Neuroactive steroids and affective disorders.

Neuroactive steroids modulate neurotransmission through modulation of specific neurotransmitter receptors such as gamma-aminobutyric acid type A (GABA(A)) receptors. Preclinical studies suggested that neuroactive steroids may modulate anxiety and depression-related behaviour and may contribute to the therapeutical effects of antidepressant drugs. Attenuations of such neuroactive steroids have been observed during major depression and in several anxiety disorders, suggesting a pathophysiological role in such psychiatric conditions. In panic disorder patients a dysequilibrium of neuroactive steroid composition has been observed, which may represent a counterregulatory mechanism against the occurrence of spontaneous panic attacks. Furthermore, alterations of 3alpha-reduced pregnane steroids during major depression were corrected by successful treatment with antidepressant drugs. However in contrast, non-pharmacological antidepressant treatment strategies did not affect neuroactive steroid composition. In addition, changes in neuroactive steroid concentrations after mirtazapine therapy occurred independently from the clinical response, thereby suggesting that changes in neuroactive steroid concentrations more likely reflect direct pharmacological effects of antidepressants rather than clinical improvement in general. Nevertheless, the effects of antidepressant pharmacotherapy on the composition of neuroactive steroids may contribute to the alleviation of certain depressive symptoms, such as amelioration of anxiety, inner tension or sleep disturbances. Moreover, first studies investigating the therapeutical effects of dehydroepiandrosterone revealed promising results in the treatment of major depression. In conclusion, neuroactive steroids are important endogenous modulators of depression and anxiety and may provide a basis for development of novel therapeutic agents in the treatment of affective disorders.

Permissive role of brain allopregnanolone content in the regulation of pentobarbital-induced righting reflex loss.

Allopregnanolone [3alpha-hydroxy-5alpha-pregnan-20-one] (ALLO), a potent neurosteroid that positively modulates gamma-aminobutyric acid (GABA) action at various GABA(A) receptor subtypes is synthesized in nanomolar concentrations and stored non uniformly in various brain structures of mammals. We have measured brain ALLO content and its precursors by negative ion chemical ionization-mass-spectrometry after purification and separation of the different steroids with HPLC and gas chromatography. Our procedure measures steroids in the femtomolar range with structural information and unsurpassed selectivity. We were able to establish an association between the decrease in content of ALLO in mouse brain cortex elicited by either long-lasting social isolation or by the administration of 17beta-17 [bis (1-methylethyl) amino carbonyl] androstane-3,5-dilene-3-carboxylic acid (SKF 105111). an inhibitor of Types I and II 5alpha reductases, and the shortening of the righting reflex loss elicited by pentobarbital (PBT). SKF 105111 added to cortical brain slices in concentrations up to 10(-5) M failed per se to alter GABAergic currents or their potentiation by PTB recorded from pyramidal neurons. Fluoxetine (1.45 or 2.9 micromol/kg i.p.) doses that fail to change the PTB-induced loss of righting reflex and the level of brain ALLO in group-housed mice normalized both parameters in socially-isolated mice. In addition, we could detect both fluoxetine actions in socially isolated mice pretreated with doses of p-chlorophenylalanine (1.2 mmol/kg i.p. at 72, 48, and 24 h) that substantially inhibit brain serotonin 5HT synthesis as shown by an 80% drop of brain 5HT content. These studies for the first time have provided evidence suggesting that the endogenous cortical stores of ALLO physiologically upregulate GABAergic tone and by such a mechanism play a permissive or facilitatory role on the PTB-induced loss of the righting reflex. In the absence of such a permissive physiological influence by endogenous ALLO, the righting reflex inhibition by PTB is down regulated.

Simultaneous detection of glutamic acid decarboxylase and reelin mRNA in adult rat neurons using in situ hybridization and immunofluorescence.

The combination of in situ hybridization and immunocytochemical technique is an important tool to detail the biochemical phenotype of individual neurons. In this work, we have developed a double fluorescence method to show the presence of reelin mRNA in GABAergic cells. This was achieved by demonstrating the colocalization of glutamic acid decarboxylase67, the synthesizing enzyme for GABA, with the mRNA for reelin, a novel factor involved in brain development and possibly the maintenance of the synaptic organization of layered structures in adult brain. The results demonstrated that reelin is expressed primarily in GABAergic cells in the adult rat cerebrum, but not in the cerebellum.

