Cerebral Cortex
Christine E. Marx, Gary E. Duncan, John H. Gilmore, Jeffrey A. Lieberman,
and A. Leslie Morrow
Background: The neurosteroid allopregnanolone (3a -hydroxy-5a-pregnan-20-one) has anxiolytic and anticon-vulsant properties, potentiating GABAAreceptor chloride channel function with 20-fold higher potency than benzo-diazepines. Behavioral studies demonstrate that olanzap-ine has anxiolyticlike properties in animals, but the mechanism responsible for these effects is not clear. We examined the effect of acute olanzapine administration on cerebral cortical allopregnanolone and its relationship to serum progesterone and corticosterone levels in rats. Methods: Male Sprague-Dawley rats were habituated to intraperitoneal (IP) saline injection for 5 days. On the day of the experiment, rats were injected with olanzapine (0, 2.5, 5.0, or 10.0 mg/kg IP, 10 –11 rats per condition). Rats were sacrificed 1 hour later, and cerebral cortical allo-pregnanolone levels and serum progesterone and cortico-sterone levels were measured by radioimmunoassay. Results: Olanzapine increases cerebral cortical allopreg-nanolone up to fourfold, depending on dose. Positive correlations were observed between cerebral cortical allopregnanolone and serum progesterone levels and between cerebral cortical allopregnanolone and serum corticosterone levels.
Conclusions: Olanzapine-induced increases in the potent GABAA receptor modulator allopregnanolone may alter GABAergic neurotransmission, possibly contributing to antipsychotic efficacy. If allopregnanolone alterations are linked to psychotic symptom relief, neurosteroids may represent molecules for pharmacologic intervention. Biol Psychiatry 2000;47:1000 –1004 © 2000 Society of Biological Psychiatry
Key Words: Allopregnanolone, olanzapine, GABA, neu-rosteroid, schizophrenia
Introduction
N
eurosteroids are potent gamma-aminobutyric acid (GABA)Aand/or weak N-methyl-D-aspartate (NMDA)receptor modulators, mediating their effects through mem-brane-bound ion-gated channel receptors rather than through classical nuclear steroid hormone receptors (Ro-bel and Baulieu 1995). They are synthesized in the central nervous system de novo from cholesterol or from periph-eral steroid precursors such as progesterone (Paul and Purdy 1992). The neurosteroid allopregnanolone (3a -hydroxy-5a-pregnan-20-one) is synthesized in the adrenal medulla, ovary, and brain by the reduction of progesterone by 5a-reductase and 3a-hydroxysteroid dehydrogenase. Although most brain allopregnanolone appears to be derived from peripheral progesterone precursor, it can also be synthesized entirely within the brain from cholesterol. Allopregnanolone demonstrates anxiolytic (Crawley et al 1986; Wieland et al 1991) and anticonvulsant effects (Belelli et al 1989; Devaud et al 1995), potentiating GABAergic neurotransmission with 20-fold higher po-tency than benzodiazepines (Morrow et al 1987, 1990). Allopregnanolone also increases with stress (Purdy et al 1991) and pregnancy (Paul and Purdy 1992) and varies across the menstrual (Genazzani et al 1998) and estrous cycles (Corpechot et al 1997).
Recent evidence suggests that neurosteroids such as allopregnanolone may be important in schizophrenia. Spe-cifically, allopregnanolone appears to affect dopaminergic neurotransmission (Grobin et al 1992; Khisti et al 1998; Motzo et al 1996). Intraventricular allopregnanolone in-duces catalepsy in mice, which can be reversed with the dopaminergic agonists bromocriptine and levodopa/carbi-dopa and the GABAergic antagonists picrotoxin and bicuculline (Khisti et al 1998). In addition, these cataleptic effects of allopregnanolone are potentiated by the dopa-minergic antagonist haloperidol (Khisti et al 1998). Allo-pregnanolone also reduces stress-induced dopamine re-lease in the prefrontal cortex and nucleus accumbens (Grobin et al 1992; Motzo et al 1996). These effects on dopaminergic function likely are mediated by GABAA
receptors because allopregnanolone potentiates
GABAer-From the Departments of Psychiatry (CEM, GED, JHG, JAL, ALM), Pharmacol-ogy (JAL, ALM), and RadiolPharmacol-ogy (JAL) and the UNC Mental Health and Neuroscience Clinical Research Center (CEM, GED, JHG, ALM), University of North Carolina at Chapel Hill, Chapel Hill.
