- Email: [email protected]
Differential accumulation of cortisol and corticosterone in the murine brain
S10
I•--•
Molecular Neuropsychopharmacology Decreased D-serine levels in CSF of schizophrenia patients
S. Amar 1, I. Bendikov2, C. Nadri 1, R.A. Panizzutti3, J. De Miranda 3, H. Wotosker2, G. Again 1. iBen
Gurion Uniuersity of the Negev, Clinical Biochemistry, Beer-Sheua, Israel," 2Technion-Israel Institute of Technology, Biochemistry, Haifa, Israel," 3Federal Uni~)ersity of Rio de Janeiro, Anatomy, Rj, Brazil Introduction: Accumulating evidence suggests that NMDA receptor hypofunction contributes to the symptomatic features of schizophrenia (Harrison et al 2005). A unique feature of the NMDA receptor is that the channel only operates when the sites of both glutamate and a coagonist are occupied. D-Serine has been shown to be a major endogenous coagonist of the NMDA receptors. Endogenous D-serine is synthesized from L-serine by serine racemase. Degradation of D-serine in the cerebellum and brainstem is mediated by D-amino acid oxidase (DAAO). Based on the NMDA receptor hypofunction hypothesis several clinical trials investigated whether administration of NMDA receptor coagonists is beneficial for the patients. When combined with conventional neuroleptics or newer atypical antipsychotics D-serine improved positive, negative and cognitive symptoms of schizophrenia patients (Heresco-Levy et al 2005). Moreover, decreased serum D-serine levels were found in schizophrenic subjects (Yamada et al 2005). The present study was carried out to further elucidate whether the metabolism of D-serine is involved in the pathophysiology and treatment of schizophrenia. Methods: Levels of D-serine, L-serine, L-glutamate and L-glutamine were measured by high-performance liquid chromatography in (i) CSF from 12 schizophrenia patients and matched controls; (ii) postmortem parietal cortex from 15 subjects of each of four matched groups of controls, schizophrenia, major depression and bipolar patients; (iii) brain samples from rats chronically treated with haloperidol, clozapine or vehicle. In addition, serine racemase and DAAO protein levels were quantified in postmortem brain samples from 15 schizophrenia patients and 15 matched controls. Results: A 25% decrease in D-serine levels and 28% decerease in D/L-serine ratio was observed in CSF of schizophrenia patients us. controls. Three L-amino acids (serine, glutamate and glutamine) levels were unaltered. Parietal cortex D-serine and the three L-amino acid levels were the same in all diagnostic groups. Chronic antipsychotic treatment did not alter frontal cortex D-serine levels in rats. Frontal cortex (39%) and hippocampal (21%) serine racemase protein levels and
hippocampal serine racemase/DAAO ratio (34%) were reduced. Hippocampal DAAO protein levels significantly correlated with duration of illness (DOI, r = 0.6, p = 0.019) but not age. DAAO levels in patients with DOI > 20 years were 77% significantly higher than in the other patients and controls. Conclusions: Decreased CSF D-serine levels in schizophrenia may reflect decrease in extracellular levels of brain D-serine, consistent with the notion of NMDA receptor hypofunction in schizophrenia. Reduction in CSF D-serine levels may result from reduced serine racemase levels and/or elevated DAAO levels so that serine racemase/DAAO ratio would be reduced. Our results partially support these possibilities since frontal cortex and hippocampal sefine racemase protein levels in schizophrenia patients were found 39% and 21%, respectively, marginally significantly reduced, and hippocampal serine racemase/DAAO ratio 38% significantly reduced. Reduced D-sefine transport may also contribute to the reduction in CSF levels of this amino acid in schizophrenia. Our results support the hypothesized dysregulation of D-serine metabolism in schizophrenia patients.