Enantiomeric resolution with a new chiral stationary phase of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide, a cognition-enhancing benzothiadiazine derivative.

The direct analytical and semipreparative high-performance liquid chromatographic (HPLC) resolution of the enantiomers of IDRA 21 [1,7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide] is reported. (+/-)-IDRA 21 administered orally to rats subjected to a water maze cognition test elicited a performance enhancing effect. Between the two enantiomers, (+)-IDRA 21 was identified as being pharmacologically active in the water maze performance test, whereas (-)-IDRA 21 was completely devoid of activity when given in doses comparable to those of the dextrorotatory (+)-enantiomer. The design and preparation of a new chiral stationary phase (CSP) employed for the liquid chromatographic resolution of the enantiomers of racemic IDRA 21 is presented. This brush-type CSP, which has not been described before, is a "mixed" (pi-donor pi-acceptor) type and is derived from (R)-N-(3,5-dinitrobenzoyl) allylglycine 2,6-dimethylanilide. It is easily prepared and possesses a relatively broad scope of applicability, as determined by its ability to resolve the enantiomers of both pi-acidic and pi-basic compounds.

Influence of mirtazapine on plasma concentrations of neuroactive steroids in major depression and on 3alpha-hydroxysteroid dehydrogenase activity.

Concentrations of 3alpha-reduced neuroactive steroids are altered in depression and normalize after antidepressant pharmacotherapy with selective serotonin re-uptake inhibitors (SSRIs). We investigated the impact of mirtazapine on the activity of a key neurosteroidogenic enzyme, the 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), and on the levels of neuroactive steroids in relation to clinical response. A total of 23 drug-free in-patients suffering from a major depressive episode (DSM-IV criteria) underwent 5-week treatment with mirtazapine (45 mg/day). Plasma samples were taken weekly at 0800 and quantified for neuroactive steroids by means of combined gas chromatography/mass spectrometry analysis. Enzyme activity was determined by assessment of steroid conversion rates. Irrespective of clinical outcome, there were significant increases in 3alpha,5alpha-tetrahydroprogesterone, 3alpha,5beta-tetrahydroprogesterone, 5alpha-dihydroprogesterone, and 5beta-dihydroprogesterone after mirtazapine treatment, whereas 3beta,5alpha-tetrahydroprogesterone levels were significantly decreased. In vitro investigations demonstrated a dose-dependent inhibitory effect of mirtazapine on the activity of the microsomal 3alpha-HSD in the oxidative direction (conversion of 3alpha,5alpha-tetrahydroprogesterone to 5alpha-dihydroprogesterone). Mirtazapine affects neuroactive steroid composition similarly as do SSRIs. The inhibition of the oxidative pathway catalyzed by the microsomal 3alpha-HSD is compatible with an enhanced formation of 3alpha-reduced neuroactive steroids. However, the changes in neuroactive steroid concentrations more likely reflect direct pharmacological effects of this antidepressant rather than clinical improvement in general.

Fluoxetine-elicited changes in brain neurosteroid content measured by negative ion mass fragmentography.

