress had a significant effect on gene expression of POMC in the anterior lobe of the pituitary (Table 3). In the intermediate lobe of the pituitary, acute imipramine treatment alone had no effect on the POMC gene expression, whereas acute stress produced a slight but not significant increase in POMC mRNA levels (Table 3).

Seven weeks of chronic administration of imipramine or Hypericum extract significantly reduced POMC mRNA levels in the anterior lobe of the pituitary of unstressed groups (Figure 4). POMC mRNA levels were reduced 27% (P < 0.05) by imipramine and 20% (P < 0.05) by Hypericum extract. Repeated immobilization significantly increased POMC mRNA levels in the anterior lobe (50%, P < 0.01) of control animals (Figure 4). The stress-induced elevated POMC mRNA expression in the anterior lobe was blocked by treatment with imipramine (22%, P < 0.05) and Hypericum extract (26%, P < 0.05) (Figure 4).

Figure 4

POMC mRNA expression in the anterior and intermediate lobes of the pituitary in the chronic conditions. Groups are: 1, chronic no stress; 2, chronic stress; 3, chronic imipramine; 4, chronic imipramine + stress, 5, chronic Hypericum extract; and 6, chronic Hypericum extract + stress. *P < 0.05; †P < 0.01.

Although less pronounced, similar effects were observed in the intermediate lobe of the pituitary. Chronic stress increased POMC mRNA expression to 124% of control (P < 0.05). This increase was prevented by long-term treatment with imipramine or the extract (Figure 4).

ACTH and corticosterone plasma levels

Acute immobilization stress produced a significant elevation (P < 0.05) of plasma ACTH and corticosterone levels compared to the non-stressed rats. Acute pre-treatment with imipramine had no effect on the stress-induced increases in plasma hormone levels. The ACTH values (pg ml−1) for the different treatment groups in the acute stress experiment were: vehicle no stress = 203 ± 38, imipramine no stress = 142 ± 14, vehicle + acute stress = 666 ± 63, imipramine + acute stress = 529 ± 15. Corticosterone values (ng ml−1) were: vehicle no stress = 83 ± 32, imipramine no stress = 94 ± 25, vehicle + stress = 459 ± 62, imipramine + stress = 573 ± 52.

The chronic stress paradigm caused a significant elevation of basal plasma hormone levels relative to the non-stressed control group. The stress-induced increases in corticosterone plasma levels were significantly reduced in animals that were pre-treated with imipramine (Figure 5), although hormone levels were not reduced to baseline. Surprisingly, Hypericum extract did not prevent the increase in basal ACTH and corticosterone levels observed after chronic stress. In fact, the plasma hormone levels in animals that received Hypericum pre-treatment and chronic stress were somewhat higher than those of the stress control rats. The difference was statistically significant for ACTH (P < 0.05).

Figure 5

Adrenocorticotropin (ACTH) and corticosterone levels in plasma in the chronic conditions. Groups are: 1, chronic no stress; 2, chronic stress; 3, chronic imipramine; 4, chronic imipramine ± stress; 5, chronic Hypericum extract; and 6, chronic Hypericum extract + stress. *P < 0.05; †P < 0.01.

GAD65 and GAD67 mRNA levels in the BST

Both GAD65 and GAD67 mRNAs were distinctly expressed throughout the BST (Figure 6). Acute imipramine treatment produced no changes in GAD65 and GAD67 mRNA levels in the anteromedial, anterodorsal, and ventral subdivisions of the BST (Table 3). Similarly, mRNA levels were not altered after acute immobilization stress in most of the subdivisions of the BST. Only GAD67 mRNA levels were increased after acute stress in the ventral subdivision of the BST (17%, P < 0.05) (Table 3).

