Extended treatment with repetitive transcranial magnetic stimulation (rTMS) has been demonstrated to ameliorate symptoms in major depression throughout the majority of controlled clinical trials (Klein et al. 1999; Berman et al. 2000; George et al. 2000; Padberg et al. 2002b). Moreover, several lines of evidence based on behavioral animal models as well as neurochemical findings support the notion that rTMS exerts an antidepressant-like action (Fleischmann et al. 1995; Zyss et al. 1997; Ben-Shachar et al. 1999; Kole et al. 1999; Keck et al. 2000).

Neuroactive steroids interacting with the -aminobutyric acid type A (GABA_A) benzodiazepine receptor complex have been suggested to be involved in the pathophysiology of major depression and the action of antidepressant pharmacotherapy (George et al. 1994; Romeo et al. 1998; Uzunova et al. 1998; Rupprecht and Holsboer 1999; Rupprecht et al. 2001). Lowered levels of 3-reduced neuroactive steroids have been found in plasma and cerebrospinal fluid (CSF) of depressed patients (Romeo et al. 1998; Uzunova et al. 1998), which normalized following successful treatment with fluoxetine (Romeo et al. 1998; Uzunova et al. 1998) and other antidepressant drugs (Romeo et al. 1998). On the other hand, dehydroepiandrosterone (DHEA) has been shown to exert beneficial effects on depressive symptoms in a placebo-controlled study (Wolkowitz et al. 1999). In rodents,3-reduced neuroactive steroids and dehydroepiandrosterone sulfate (DHEAS) have been found to exert antidepressant-like effects in the forced swim test (Khisti et al. 2000; Urani et al. 2001). In addition, the formation of 3-reduced neuroactive steroids is enhanced by treatment with selective serotonin reuptake inhibitors (SSRI) (Uzunov et al. 1996; Griffin and Mellon 1999). Currently it is not known whether the normalization of altered neuroactive steroid concentrations is a prerequisite for the alleviation of depressive symptoms or due to a specific pharmacological action of antidepressant drugs. In the present study we therefore investigated whether repetitive transcranial magnetic stimulation (rTMS) as a novel non-pharmacological treatment in major depression affects plasma concentrations of neuroactive steroids.

Thirty-seven inpatients were included in an open-label protocol (age: 51.5 ± 14.8 years, 23 women, 14 men). Patients met DSM-IV criteria for a major depressive episode. All patients gave their written informed consent for this study after the procedure had been fully explained. The study was approved by the local ethical committee. Prior to the study, antidepressant medication and benzodiazepines were washed out for at least seven days and patients remained medication-free during the entire study.

Repetitive transcranial magnetic stimulation was applied as reported elsewhere (Padberg et al. 2002a). Patients underwent 10 sessions of rTMS (10 Hz, 15 trains of 10 s each, 30 s inter-train interval) of the left dorsolateral prefrontal cortex within two weeks at 100% stimulation intensity related to the individual motor threshold. Severity of depression was assessed using the 21-item Hamilton Rating Scale for Depression (HRSD) (Hamilton 1960). Response after rTMS was defined as 50% reduction of the baseline HRSD score and remission by a HRSD score of 9.

Plasma samples were obtained at 8:00 A.M. before the first rTMS (day 0) and the day after the last rTMS session (day 14). After extraction with ethyl acetate, 4-pregnene-3,20-dione (progesterone), 5-pregnan-3-ol-20-one (3,5-tetrahydroprogesterone; 3,5-THP), 5-pregnan-3-ol-20-one (3,5-tetrahydroprogesterone; 3,5-THP), 5-pregnan-3-ol-20-one (3,5-tetrahydroprogesterone; 3, 5-THP) and 5-androsten-3-ol-17-one (dehydroepiandrosterone; DHEA) were quantified using a highly sensitive and specific combined gas chromatography/mass spectrometry analysis as previously described (Strohle et al. 2002). A Finningham Trace gas chromatography/mass spectrometry unit equipped with a capillary column was used to analyze the derivatized steroids in the negative ion chemical ionization mode. The detection limit was approximately 10 fmol.

Results are expressed as mean ± SEM. Two-tailed t-tests were used to compare HRSD scores before and after rTMS and responders versus non-responders regarding their demographic variables and HRSD scores at baseline. The treatment effect of rTMS on steroid concentrations was tested about significance using a one-factorial multivariate analysis of variance (MANOVA) with treatment as within-subjects factor. Responders and non-responders were compared before and after rTMS by Wilks' multivariate test applying in each case a one-factorial MANOVA with group as between-subjects factor. Correlation analysis was performed using Pearson's correlation coefficients. The level of significance was set at p < .05.

