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P.1.24 A transmembrane calcium flux is requiredfor circadian clock gene rhythmicity in mammalian pacemaker neurons
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Molecular Neuropsychopharmacology
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IP.1.231 The long-term paraquat administration produces a slowly progressing degeneration of the nigrostriatal system in rats K. KuteP, J. Wardas 1, R Nowak s, J. D~browskas, A. Bortel s, L. Labus s, T. Lenda 1, M, Smiatowska 2, J. Wieroflska 2, B. Zi~ba2, S. Wolfarth1, K. Ossowska 1.
1Department of Neuropsychopharmacology, 2Department of Neurobiology, Institut~ of Pharmacology, Polish Academy of Science, Krakdw, Poland; 3Department of Pharmacology, Medical Unioersity of Silesia, Zabrze, Poland Slowly developing degeneration of dopaminergic neurons in the substantia nigra (SN) underlies Parkinson's disease, although factors responsible for this process have not been established. Positive correlation was observed between long-term exposure to pesticides, especially paraquat and an incidence of Parkinson's disease (PD). Paraquat shares structural similarity with MPTP (a parkinsonism-evoking toxin) and its short-term administration induces parkinsonian-like effects in mice. The goal of the present study was to investigate if chronic administration of paraquat produces progressive de~neration of dopaminergic neurons in rats. Paraquat was administered ip to male Wistar rats, at a dose of 10 mg/kg, once a week for 4, 8, 12 or 24 weeks. Animals were sacrificed 7 days after the last dose. Levels of dopamine (DA), its metabolites, noradrenaline (NA) and serotonin (5-HT) were assessed using HPLC with electrochemical detection. Autoradiographic analysis of binding of [SH]GBR12.935 to dopamine transporter (DAT) and immunohistochemical detection of T h immunoreactivity (I'H-IR) were also performed. Paraquat induced 30% loss of the number of T h - I R neurons in the anterior SN after 12 weeks of treatment and 25% loss of neurons in the whole SN after 24 weeks. Paraquat increased the levels of DA and its metabolites in the striatum after 4 and 8 weeks. After 12 weeks DA level was normalized, and then decreased by 31% in the anterior and by 26% in the posterior striatum after 24 weeks. After 8 and 24 weeks DA turnover was significantly raised. The increase in the DA level was also seen in the SN after 8 weeks of treatment but no changes in DA turnover were detected. No deficits of DA in the SN and frontal cortex as well as of N A and 5-HT in any of the examined structures were observed. Binding of [SH]GBR12.935 to DAT was decreased after 4 and 8 weeks of treatment, after 12 weeks it was normalized and after 24 weeks it was again reduced by 11% and 33% in the dorsal and ventral striatum,
respectively. Paraquat elicited also decreases in binding of the ligand to DAT in the SN after 4, 8 and 12 weeks of injections. These data show three succeeding stages of paraquat toxicity: (i) a period of enhanced dopaminergic transmission and metabolism, with reduced binding to DAT, (ii) reversion to control DA levels, (iii) a stage of decreased dopaminergic transmission and binding to DAT. We postulate that long-lasting exposure of rats to paraquat leads to the loss of striatal dopaminergic transmission due to degeneration of nigral neurons which is preceded by a compensatory activation of this system. Acknowledgements: This study was supported by the State Committee for Scientific Research, project PB2-MIN-001/PO5/18 which is a part of a research project PB2-MIN-001/PO5/2002 entitled: Polish-German solicited research projects in the field of neurological sciences.
IP.1.241A transmembrane calcium flux is required for circadian clock gene rhythmicity in mammalian pacemaker neurons G. Lundkvist, Y. Kwak, E. Davis, H. Tel, G. Block.