Figure 6

Autoradiographic image of GAD65 mRNA expression in the bed nucleus of the stria terminalis (BST). A similar pattern and density of expression was seen for GAD67. The parcellation of BST was based on Bowers et al28 and the Paxinos and Watson atlas32 plates 19 and 20 as follows: anteromedial BST (AM, encompassing atlas divisions BSTMA); anterodorsal BST (AD, encompassing atlas divisions BSTLD, BSTLJ, and BSTI), and ventral (V, BSTV). Bar measures 1 mm.

Chronic treatment with either imipramine or Hypericum extract did not affect expression of GAD65 and GAD67 in any subdivisions of the BST, but chronic stress increased GAD65 and GAD67 mRNA expression (Figure 7). GAD65 mRNA expression was increased in the anteromedial (27%, P < 0.05), anterodorsal (26%, P < 0.01), and ventral BST (34%, P < 0.01) subdivisions. The stress-induced increases in GAD65 mRNA expression were reduced by treatment with imipramine (anteromedial = 15%, P < 0.05; anterodorsal = 23%, P < 0.01; ventral = 18%, P < 0.01) or Hypericum extract (anteromedial = 25% P < 0.01; anterodorsal = 18%, P < 0.01; ventral = 22%, P < 0.01), respectively (Figure 7). Similarly, chronic stress-induced increases in GAD67 mRNA expression levels were observed in the anteromedial (24%, P < 0.05), anterodorsal (19%, P < 0.05) and ventral (26%, P < 0.01) subdivisions of the BST. After chronic treatment with imipramine or Hypericum extract, stress-induced elevations in GAD67 mRNA levels were no longer apparent, and the stress-blocking effect was significant in the anterodorsal and ventral subdivisions (Figure 7).

Figure 7

GAD65 and 67 mRNA expression in the bed nucleus of the stria terminalis (BST) in the chronic conditions. BST subdivisions are anteromedial (AM), anterodorsal (AD), and ventral (V). Groups are: 1, chronic no stress; 2, chronic stress; 3, chronic imipramine; 4, chronic imipramine + stress; 5, chronic Hypericum extract; and 6, chronic Hypericum extract + stress. *P < 0.05; †P < 0.01.

5-HT1A mRNA levels in the hippocampus

All cell layers of the hippocampus expressed moderate levels of 5-HT1A mRNA (Figure 8). Neither acute stress nor acute imipramine had effects on 5-HT1A mRNA levels (Table 3).

Figure 8

Autoradiographic film images of mRNA expression of 5-HT1A (a and b), BDNF (c and d), and CREB (e and f) in the hippocampus. Notice the differences in density of labeling between the chronic control (no stress) at the top with chronic stress at the bottom. Images were selected as described in Figure 1 caption. Hippocampal fields labeled are Cornu Ammonis CA1 and CA3 and the dentate gyrus (DG). Bar measures 1 mm.

Long-term treatment with either imipramine or Hypericum extract caused a significant decrease in 5-HT1A mRNA levels across all subfields of the hippocampus relative to control (Table 4). The decreases were 17–32% in the unstressed imipramine group and 19–28% in the Hypericum group. Animals receiving chronic immobilization stress also showed a decrease in 5-HT1A mRNA levels across all hippocampal subfields (Figure 8b). The decreases were 18–37% (P < 0.01) in the stressed control group relative to unstressed control. The stressed groups treated with imipramine or Hypericum extract for 7 weeks had 5-HT1A mRNA levels that were not different from those of the untreated stressed control group (Table 4).

Table 4 Expression of mRNA in the hippocampus in chronic conditions

BDNF mRNA levels in the hippocampus

All changes in BDNF mRNA expression in the hippocampus were confined to the dentate gyrus granule cell layer (Tables 3 and 4). Both acute imipramine treatment and acute immobilization stress produced decreases in BDNF mRNA levels in the dentate gyrus relative to the unstressed control group (17%, P < 0.01 and 32%, P < 0.01, respectively) (Table 3). A single imipramine administration prior to acute stress further down-regulated BDNF mRNA expression relative to the stressed control (15%, P < 0.05).