In the overall patient group HRSD scores were significantly reduced from 24.2 ± 0.9 at baseline to 15.9 ± 1.8 after rTMS (t = 4.8; df = 36; p < .001; Figure 1). After rTMS treatment, 49% of patients were responders and 32% were remitters. Demographic variables and baseline HRSD scores did not differ between responders and non-responders.

Plasma concentrations of neuroactive steroids are shown in Figure 2. There was no change in either neuroactive steroid studied after rTMS compared with baseline. This was the case in the overall patient group and when male and female patients were analyzed separately (data not shown). Moreover, MANOVA revealed no significant group effect between responders and non-responders at baseline or after rTMS treatment. Similarly, there were no significant differences between remitters and non-remitters (data not shown). No significant correlation was found between percent reduction of HRSD scores after rTMS and baseline levels of neuroactive steroids or their changes after treatment (r ranging from -0.229 to 0.285). Baseline HRSD scores and levels of neuroactive steroids at baseline were not correlated either (r ranging from -0.196 to 0.267).

In contrast to antidepressant pharmacotherapy (Romeo et al. 1998; Uzunova et al. 1998), rTMS did not affect plasma concentrations of neuroactive steroids. There was no change in neuroactive steroid levels associated with clinical recovery in spite of moderate to substantial antidepressant effects of rTMS treatment.

Recently, 3-reduced neuroactive steroids (3,5-THP and 3,5-THP) have been proposed to play a causal role in the pathogenesis of major depression and its treatment (Romeo et al. 1998; Rupprecht and Holsboer 1999; Rupprecht et al. 2001). 3,5-THP and 3,5-THP are potent positive allosteric modulators of various GABA_A-receptor subtypes (Rupprecht et al. 2001). Moreover, 3,5-THP exerts antidepressant like effects in the forced swim test which can be potentiated by serotonergic agents (Khisti et al. 2000; Khisti and Chopde 2000).

Concentrations of 3,5-THP and 3,5-THP are decreased in plasma and cerebrospinal fluid during depression and normalize during treatment with fluoxetine and other antidepressant drugs (Romeo et al. 1998; Uzunova et al. 1998). In contrast, plasma concentrations of 3,5-THP, an antagonistic isomer of GABAergic steroids, are increased in depression (Romeo et al. 1998). In previous studies it has not been possible to differentiate effects due to amelioration of clinical symptoms from direct pharmacological effects of antidepressants. In view of recent molecular data showing that selective serotonin reuptake inhibitors may shift the activity of the 3-hydroxysteroid oxidoreductase toward the reductive direction, thereby increasing the formation of endogenous 3-reduced neuroactive steroids (Griffin and Mellon 1999), it is of particular interest to investigate the action of non-pharmacological antidepressant interventions on the concentrations of neuroactive steroids. Increased concentrations of DHEAS, the sulfate derivative of DHEA, have been suggested to predict non-response to electroconvulsive therapy (ECT) (Maayan et al. 2000). However, concentrations of 3-reduced neuroactive steroids during ECT have not been investigated to date.

In the present study, no changes in neuroactive steroid levels were found after clinical improvement following rTMS treatment. Therefore, the previously reported changes in neuroactive steroid concentrations following antidepressant pharmacotherapy (Romeo et al. 1998; Uzunova et al. 1998) more likely reflect specific pharmacological effects of antidepressant drugs (Griffin and Mellon 1999) that may contribute to symptom improvement, rather than a common mechanism leading to the alleviation of depressive symptoms following successful treatment in general, regardless of the treatment modality. The open label study design and the fact that rTMS is not yet a clinically established antidepressant intervention are limitations for the interpretation of our findings. Clinical improvement after rTMS may be due to a specific effect of rTMS or a placebo effect, or more likely a combination of both. Nevertheless, changes in neuroactive steroid levels are not mandatory for the alleviation of depressive symptoms. Future studies should investigate the influence of other non-pharmacological treatments of depression, e.g. sleep deprivation, ECT, or vagus nerve stimulation (VNS), on the concentrations of neuroactive steroids. Although changes in neuroactive steroids apparently do not explain the antidepressant efficacy of rTMS, these molecules nevertheless may contribute to the efficacy of treatment with antidepressant drugs in various psychiatric disorders.

Acknowledgements

The authors wish to thank Dr. Patrick Mikhaiel and Dr. Heike Thoma for conducting the rTMS treatment. They further thank Mr. J. Scandone and Mr. Peter Houseman from the Magstim Company for their technical support. This project was supported by the German Ministry for Education and Research within the promotional emphasis "German Research Network on Depression" (Subproject 4.3, R.R., Subproject 6.5, F.P.), the Gerhard Hess Program of the German Research Foundation and the Max-Planck Society.