Department of Neuroscience, Karolinska Instituter, Stockholm, Sweden Calcium (Ca 2+) homeostasis have in numerous studies been suggested to play a role in the pathophysiology of human major depression, and Ca 2+ channel antagonists have been ascribed antidepressant effects (Paul and Skolnick, 2003). One feature of major depression is sleep disturbances and disruption of the sleep-wake cycle (Paul and Skolnick, 2003), possibly via brain circuits involved in the generation of circadian rhythms, daily rhythms in physiological and behavioral processes with a period of approximately 24 h, which in mammals are primarily driven by the hypothalamic suprachiasm atic nucleus (SCN; Hastings and Maywood, 2000). The SCN expresses circadian rhyttnns in spontaneous electrical impulse activity and neuropeptide release. Rhythm generation within the SCN is a cell-autonomous property. It is generally believed that circadian rhythms are driven by molecular feedback loops of rhythmically expressed 'clock genes' and their protein products (Hastings and Maywood, 2000). Membrane phenomena have not been generally recognized as part of the core clock mechanism but rather as pathways by which information reaches the clock for light synchronization and by which the clock regulates tissue and organ targets. This view has been challenged recently in Drosophila, suggesting that membrane potential and Ca 2+ flux may play a more central role in circadian rhythm generation (Nitabach et al., 2002).
Molecular Neuropsychopharmacology Purpose of the study: The aim of this study was to address the question if membrane potential and Ca 2+ flux can impact the expression of clock genes in mammalian pacemaker neurons, and if Ca 2+ flux is acritical component of the circadian timing mechanism. Methods: The rhythmic expression of mPerl was monitored in vitro by continuously recording light emission in cultured SCN tissue from transgenic rats and mice, in which a reporter for luciferase has been linked to the mouse Perl promoter region. In addition, an roPer2L~ knock-in mouse was used to monitor expression of PER2. The SCN was cultured in media containing different concentrations of K + and Ca 2+ concentrations. Patch clamp recordings were performed to estimate membrane potentials. Results: Low [K+] hyperpolarized the membrane potential of SCN neurons and immediately reduced the amplitude or stopped the rhythmic expression of mPerl. Low [Ca 2+] similarly abolished the rhythm in mPerland mPER2 expression but depolarized the membrane potential. Per rhythms were restored after washout with control medium. The amplitude of the Perl expression was markedly decreased by voltage-gated Ca 2+ channel blockers. Conclusion: These results strongly suggest that a transmembrane Ca 2+ flux is necessary for sustained molecular rhythmicity in the SCN, and that Ca 2+ influx, due to circadian variations in membrane potential, is a critical process for circadian pacemaker function.
References [l] Hastings M, Maywood ES (2000). Circadian clocks in the mammalian brain. 13ioEssays 22: 23-31. [2] Nitabach MN, Blau J, Holmes TC (2002). Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock. Cell 109: 485-95. [3] Paul IA, Skolnick P (2003). Glutamate and depression: clinical and preclinical studies. Ann N Y Acad Sci 1003: 250-72.
IP.1.251 Galantamine increases firing activity of dopaminergic neurons in the ventral tegmental area 13. Schilstr6m, V. Ivanov, C. Wiker, T.H. Svensson. Department of Physiology and Pharmacology, Section of Neuropsyehopharmaeology, Karolinska Instituter, Stockholm, Sweden. Galantamine is currently used in the treatment of Alzheimer's disease. Recent clinical studies suggest that galantamine may also be useful as adjunct therapy in