Address reprint requests to Christine E. Marx, M.D., UNC School of Medicine, Department of Psychiatry, Campus Box #7160, Chapel Hill NC 27599-7160. Received August 11, 1999; revised November 17, 1999; accepted December 2,
1999.
© 2000 Society of Biological Psychiatry 0006-3223/00/$20.00
gic neurotransmission and does not act at dopamine receptors. GABAA receptor activity may be particularly
relevant to schizophrenia because converging evidence implicates altered GABAergic neurotransmission in the illness, and GABAAreceptor agonists, such as
benzodiaz-epines, are helpful in alleviating symptoms of the disorder in certain settings (Carpenter et al 1999). Finally, allopreg-nanolone levels in plasma and brain are present in greater quantities in women and may be important in the neuro-biology of gender differences in schizophrenia.
Similar to allopregnanolone, the antipsychotic olanzap-ine also demonstrates anxiolyticlike effects in animals. For example, animals treated with olanzapine demonstrate anticonflict effects (Moore et al 1994; Nanry et al 1995), and olanzapine decreases the acquisition of conditioned freezing (Inoue et al 1996). These anxiolyticlike effects of olanzapine suggest that this agent may alter neurosteroid biosynthesis. Interestingly, the benzodiazepine antagonist flumazenil reduces the anticonflict effect of chlordiazep-oxide but not olanzapine (Moore et al 1994), suggesting that the anxiolytic activity of olanzapine may not be mediated by the GABAA receptor benzodiazepine site.
Because allopregnanolone does not act at the benzodiaz-epine recognition site on GABAAreceptors (Morrow et al
1990), it is possible that the anxiolytic effects of olanza-pine may be mediated by the induction of allopreg-nanolone in brain. The purpose of this study was to determine if olanzapine modulates the endogenous GABAA receptor-active neurosteroid allopregnanolone,
possibly contributing to its clinical efficacy.
Methods and Materials
All animal care and use was conducted in accordance with IACUC guidelines. Male Sprague-Dawley rats weighing 200 – 250 g were obtained from Harlan (Indianapolis, IN). Rats were habituated to intraperitoneal saline injection for 5 days prior to the day of the experiment to minimize possible stress-induced increases in brain allopregnanolone. Rats were housed three per cage and placed on a 12-hour, light-dark cycle. Food and water were available ad libitum. To limit circadian variations in steroid levels, all animals were sacrificed between the hours of 9AMand noon.
Olanzapine, kindly provided by Eli Lilly and Company (Indi-anapolis, IN), was dissolved in acetic acid then buffered with NaOH (pH56). On the day of the experiment, intraperitoneal olanzapine was administered at doses of 0, 2.5, 5.0, and 10.0 mg/kg, 10 –11 rats per condition. Rats were sacrificed by decapitation 1 hour later. Cerebral cortex was rapidly dissected on ice and stored at280°C until assayed for allopregnanolone. Trunk blood was collected for serum progesterone and cortico-sterone level determination and stored on ice until centrifugation for serum collection.
Serum progesterone and corticosterone levels were measured in duplicate with radioimmunoassay kits using125
I-progesterone
and125
I-corticosterone (ICN Biomedicals, Inc. Costa Mesa, CA). The limits of detection for the progesterone and corticosterone assays were 0.1 and 10 ng/mL, respectively. Allopregnanolone levels were measured in cerebral cortex by radioimmunoassay as described by Janis et al (1998), with a modification of the methods of Purdy et al (1990) using a sheep polyclonal antibody kindly provided by CoCensys Inc. (Irvine, CA). Sample extracts in sodium phosphate-BSA buffer were incubated with antiserum and [3
H]allopregnanolone for a minimum of 60 min. Bound steroids were separated from free steroids by the addition of ice-cold, dextran-coated charcoal, followed by centrifugation for 20 min at 2000 g. Supernatants were collected and counted by liquid scintillation spectroscopy. Sample values were compared with a concurrently run allopregnanolone standard curve pro-duced using a one-site competition model (Prism2, GraphPad, San Diego, CA). Sample values were adjusted to account for the previously determined extraction efficiency. The sensitivity of the assay was 25 pg. The allopregnanolone antiserum used in this study has been shown to produce minimal cross-reactivity with other circulating steroids (Finn and Gee 1994).