Similarly, both chronic antidepressant treatment as well as chronic stress decreased BDNF mRNA expression in the dentate gyrus. Chronic antidepressant treatment alone decreased BDNF mRNA levels by 15–20% (P < 0.01) (Table 4). Chronic stress also markedly reduced mRNA expression, by 44% (P < 0.01) (Figure 8c, d). Chronic administration of imipramine or Hypericum extract did not prevent the stress-induced decrease in BDNF mRNA. In fact, BDNF levels were comparable to those of the stressed control group (Table 4).

CREB mRNA levels in the hippocampus

No significant change in CREB mRNA expression was seen in the hippocampus with the acute stress paradigm (Table 3). After long-term treatment with imipramine and Hypericum extract, CREB mRNA levels remained unchanged in all subdivisions of the hippocampus (Table 4). CREB mRNA expression was increased after chronic stress in the CA1 field (16%, P < 0.05) (Figure 8e, f) and in the dentate gyrus (13%, P < 0.05). Chronic administration of imipramine and Hypericum extract resulted in CREB mRNA levels in stressed animals that were close to the levels of the unstressed control group (Table 4).

TH mRNA levels in the locus coeruleus

Acute exposure to immobilization stress increased (25%, P < 0.05) TH mRNA levels in the locus coeruleus (Table 3). Acute treatment with imipramine alone had no effect on TH gene expression. The stress-induced acute increase of TH mRNA levels was normalized by acute pre-treatment with imipramine (17%, P < 0.05) (Table 3). Chronic exposure to immobilization stress significantly increased TH mRNA levels in the locus coeruleus (19%, P < 0.05). Pre-treatment with imipramine but not the plant extract normalized the stress-induced elevation of TH mRNA (P < 0.01) (Figures 9 and 10).

Figure 9

Autoradiographic film images of TH mRNA expression in the locus coeruleus (LC) on the right side of the brain. The three conditions shown are chronic control (a), chronic stress (b), and chronic imipramine + stress (c). Images were selected as described in Figure 1 caption. Insets show the same image after blurring in NIH Image and coding with a five-shades-of-gray scale in order to highlight the density differences. All images were manipulated identically. Bar measures 0.2 mm.

Figure 10

TH mRNA expression in the locus coeruleus in the chronic conditions. Groups are: 1, chronic no stress; 2, chronic stress; 3, chronic imipramine; 4, chronic imipramine + stress; 5, chronic Hypericum extract; and 6, chronic Hypericum extract + stress. *P < 0.05; †P < 0.01.


Experiment 1—chronic administration effects

The major finding of the first study is that St John's wort (Hypericum extract) altered gene expression levels in brain areas involved in HPA axis control after long-term administration, similar to the changes elicited by the prototypic synthetic antidepressant imipramine—a finding that has not been previously reported in an in vivo model. The neurochemical actions of Hypericum extract could also be demonstrated with pure hypericin. It appears, therefore, that this naphthodianthrone is a major active principle of St John's wort that may contribute to its therapeutic effect. Hypericin is a stable compound that is convenient and inexpensive to bioextract, making it particularly attractive for further exploitation and characterization for potential use in CNS mood disorders.

The drug administration paradigm and the selection of molecular targets was as described by Brady et al.15 All three drugs—Hypericum extract, hypericin, and imipramine—decreased CRH mRNA expression in the hypothalamus after long-term (8 weeks) but not short-term (2 weeks) administration. The imipramine-induced delayed decrease in CRH mRNA levels in the PVN reported previously15 was with 5 mg kg−1 given intraperitoneally, whereas in the present study 15 mg kg−1 were given orally by gavage. The time-dependent decrease in CRH mRNA levels was also induced by fluoxetine, idazoxan and phenelzine in the same paradigm.16 Thus, Hypericum, hypericin, and several synthetic antidepressants act in a similar delayed fashion to affect expression levels of genes controlling activity of the HPA axis.