Fluoxetine administered intraperitoneally to sham-operated or adrenalectomized/castrated (ADX/CX) male rats dose-dependently (2.9-58 mumol/kg i.p.) increased the brain content of the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, 3 alpha, 5 alpha-TH PROG). The increase of brain 3 alpha, 5 alpha-TH PROG content elicited by 58 mumol/kg fluoxetine lasted more than 2 hr and the range of its extent was comparable in sham-operated (approximately 3-10 pmol/g) and ADX/CX rats (2-9 pmol/g) and was associated with a decrease (from 2.8 to 1.1 pmol/g) in the 5 alpha-pregnan-3,20-dione (5 alpha-dihydroprogesterone, 5 alpha-DH PROG) content. The pregnenolone, progesterone, and dehydroepiandrosterone content failed to change in rats receiving fluoxetine. The extent of 3 alpha, 5 alpha-TH PROG accumulation elicited by fluoxetine treatment differed in various brain regions, with the highest increase occurring in the olfactory bulb. Importantly, fluoxetine failed to change the 3 alpha, 5 alpha-TH PROG levels in plasma, which in ADX/CX rats were at least two orders of magnitude lower than in the brain. Two other serotonin re-uptake inhibitors, paroxetine and imipramine, in doses equipotent to those of fluoxetine in inhibiting brain serotonin uptake, were either significantly less potent than fluoxetine (paroxetine) or failed to increase (imipramine) 3 alpha, 5 alpha-TH PROG brain content. The addition of 10 microM of 5 alpha-DH PROG to brain slices of ADX/CX rats preincubated with fluoxetine (10 microM, 15 min) elicited an accumulation of 3 alpha, 5 alpha-TH PROG greater than in slices preincubated with vehicle. A fluoxetine stimulation of brain 3 alpha, 5 alpha-TH PROG biosynthesis might be operative in the anxiolytic and antidysphoric actions of this drug.

Reelin is preferentially expressed in neurons synthesizing gamma-aminobutyric acid in cortex and hippocampus of adult rats.

During embryonic development of brain laminated structures, the protein Reelin, secreted into the extracellular matrix of the cortex and hippocampus by Cajal-Retzius (CR) cells located in the marginal zone, contributes to the regulation of migration and positioning of cortical and hippocampal neurons that do not synthesize Reelin. Soon after birth, the CR cells decrease, and they virtually disappear during the following 3 weeks. Despite their disappearance, we can quantify Reelin mRNA (approximately 200 amol/ g of total RNA) and visualize it by in situ hybridization, and we detect the translated product of this mRNA by immunocytochemistry preferentially in gamma-aminobutyric acid (GABA)ergic neurons of adult rat cortex and hippocampus. In adult rat cerebellum, Reelin is expressed in glutamatergic neurons (granule cells). The translated product of this mRNA is readily exported from the granule cell somata to the parallel fibers, where it has been detected by electron microscopy in axon terminals located presynaptically to Purkinje cell dendrites.

Increase in the cerebrospinal fluid content of neurosteroids in patients with unipolar major depression who are receiving fluoxetine or fluvoxamine.

We recently reported that fluoxetine or paroxetine, two selective serotonin reuptake inhibitors (SSRIs), when administered to rats, increase the brain content of the neurosteroid 3alpha-hydroxy-5alpha-pregnane-20-one (3alpha5alpha-ALLO) without altering the brain content of other neurosteroids. ALLO (3alpha5alpha and 3alpha5beta isomers) binds with high affinity to various gamma-aminobutyric acid (GABA) receptor A subtypes and facilitates the action of GABA at these receptors. We hypothesized that the increase of ALLO brain content induced by treatment with SSRIs could contribute to alleviating the anxiety and dysphoria associated with the symptomatology of major unipolar depression. We measured ALLO content in four cisternal-lumbar fractions of cerebrospinal fluid (CSF) before and 8-10 weeks after treatment with fluoxetine or fluvoxamine in 15 patients with unipolar major depression. The concentration of ALLO ( approximately 40 fmol/ml in each CSF fraction of three control subjects) was about 60% lower in patients with major unipolar depression. However, in the same patients, fluoxetine or fluvoxamine treatment normalized the CSF ALLO content. Moreover, a statistically significant correlation (r = 0.58; P < 0.023; n = 15) existed between symptomatology improvement (Hamilton Rating Scale for Depression scores) and the increase in CSF ALLO after fluoxetine or fluvoxamine treatment. The CSF content of PREG and PROG remained unaltered after treatment and failed to correlate with the SSRI-induced increase of CSF ALLO. The normalization of CSF ALLO content in depressed patients appears to be sufficient to mediate the anxiolytic and antidysphoric actions of fluoxetine or fluvoxamine via its positive allosteric modulation of GABA type A receptors.

A decrease of reelin expression as a putative vulnerability factor in schizophrenia.

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.