References [1] Harrison, RJ., and Weinberger, D.R., 2005. Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence. Mol Psychiatr 10, 40-68. [2] Heresco-Levy, U., Javitt, D.C., Ebstein, R., et al., 2005. D-serine efficacy as add-on phammcotherapy to risperidone and olanzapine for treatment-refractory schizophrenia. Biol Psychiatr 57, 577-85. [3] Yamada, K., Ohnishi, T., Hashimoto, K., et al., 2005. Identification of multiple serine racemase (SRR) mRNA isoforms and genetic analyses of SRR and DAO in schizophrenia and D-serine levels. Biol Psychiatr 12, 1493 503.
IP_~
Differential accumulation of cortisol and corticosterone in the murine brain
B. Mason l, C.M. Pariante l, L. Sandersonz, S.A. Thomas z. ]Institute of Psychiatry, Clinical
Neuropharmacology, London, United Kingdom," 2King' s College London, Wolfson Centre for Age-Related Disease, London, United Kingdom Background: Hyperactivity of the hypothalamic pituitary adrenal (HPA) axis has been implicated in the pathogenesis of depression. Glucocorticoids released from the adrenal medulla regulate the negative feedback of the HPA axis. The level of glucocorticoids that cross the
Molecular Neuropsychopharmacology blood-brain barrier (BBB) and enter the brain is currently unclear. By increasing the level of glucocorticoids that enter the brain, the self-regulation of the HPA axis could be improved. The present study seeks to clarify the levels of glucocorticoids that pass into the brain. Aim: To quantify the ability of glucocorticoids to cross the murine BBB. Methods: Male FVB mice (25-40g) were anaesthetized intraperitoneally using a medetomindine hydrochloride (2mg/kg) and ketamine solution (150mg/kg) and heparinised (250 Units, i.p.). The brain was perfused using an in situ heart perfusion through cannulation of the left ventricle and sectioning of the right atrium to create an open circuit. The perfusion fluid consisted of a warmed (37~ oxygenated artificial plasma. Endogenous glucorticoids were removed from the cerebral circulation by a 2.5 rain pre-isotope perfusion. [3H]cortisol (3.6 nM) or [3H]corticosterone (3.8 nM), in addition to the vascular space marker, [14C]sucrose (0.5 microM), were then infused for a further 10rain. Mice were then decapitated and the pituitary gland and frontal cortex were removed. Brain and plasma samples were prepared for liquid scintillation counting (Packard TriCarb counter). The amount of radioactivity in the brain samples was expressed as a percentage of that in the plasma, corrected for sucrose space and termed uptake %. Data is presented as mean+standard error.
Sll
of an influx transporter for corticosterone and/or an efflux transporter for cortisol at the level of the BBB. Karssen et al. (2001) has demonstrated that the efflux transporter, P-glycoprotein (P-gp), limits the accumulation of cortisol but not corticosterone in the mouse brain. P-gp is expressed at the BBB and could be one mechanism by which glucocorticoids are regulated as they enter the brain. This research is funded by the Medical Research Council, UK.
References [1] Karssen, A.M., Meijer, O.C., van der Sandt, I., Lucassen, RJ., de Lange, E.C., de Boer, A.G., and de Kloet, E.R., 2001. Multidrug resistance P-glycoprotein hampers the access of cortisol but not of corticosterone to mouse and human brain. Endocrinology 142, 2686-94. [2] Pariante, C.M., Thomas, S.A., Lovestone, S., Makott, A., and Kerwin, R.W., 2004. Do antidepressants regulate how cortisol affects the brain? Psychoneuroendocrinology 29, 423-47.