Imipramine and hypericin significantly reduced POMC mRNA levels in the anterior lobe of the pituitary at both 2 and 8 weeks. Inconsistent effects have been reported in previous studies measuring POMC mRNA levels after administration with imipramine, fluoxetine, idazoxan, fluoxetine, and citalopram.15,16,34 It is surprising that at 2 weeks, POMC mRNA and plasma ACTH and corticosterone levels were decreased while CRH and AVP mRNA expression levels were unchanged. At 8 weeks, anterior pituitary POMC mRNA levels and hypothalamic CRH mRNA levels were reduced, and plasma ACTH and corticosterone levels were back to baseline. The functional consequences of these findings are not clear. The relatively high baseline levels of ACTH might be a reason for these dissociative effects after 2 and 8 weeks. It can be speculated that different adrenal responsiveness after both time points might play an important role in mediating the ACTH effects.

5-HT has long been implicated in the biological basis of depression as well as in the mechanism of action of antidepressant drugs.35 Animal studies suggest that increases in neurotransmission at postsynaptic 5-HT1A receptors may mediate the therapeutic effects of some antidepressant drugs.36 The 5-HT1A receptor has been identified as an inhibitory somatodendritic autoreceptor in the raphe serotonergic cells and as a postsynaptic receptor in serotonergic terminal fields.37 The highest density of 5-HT1A receptor binding sites occurs in the hippocampus and other limbic regions.38 A role for hippocampal 5-HT1A in the pathophysiology of mood disorders has been explored, but data from postmortem human brain studies have been inconsistent, showing unchanged,39 increased,40 or reduced 5-HT1A receptor binding in the hippocampus of patients with major depressive disorders who died by suicide.29

We found that 2-week imipramine administration had no effect on 5-HT1A receptor mRNA expression in the hippocampus, consistent with Lopez et al.29 However, long-term treatment with all three agents significantly decreased 5-HT1A receptor mRNA expression in CA1 of the hippocampus. Other studies have shown small decreases41,42 in 5-HT1A receptor number after long-term antidepressant administration. The mechanisms responsible for the delayed decrease in 5-HT1A receptor mRNA expression after daily treatment with Hypericum extract, and hypericin, and imipramine are unknown. It can be speculated that alterations in central catecholaminergic, serotonergic and/or cholinergic systems are involved. Chronic antidepressants increase serotonergic neurotransmission in the hippocampus43 and elevate extracellular 5-HT concentrations in the nerve endings,44 possibly causing a postsynaptic down-regulation of the 5-HT1A receptors.

Long-term treatment with imipramine significantly decreased TH mRNA levels in the locus coeruleus, whereas Hypericum extract and hypericin had no effect on TH message. Previous studies also showed that chronic imipramine treatment decreased TH expression,15,45 whereas chronic fluoxetine, idazoxan and phenelzine increased TH mRNA levels.16 Brady et al16 suggested that the therapeutic efficacy of activating drugs such as fluoxetine and phenelzine in the treatment of atypical depression may result from their ability to increase mRNA levels of TH in the locus coeruleus. Because Hypericum extract and hypericin had no effect on TH mRNA levels, it can be speculated that the natural substances have an action profile that is different from that of classical antidepressants.