Statistical analysis of the results was conducted by analyses of variance (ANOVA; with post-hoc Dunnett tests) or Linear Regression Analysis.
Results
Intraperitoneal olanzapine injection dose-dependently in-creased cerebral cortical allopregnanolone levels up to fourfold [Figure 1A, ANOVA F(3,38) 5 3.828, p 5
.0172, post hoc Dunnett p,.05 for olanzapine 10 mg/kg dose]. Maximal allopregnanolone response to 4 ng/g was observed with 10 mg/kg olanzapine. Serum progesterone levels increased after acute olanzapine injection in a dose-dependent manner [Figure 1B, ANOVA F(3,38)5
8.020, p5.0003, post hoc Dunnett p,.01 for olanzapine doses 5 and 10 mg/kg]. A positive correlation was ob-served between cerebral cortical allopregnanolone levels and serum progesterone levels [Figure 2A, Linear Regres-sion Analysis, Pearson correlation coefficient r 5 .8911, F(1,40)5154.18, p,.0001]. Serum corticosterone levels also increased dose-dependently after acute olanzapine injection [Figure 1C, ANOVA F(3,38) 5 13.608, p ,
.0001, post hoc Dunnett p, .01 for olanzapine doses 5 and 10 mg/kg]. A positive correlation was observed between cerebral cortical allopregnanolone levels and serum corticosterone levels [Figure 2B, Linear Regression Analysis, Pearson correlation coefficient r 5 .7503, F(1,40)551.527, p ,.0001].
Discussion
GABAergic neurotransmission (Morrow et al 1987). Com-parable effects on allopregnanolone levels have been observed in hypothalamus using a swim-stress paradigm (Purdy et al 1991). It therefore is likely that allopreg-nanolone affects GABA neurotransmission at the concen-trations observed in this study.
As a potent GABAA receptor modulator,
allopreg-nanolone may be particularly relevant to the apparent GABAergic deficit observed in schizophrenia. Postmor-tem studies of patients with schizophrenia have demon-strated a reduction in GABA interneurons, an upregulation of GABAAreceptor binding activity, and a decrease in the
synthetic GABA enzyme GAD67, supporting the
hypoth-esis that GABAergic neurotransmission is altered in schizophrenia (Akbarian et al 1995; Benes et al 1991,
1992, 1997). A deficit in GABAergic neurons expressing the calcium-binding protein parvalbumin (Beasley and Reynolds 1997; Reynolds et al 1999) and a decrease in the density of axon terminal “cartridges” for the GABA membrane transporter GAT-1 (Woo et al 1998) in the prefrontal cortex of schizophrenia have also been demon-strated. An acute olanzapine-induced increase in the potent GABAAreceptor modulator allopregnanolone may
repre-sent a mechanism by which this antipsychotic alters GABAergic neurotransmission, possibly contributing to the efficacy of this drug.
Clinically, studies examining GABAAmodulators, such
as benzodiazepines, in the treatment of schizophrenia symptoms have yielded mixed results. A recent study, however, found that diazepam was superior to placebo and comparable to fluphenazine in preventing symptom pro-gression during early symptom exacerbation in schizo-phrenia (Carpenter et al 1999). Because allopregnanolone,
Figure 1. (A) Cerebral cortical allopregnanolone levels in-creased following acute olanzapine injection in a dose-dependent manner, analysis of variance (ANOVA) F(3,38)53.828, p5
.0172, post hoc Dunnett p ,.05 for olanzapine 10 mg/kg. (B) Serum progesterone levels increased following acute olanzapine injection in a dose-dependent manner, ANOVA F(3,38)58.020, p5.0003, post hoc Dunnett p, .01 for olanzapine 5 and 10 mg/kg. (C) Serum corticosterone levels increased following acute olanzapine injection in a dose-dependent manner, ANOVA F(3,38) 5 13.608, p , .0001, post hoc Dunnett p, .01 for olanzapine 5 and 10 mg/kg.