I - P _ ~ GABAB receptor positive modulation attenuates selected molecular markers relevant to cocaine and nicotine dependence L. Lhuillier l, C. Mombereau 1, J.E Cryan l, K. Kaupmann 1. 1Novartis Pharma AG,
Table 1. Cortisol uptake Corticosterone uptake Significance % ( n - 4-5) % ( n - 4)
NIBR-Neuroscience, Basel, Switzerland
There is a strong body of evidence indicating that GABAB receptor activation could be beneficial in the treatment of drug addiction. Baclofen, the prototypical GABA-B receptor agonist reduces craving and self-administration Results: Preliminary data is presented in Table 1. of a variety of drugs of abuse, including psychostimulants Both glucocorticoids reached the frontal cortex at levels such as cocaine, alcohol, opiates and nicotine, as higher than the vascular marker. The accumulation of supported by both preclinical and clinical evidence [3H]cortisol was significantly lower than that achieved for (Brebner et al., 2002). The recent discovery of GABA-B [3H]corticosterone in the frontal cortex (Mann-Whitney positive allosteric modulators such as GS39783 (Urwyler Rank Sum test). In contrast, [3H]cortisol accumulation et al., 2003), lacking sedative properties of baclofen now was not significantly different to that achieved for allows the pursuit of a potential pharmacotherapeutic [3H]corticosterone in the pituitary gland (Student' s t-test). strategy. Very few studies however have focused on Conclusions: Corticosterone and cortisol can cross the molecular mechanisms of action of GABA-B the murine BBB and reach the frontal cortex. The receptor agonists/modulators in preclinical models of drug accumulation of corticosterone in this region is signif- dependence. Nearly all drugs of abuse (including cocaine icantly higher than that for cortisol. In contrast, the and nicotine) trigger an increase of dopamine levels in accumulation of corticosterone in a non-barrier brain mesolimbic structures (including the Nucleus Accumbens, region (the pituitary gland) was not significantly different NAc and Caudate Putamen, CPu). Several intracellular to cortisol. The difference between cortisol accumulation signaling correlates underlying acute or sustained addictive in the frontal cortex and the pituitary gland has previously behaviors have been identified in these structures (Nestler been observed in the guinea-pig (Pariante et al., 2004). and Aghajanian, 1997). These include members of the APOverall this preliminary data would suggest the presence 1 family of transcription factors, such as Fos, the product Frontal cortex 6.4• Pituitary gland 215.1•
85.2• 395.6•
P-0.016 not significant
I•--•
Molecular Neuropsychopharmacology Decreased D-serine levels in CSF of schizophrenia patients
S. Amar 1, I. Bendikov2, C. Nadri 1, R.A. Panizzutti3, J. De Miranda 3, H. Wotosker2, G. Again 1. iBen
Gurion Uniuersity of the Negev, Clinical Biochemistry, Beer-Sheua, Israel," 2Technion-Israel Institute of Technology, Biochemistry, Haifa, Israel," 3Federal Uni~)ersity of Rio de Janeiro, Anatomy, Rj, Brazil Introduction: Accumulating evidence suggests that NMDA receptor hypofunction contributes to the symptomatic features of schizophrenia (Harrison et al 2005). A unique feature of the NMDA receptor is that the channel only operates when the sites of both glutamate and a coagonist are occupied. D-Serine has been shown to be a major endogenous coagonist of the NMDA receptors. Endogenous D-serine is synthesized from L-serine by serine racemase. Degradation of D-serine in the cerebellum and brainstem is mediated by D-amino acid oxidase (DAAO). Based on the NMDA receptor hypofunction hypothesis several clinical trials investigated whether administration of NMDA receptor coagonists is beneficial for the patients. When combined with conventional neuroleptics or newer atypical antipsychotics D-serine improved positive, negative and cognitive symptoms of schizophrenia patients (Heresco-Levy et al 2005). Moreover, decreased serum D-serine levels were found in schizophrenic subjects (Yamada et al 2005). The present study was carried out to further elucidate whether the metabolism of D-serine is involved in the pathophysiology and treatment of schizophrenia. Methods: Levels of D-serine, L-serine, L-glutamate and L-glutamine were measured by high-performance liquid chromatography in (i) CSF from 12 schizophrenia patients and matched controls; (ii) postmortem parietal cortex from 15 subjects of each of four matched groups of controls, schizophrenia, major