Experiment 2—actions in a stress paradigm

Analysis of relative hybridization densities demonstrated that chronic stress more so than acute stress induced pronounced changes in levels of gene expression of stress-related molecules and that chronic oral pre-treatment with imipramine or St John's wort (Hypericum extract) countered a number of these changes in an anatomically specific manner. The stress axis, comprised of the CNS nuclei and pathways that control glucocorticoid secretion elicited by psychological stressors such as immobilization, is principally centered on the hypothalamic parvocellular PVN and its neurotransmitters CRH and AVP. Neural circuits that control PVN CRH activity have been demonstrated by tract-tracing, lesion, and electrophysiology studies and by studies showing gene or protein regulation in the afferent pathways (summarized in Herman and Cullinan46). Acute and/or repeated stress elevates levels of transmitters, enzymes, and/or immediate-early genes in the PVN and its major afferent control centers such as the brainstem catecholamine cell groups, the amygdala, and the BST. Inputs from hippocampus and prefrontal cortex may inhibit the stress response. We focused our efforts on well-studied stress-responsive brain areas involved in HPA axis control, but it is likely that changes in gene expression levels occurred in other neurotransmitter systems and locations.

CRH and AVP mRNA expression

In this study, chronic immobilization stress increased CRH gene expression in the PVN. The stress-induced increase in CRH mRNA levels was blocked by long-term (7 weeks) pre-treatment with imipramine but not Hypericum extract. AVP mRNA density and numbers of expressing cells in the PVNpc were not affected by either stress or by antidepressant treatments. It is well established that the levels of CRH mRNA in the PVNpc increase in response to stress.24,47,48,49,50 However, the situation is less clear for AVP mRNA, with some studies showing no response and others showing a small increase either in message levels or in numbers of cells expressing message.25,48,50,51,52,53,54 The different transcript responses of CRH and AVP to stress imply different regulatory mechanisms for these genes.55,56 Evidence for progressive changes in AVP and CRH mRNA levels after repeated stress has been reported.57

Our present data suggest that imipramine but not Hypericum extract exerts its antidepressant activity by blocking the stress-induced changes in CRH gene expression, which might be relevant to its therapeutic actions. This interpretation is tempered by the facts that we do not know whether changes in CRH mRNA expression levels are reflected in changes in the amount of CRH released from nerve terminals, or whether these changes are affecting the hormonal output of the pituitary and adrenal glands. We also cannot exclude the possibility that isolated substances of Hypericum might have stress-blocking properties that are masked in the whole preparation.

POMC mRNA levels in the pituitary and plasma hormone levels

The elevation in CRH mRNA levels after repeated immobilization stress in the PVN was accompanied by significant elevation of pituitary POMC mRNA levels in the anterior pituitary, as reported previously.24 Chronic stress-related changes in POMC mRNA levels in the anterior pituitary were accompanied by significant elevations of plasma ACTH and corticosterone levels. When imipramine and Hypericum extract were administered concomitantly with chronic immobilization stress, up-regulation of POMC mRNA levels was prevented. These observations are in good correlation with our previous findings.

Although both antidepressants alone reduced POMC gene expression in the anterior pituitary, plasma ACTH and corticosterone levels were not different from control. The same pattern of data occurred in experiment 1. The functional consequences of these findings are not clear. The relatively high basal levels of ACTH might be a reason for the dissociative effects after chronic treatment. It can be also speculated that different adrenal responsiveness after chronic treatment might feed back to affect ACTH output.

Immobilization stress induced a strong ACTH and corticosterone response. Long-term administration of imipramine reduced stress-induced increases in corticosterone plasma levels, a finding which was reported recently.29 However, Hypericum extract was unable to prevent the stress-induced elevation in plasma hormone levels. A similar result was observed for zimelidine and fluoxetine, two specific serotonin reuptake inhibitors (SSRIs).29 Hypericum might have an influence on the serotonergic system comparable to that of the SSRIs. There is evidence from the literature that different compounds from Hypericum differentially affect neurotransmitter systems, warranting further study of individual extracts.