similar to benzodiazepines, diminishes dopaminergic neu-rotransmission by modulation of dopamine release (Grobin et al 1992; Motzo et al 1996; van Kammen 1977), it is possible that these GABAergic effects could have therapeutic actions. Allopregnanolone may have more pronounced antidopaminergic effects than benzodiaz-epines because it induces catalepsy in mice (Khisti et al 1998). Allopregnanolone may also have a broader spec-trum of action than benzodiazepines because benzodiaz-epines bind a limited population of GABAAreceptors that
containaandgsubunits (Pritchett et al 1989). Allopreg-nanolone, in contrast, demonstrates equal potency and efficacy on several GABAAreceptor subtypes assembled
only withb, withaorb, or witha,b, andgsubunits (Puia et al 1990). Given evidence suggesting a GABAergic deficit in the pathophysiology of schizophrenia, it is particularly interesting that an antipsychotic such as olan-zapine increases cerebral cortical allopregnanolone, which potentiates GABAergic neurotransmission. Future experi-ments utilizing antipsychotics other than olanzapine, both typical and atypical, will be required to determine the specificity of this effect.
Our data suggest that olanzapine-induced increases in cerebral cortical allopregnanolone levels are correlated with increases in serum progesterone (r 5 .89). The degree to which serum progesterone serves as a precursor to olanzapine-induced allopregnanolone biosynthesis in brain is currently not known and will require further study using adrenalectomized animals. It is possible that serum progesterone is a peripheral indicator of altered cerebral cortical allopregnanolone levels in response to acute olan-zapine administration.
The neurochemical mechanisms responsible for olanza-pine-induced HPA axis activation resulting in dose-depen-dent serum progesterone and corticosterone elevations are currently unknown. Because brain allopregnanolone and serum corticosterone levels were correlated in our exper-iments, the potential actions of allopregnanolone on the HPA axis may be important. It is known that corticotropin-releasing factor (CRF) release from the hypothalamus is decreased by activation of GABAAreceptors in the
hypo-thalamus (Calogero et al 1988). Allopregnanolone de-creases CRF (Patchev et al 1994), adrenocorticotropin hormone (ACTH; Patchev et al 1996), and corticosterone (Guo et al 1995; Patchev et al 1996) release in rats. Stress-induced increases in brain allopregnanolone there-fore would ultimately suppress the HPA axis. This process may represent an endogenous autoregulatory mechanism to restore homeostasis after a stressful event (Morrow et al 1995). It is possible that a similar autoregulatory mecha-nism involving allopregnanolone may be activated by treatment with olanzapine (or other antipsychotics) and that this mechanism is important in the clinical actions of
olanzapine. This action may be particularly relevant to schizophrenia pathophysiology because recent evidence indicates that suppression of glucocorticoid secretion and antipsychotic drugs have similar effects on mesolimbic dopaminergic transmission (Piazza et al 1996).
In summary, these preclinical results demonstrating that olanzapine alters cerebral cortical allopregnanolone may be clinically relevant, and it is possible that allopreg-nanolone may play a role in mediating the therapeutic actions of olanzapine and its consequent symptom reduc-tion. Olanzapine also may alter other neurosteroids in addition to allopregnanolone, and this possibility will require further investigation. Clinical studies will be needed to elucidate a possible relationship between olan-zapine-induced changes in neurosteroids and the patho-physiology of schizophrenia. If neurosteroid alterations are linked to psychotic symptom relief, neuroactive ste-roids may represent molecules for pharmacologic intervention.
This research was supported by NIH Grants Nos. 5-ROI-AA10564 and 2-P30-MH33127. The authors thank Dr. Chistina Grobin for helpful discussion, Tejas Patel for excellent technical assistance with allopreg-nanolone assays, and Jeremy Leipzig for assistance in animal handling. Presented as an abstract at the VIIth International Congress on Schizo-phrenia Research, April 1999, Santa Fe, New Mexico.
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