depression and bipolar patients; (iii) brain samples from rats chronically treated with haloperidol, clozapine or vehicle. In addition, serine racemase and DAAO protein levels were quantified in postmortem brain samples from 15 schizophrenia patients and 15 matched controls. Results: A 25% decrease in D-serine levels and 28% decerease in D/L-serine ratio was observed in CSF of schizophrenia patients us. controls. Three L-amino acids (serine, glutamate and glutamine) levels were unaltered. Parietal cortex D-serine and the three L-amino acid levels were the same in all diagnostic groups. Chronic antipsychotic treatment did not alter frontal cortex D-serine levels in rats. Frontal cortex (39%) and hippocampal (21%) serine racemase protein levels and
hippocampal serine racemase/DAAO ratio (34%) were reduced. Hippocampal DAAO protein levels significantly correlated with duration of illness (DOI, r = 0.6, p = 0.019) but not age. DAAO levels in patients with DOI > 20 years were 77% significantly higher than in the other patients and controls. Conclusions: Decreased CSF D-serine levels in schizophrenia may reflect decrease in extracellular levels of brain D-serine, consistent with the notion of NMDA receptor hypofunction in schizophrenia. Reduction in CSF D-serine levels may result from reduced serine racemase levels and/or elevated DAAO levels so that serine racemase/DAAO ratio would be reduced. Our results partially support these possibilities since frontal cortex and hippocampal sefine racemase protein levels in schizophrenia patients were found 39% and 21%, respectively, marginally significantly reduced, and hippocampal serine racemase/DAAO ratio 38% significantly reduced. Reduced D-sefine transport may also contribute to the reduction in CSF levels of this amino acid in schizophrenia. Our results support the hypothesized dysregulation of D-serine metabolism in schizophrenia patients.
References [1] Harrison, RJ., and Weinberger, D.R., 2005. Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence. Mol Psychiatr 10, 40-68. [2] Heresco-Levy, U., Javitt, D.C., Ebstein, R., et al., 2005. D-serine efficacy as add-on phammcotherapy to risperidone and olanzapine for treatment-refractory schizophrenia. Biol Psychiatr 57, 577-85. [3] Yamada, K., Ohnishi, T., Hashimoto, K., et al., 2005. Identification of multiple serine racemase (SRR) mRNA isoforms and genetic analyses of SRR and DAO in schizophrenia and D-serine levels. Biol Psychiatr 12, 1493 503.
IP_~
Differential accumulation of cortisol and corticosterone in the murine brain
B. Mason l, C.M. Pariante l, L. Sandersonz, S.A. Thomas z. ]Institute of Psychiatry, Clinical
Neuropharmacology, London, United Kingdom," 2King' s College London, Wolfson Centre for Age-Related Disease, London, United Kingdom Background: Hyperactivity of the hypothalamic pituitary adrenal (HPA) axis has been implicated in the pathogenesis of depression. Glucocorticoids released from the adrenal medulla regulate the negative feedback of the HPA axis. The level of glucocorticoids that cross the
Molecular Neuropsychopharmacology blood-brain barrier (BBB) and enter the brain is currently unclear. By increasing the level of glucocorticoids that enter the brain, the self-regulation of the HPA axis could be improved. The present study seeks to clarify the levels of glucocorticoids that pass into the brain. Aim: To quantify the ability of glucocorticoids to cross the murine BBB. Methods: Male FVB mice (25-40g) were anaesthetized intraperitoneally using a medetomindine hydrochloride (2mg/kg) and ketamine solution (150mg/kg) and heparinised (250 Units, i.p.). The brain was perfused using an in situ heart perfusion through cannulation of the left ventricle and sectioning of the right atrium to create an open circuit. The perfusion fluid consisted of a warmed (37~ oxygenated artificial plasma. Endogenous glucorticoids were removed from the cerebral circulation by a 2.5 rain pre-isotope perfusion. [3H]cortisol (3.6 nM) or [3H]corticosterone (3.8 nM), in addition to the vascular space marker, [14C]sucrose (0.5 microM), were then infused for a further 10rain. Mice were then decapitated and the pituitary gland and frontal cortex were removed. Brain and plasma samples were prepared for liquid scintillation counting (Packard TriCarb counter). The amount of radioactivity in the brain samples was expressed as a percentage of that in the plasma, corrected for sucrose space and termed uptake %. Data is presented as mean+standard error.