GAD65/67 mRNA expression

There is an increasing body of evidence suggesting that GABA plays an important role in the therapeutic effects of antidepressant/antipanic drugs. In clinical studies, the levels of GABA in plasma have been found to be significantly lower in depressed patients than in controls.17,18 GABA agonists have been demonstrated to possess antidepressant and antipanic effects in animal and clinical studies.19,20 Phenelzine, a non-selective monoamine oxidase inhibitor, is efficacious in the treatment of depression and panic disorder, and it has been shown to elevate brain GABA levels.58,59 Similarly, imipramine has been reported to acutely enhance GABA release in the brain.60

We found that neither of the long-term treatments gave rise to significant changes in levels of mRNA encoding GAD65 and GAD67 in the BST, one of the major sources of afferent inputs to the PVN.61 Our results are in good correlation with the results of Lai et al62 who observed no changes in levels of GAD65 and GAD67 mRNAs in cortex after short-term and long-term phenelzine and imipramine treatment. Therefore, it appears that antidepressants elevate GABA levels without altering mRNA levels of GAD65 and GAD67 in the places measured.

GABA is known to inhibit the release of ACTH and corticosterone in vivo and reduce CRH release from hypothalamic explants.63 It has been noted that PVN projecting GABAergic BST cell populations are part of a feedback circuit that serves to inhibit the stress responses, suggesting that these cell groups should be activated by stressful stimuli.28 Stress-induced GAD65 and GAD67 mRNA increases in the BST subdivisions comport with the results of Bowers et al.28

Interestingly, the stress-induced increases in GAD65 and GAD67 gene expression were reduced by long-term pre-treatment with either imipramine or Hypericum extract. Whereas a decrease in the inhibition of the PVN by the BST seems paradoxical given that the CRH mRNA levels in the PVN are also decreased by antidepressants, a overall return to homeostasis may be the more important aspect of the consequence of chronic administration. The data suggest that inhibition of the HPA axis by a circuit originating in the hippocampus and relayed through the BST is not crucial for restraining the stress response during chronic antidepressant administration. Alternatively, changes in activity of local GABAergic interneurons in the hypothalamus28 may ‘reverse’ the sign of the BST input to the PVN.

5-HT1A mRNA expression

Confirming experiment 1, long-term treatment with imipramine and Hypericum extract significantly decreased 5-HT1A receptor mRNA expression in the hippocampus. Chronic but not acute immobilization stress also reduced levels of 5-HT1A mRNA. The decrease in 5-HT1A gene expression is consistent across various stress experiments.29,64 Imipramine and Hypericum extract were unable to prevent the stress-induced decrease in 5-HT1A mRNA levels in the hippocampus. Our results are different from those of Lopez et al29 who showed that imipramine administered concomitantly with chronic unpredictable stress prevented the reduction in 5-HT1A mRNA expression. The authors suggest that this effect might be steroid-mediated because elevated levels of glucocorticoids have been shown to downregulate 5-HT1A mRNA expression and binding in the hippocampus,65 and imipramine was able to decrease corticosterone levels in chronically stressed rats. In our study, imipramine was also able to reduce the stress-induced increase in corticosterone levels, casting doubt on this explanation. It is possible that differences in the nature of the stressor may underlie the different outcomes.

Imaging studies of primary mood disorder have shown a decrease in 5-HT1A binding capacity in the human mesiotemporal cortex.66,67 In the study by Sargent et al,67 antidepressants did not restore the reduction.

CREB and BDNF mRNA expression

Recent work has raised the possibility that affective disorders may have an organic component that involves cell survival.68 Among the many long-term targets of antidepressant treatments, neurotrophins may play an important role. Neurotrophins promote the growth and development of immature neurons and enhance the survival and function of adult neurons.69 Therefore it can be speculated that antidepressants mediate their actions partially via BDNF, the most abundant neurotrophin in the brain. Stress-induced atrophy of hippocampal neurons may contribute to the loss of hippocampal control of the HPA axis and hypercortisolism that often occurs in depression.70,71 It has been proposed that BDNF expression in the hippocampus may oppose the damage and may be part of a critical link between stress-induced hypercortisolism and antidepressant drug actions.72 Finally, BDNF mRNA expression is regulated by CREB,73 whose activity could thereby underlie some of the long-term effects of antidepressant treatment.74

In this study, acute immobilization stress decreased levels of BDNF mRNA selectively in the dentate gyrus, confirming the results of Smith et al.75 Chronic stress also reduced BDNF gene expression selectively in the dentate gyrus; no effects were observed in the CA fields of the hippocampus. Chronic but not acute stress increased CREB mRNA levels in CA1 and the dentate gyrus. Therefore, the two markers were not linked.

Surprisingly, antidepressant treatment alone (acutely and chronically, with imipramine and Hypericum) also decreased BDNF mRNA levels selectively in the dentate gyrus. In contrast, Nibuya et al72 showed that the antidepressants imipramine, desipramine, sertraline, and mianserin significantly increased BDNF gene expression in the hippocampus after a treatment period of 21 days, whereas Kuroda et al76 found no effect on BDNF mRNA levels after chronic tianeptine treatment. One reason for those discrepancies might be the use of different treatment paradigms. In our present study, antidepressants were administered over a period of 7 weeks, whereas Nibuya et al72 treated rats for 3 weeks. Alternatively, dose differences might underlie the opposite outcomes. Nibuya et al72 administered imipramine at a dose of 15 mg kg−1 i.p., whereas we administered 15 mg kg−1 by gavage. The comparatively high i.p. dose could have side effects that induce BDNF mRNA expression. Further studies are necessary to clarify the role of antidepressants on BDNF gene expression and the relationship between mRNA levels and neurotrophin function vis-à-vis cell proliferation, maintenance, and survival.

Long-term antidepressant pre-treatment had no effect on the stress-induced decrease in BDNF gene expression in the dentate gyrus. Nibuya et al72 showed that long-term pre-treatment (21 days) with several classes of antidepressants was able to antagonize the significant decrease in BDNF mRNA levels after acute (45 min) restraint stress. Together with the data from our study, it can be speculated that chronic antidepressant pre-treatment can prevent the decrease of BDNF gene expression after acute but not chronic stress.

It has been shown that administration of corticosterone (10 mg per rat) reduced the level of BDNF mRNA in the dentate gyrus.75 In our present study, glucocorticoids were significantly elevated after acute and chronic stress. Although chronic treatment with imipramine was able to reduce the increase in plasma corticosterone, baseline levels were not reached. Pre-treatment with Hypericum extract even increased plasma hormone levels. Therefore our data suggest that the decreased induction of BDNF mRNA in the dentate gyrus after chronic stress is mediated via elevated glucocorticoid levels.

In the present study, none of the antidepressants alone had a significant effect on CREB mRNA in the hippocampus, whereas chronic but not acute stress slightly increased its expression. The increased expression was not maintained in the stressed animals receiving chronic imipramine or plant extract. The functional significance of this small effect is not clear, though it supports the hypothesis that hippocampal activity, as reflected by CREB mRNA expression, is positively linked to the activity of the HPA axis.

Nibuya et al74 showed that chronic (21 days) administration of different types of antidepressant drugs significantly increased levels of CREB mRNA in the rat hippocampus. Moreover, increased expression of CREB mRNA was observed after 10 days, but not 3 days, of antidepressant treatment, suggesting that upregulation of CREB is dependent on repeated antidepressant treatment. However, we did not see any drug-induced upregulation at 7 weeks. Therefore, it can be speculated that increases in CREB mRNA levels are related to shorter-term or high-dose effects of antidepressants and that they are not of important relevance for the long-term treatment with therapeutic doses.

TH mRNA expression

The norepinephrine-locus coeruleus and CRH-PVN systems often respond in similar directions in response to acute and chronic stress and to chronic antidepressant drug treatment (reviewed in Brady77). The results of the present study confirm previous reports that chronic stress and antidepressant treatments regulate the norepinephrine system by increasing or decreasing, respectively, the expression of TH in the locus coeruleus.24,45,78 Moreover, the data demonstrate that the increased TH mRNA levels in response to chronic stress could be normalized by pre-treatment with imipramine for 7 weeks. Interestingly, chronic pre-treatment with Hypericum extract again had no effect on TH gene expression alone (as in experiment 1) or in combination with chronic stress, suggesting that the natural antidepressant might have a unique mode of action and a unique therapeutic indication.