Sll
of an influx transporter for corticosterone and/or an efflux transporter for cortisol at the level of the BBB. Karssen et al. (2001) has demonstrated that the efflux transporter, P-glycoprotein (P-gp), limits the accumulation of cortisol but not corticosterone in the mouse brain. P-gp is expressed at the BBB and could be one mechanism by which glucocorticoids are regulated as they enter the brain. This research is funded by the Medical Research Council, UK.
References [1] Karssen, A.M., Meijer, O.C., van der Sandt, I., Lucassen, RJ., de Lange, E.C., de Boer, A.G., and de Kloet, E.R., 2001. Multidrug resistance P-glycoprotein hampers the access of cortisol but not of corticosterone to mouse and human brain. Endocrinology 142, 2686-94. [2] Pariante, C.M., Thomas, S.A., Lovestone, S., Makott, A., and Kerwin, R.W., 2004. Do antidepressants regulate how cortisol affects the brain? Psychoneuroendocrinology 29, 423-47.
I - P _ ~ GABAB receptor positive modulation attenuates selected molecular markers relevant to cocaine and nicotine dependence L. Lhuillier l, C. Mombereau 1, J.E Cryan l, K. Kaupmann 1. 1Novartis Pharma AG,
Table 1. Cortisol uptake Corticosterone uptake Significance % ( n - 4-5) % ( n - 4)
NIBR-Neuroscience, Basel, Switzerland
There is a strong body of evidence indicating that GABAB receptor activation could be beneficial in the treatment of drug addiction. Baclofen, the prototypical GABA-B receptor agonist reduces craving and self-administration Results: Preliminary data is presented in Table 1. of a variety of drugs of abuse, including psychostimulants Both glucocorticoids reached the frontal cortex at levels such as cocaine, alcohol, opiates and nicotine, as higher than the vascular marker. The accumulation of supported by both preclinical and clinical evidence [3H]cortisol was significantly lower than that achieved for (Brebner et al., 2002). The recent discovery of GABA-B [3H]corticosterone in the frontal cortex (Mann-Whitney positive allosteric modulators such as GS39783 (Urwyler Rank Sum test). In contrast, [3H]cortisol accumulation et al., 2003), lacking sedative properties of baclofen now was not significantly different to that achieved for allows the pursuit of a potential pharmacotherapeutic [3H]corticosterone in the pituitary gland (Student' s t-test). strategy. Very few studies however have focused on Conclusions: Corticosterone and cortisol can cross the molecular mechanisms of action of GABA-B the murine BBB and reach the frontal cortex. The receptor agonists/modulators in preclinical models of drug accumulation of corticosterone in this region is signif- dependence. Nearly all drugs of abuse (including cocaine icantly higher than that for cortisol. In contrast, the and nicotine) trigger an increase of dopamine levels in accumulation of corticosterone in a non-barrier brain mesolimbic structures (including the Nucleus Accumbens, region (the pituitary gland) was not significantly different NAc and Caudate Putamen, CPu). Several intracellular to cortisol. The difference between cortisol accumulation signaling correlates underlying acute or sustained addictive in the frontal cortex and the pituitary gland has previously behaviors have been identified in these structures (Nestler been observed in the guinea-pig (Pariante et al., 2004). and Aghajanian, 1997). These include members of the APOverall this preliminary data would suggest the presence 1 family of transcription factors, such as Fos, the product Frontal cortex 6.4• Pituitary gland 215.1•
85.2• 395.6•
P-0.016 not significant