Norepinephrine is a potent stimulus of hypothalamic CRH release.79 Because tricyclic antidepressants decrease the firing rate of locus coeruleus neurons,77 the imipramine-induced decrease in TH gene expression may be involved in mediating the decrease in CRH gene expression in the PVN. However, the PVN and the locus coeruleus are only weakly linked anatomically, and a dissociation between gene expression activity in the locus coeruleus (TH) and the PVN (CRH) has been shown in the chronic drug administration paradigm. The ability of antidepressant treatments to maintain control levels of TH in the presence of stress may be relevant to their therapeutic action.


Long-term but not short-term daily administration of all three drugs decreased mRNA expression of CRH mRNA in the hypothalamic PVN. The delayed action of the drugs on the key central determinant of HPA axis activity replicates the earlier work15,16 and validates this model as a test of antidepressant drug actions relevant to clinical efficacy. Long-term administration of the three drugs also reduced POMC mRNA levels in the anterior lobe of the pituitary, though plasma hormone levels of ACTH and corticosterone returned to control levels. These findings are consistent with the concept of a resetting of basal activity in the HPA axis by chronic antidepressant drugs. Long-term administration of all three drugs decreased mRNA levels of the serotonin 5-HT1A receptor in the hippocampus, suggesting common actions on aspects of serotonin transmission. The tested drugs had different effects on TH mRNA expression in the locus coeruleus. The individual neurochemical profiles of imipramine, Hypericum (St John's wort), and hypericin may be relevant to their particular therapeutic efficacies in mood disorders.

Repeated immobilization stress produced a persistent alteration of mRNA expression of CRH in the PVN, POMC in the anterior pituitary, TH in the locus coeruleus, GAD65/67 in the BST, and CREB, BDNF and 5-HT1A in the hippocampus; and it increased plasma hormone levels of ACTH and corticosterone. Long-term pre-treatment with either imipramine or St John's wort reduce to control levels the stress-induced increases in gene expression of GAD65 and GAD67 in the BST and POMC in the anterior pituitary. The stress-induced decreases in BDNF and 5-HT1A mRNA levels in hippocampus were not prevented by either imipramine or St John's wort. The stress-induced increases in mRNA levels of CRH in the PVN and TH in the locus coeruleus were reduced by imipramine but not by the plant extract, indicating that St John's wort only partially affects the stress circuits. Our results, therefore, show that there are differences between the tricyclic antidepressant imipramine and the ‘natural’ herbiceutical St John's wort in their ability to modify the effects of chronic stress. Imipramine appears to be more effective in blocking stress effects on the HPA axis, but both drugs counter gene expression changes induced in select nuclei of the brain's stress circuitry. Imipramine, therefore, may be a more effective antidepressant drug for depressive disorders that share features with chronic stress and activation of the HPA axis.


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Funding was supplied by the NIMH Intramural Research Program and by Lichtwer Pharma (for VB).

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Correspondence to M Herkenham.

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Butterweck, V., Winterhoff, H. & Herkenham, M. St John's wort, hypericin, and imipramine: a comparative analysis of mRNA levels in brain areas involved in HPA axis control following short-term and long-term administration in normal and stressed rats. Mol Psychiatry 6, 547–564 (2001).

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  • antidepressant
  • hypothalamic-pituitary-adrenal
  • corticotropin-releasing hormone
  • paraventricular hypothalamic nucleus
  • norepinephrine
  • locus coeruleus
  • imipramine
  • Hypericum
  • in situhybridization
  • gene expression regulation

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