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schizophrenia, treating negative and cognitive symptoms (Rosse and Deutch, 2002). Galantarnine has two separate mechanisms of action which both enhance cholinergic neurotransmission in the brain. At low doses it binds allosterically to nicotinic acetylcholine receptors (nAChRs) and potentiates their function and at higher doses it also becomes an inhibitor of aeetylcholine esterase (ACHE; Maelicke et al., 2000). Given that several lines of evidence suggest a role for ~7 nAChRs in schizophrenia and that the selective ACHE-inhibitor donepezil has not proven effective in schizophrenia, galantamine's beneficial effect may be mediated, largely, through potentiation of nAChRs. Cognitive deficits in schizophrenia, such as impairments in working memory, are thought to be linked to an impaired doparninergic neurotransmission in the prefrontal cortex. Nicotine, which is known to enhance working memory, activates dopaminergic neurons by stimulating nAChRs in the cell body region (Schilstr6m et al., 2003). Thus, we hypothesized that the beneficial effects of galantamine on cognition in schizophrenia might be due to potentiation of those somatodendritic nAChRs. To test this hypothesis, the effects of galantamine (0.011.0 mg/kg s.c.) on doparnine cell firing were tested, using in vivo extracellular single cell recordings in anaesthetized rats. Galantamine increased both firing rate and burst firing of dopaminergic cells in the ventral tegmental area. The effect was observed already at a low dose, unlikely to result in significant ACHE-inhibitiort Moreover, the effect of galantamine was not mimicked by the selective ACHE-inhibitor donepezil (1.0 mg/kg s.c.) and it was blocked by the nAChR antagonist mecamylamine (1.0 mg/kg s.c.) but not the muscarinic receptor antagonist scopolamine (0.1 mg/kg s.c.). Thus, our data indicate that galantamine enhances dopaminergic activity through potentiation of nAChRs and supports the notion that allosteric potentiation of nAChRs may improve cognitive function in schizophrenia. Supported by Janssen-Cilag A13, Sweden, the Lundbeck Foundation, Denmark, the Swedish Research Council and the Karolinska Institutet.
References [1] Maelicke, A., Schrattenholz, A., Samochocki, M., Radina, Iv[ and Albuquerque E.X., 2000. Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer's disease. 13char. Brain Res 113, 199-206. [2] Rosse, tL13., and Deutsch, S.I., 2002. Adjuvant galantamine administration improves negative symptoms in a patient with treatment-refractory schizophrenia. Clin Neuropharmacol. 25, 272-275. [3] Schilstr6m, 13., Rawal, N., Mameli-Engvall, M., Nomikos, G.G., and Svensson, T.H., 2003. Dual
Molecular Neuropsychopharmacology
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IP.1.231 The long-term paraquat administration produces a slowly progressing degeneration of the nigrostriatal system in rats K. KuteP, J. Wardas 1, R Nowak s, J. D~browskas, A. Bortel s, L. Labus s, T. Lenda 1, M, Smiatowska 2, J. Wieroflska 2, B. Zi~ba2, S. Wolfarth1, K. Ossowska 1.
1Department of Neuropsychopharmacology, 2Department of Neurobiology, Institut~ of Pharmacology, Polish Academy of Science, Krakdw, Poland; 3Department of Pharmacology, Medical Unioersity of Silesia, Zabrze, Poland Slowly developing degeneration of dopaminergic neurons in the substantia nigra (SN) underlies Parkinson's disease, although factors responsible for this process have not been established. Positive correlation was observed between long-term exposure to pesticides, especially paraquat and an incidence of Parkinson's disease (PD). Paraquat shares structural similarity with MPTP (a parkinsonism-evoking toxin) and its short-term administration induces parkinsonian-like effects in mice. The goal of the present study was to investigate if chronic administration of paraquat produces progressive de~neration of dopaminergic neurons in rats. Paraquat was administered ip to male Wistar rats, at a dose of 10 mg/kg, once a week for 4, 8, 12 or 24 weeks. Animals were sacrificed 7 days after the last dose. Levels of dopamine (DA), its metabolites, noradrenaline (NA) and serotonin (5-HT) were assessed using HPLC with electrochemical detection. Autoradiographic analysis of binding of [SH]GBR12.935 to dopamine transporter (DAT) and immunohistochemical detection of T h immunoreactivity (I'H-IR) were also performed. Paraquat induced 30% loss of the number of T h - I R neurons in the anterior SN after 12 weeks of treatment and 25% loss of neurons in the whole SN after 24 weeks. Paraquat increased the levels of DA and its metabolites in the striatum after 4 and 8 weeks. After 12 weeks DA level was normalized, and then decreased by 31% in the anterior and by 26% in the posterior striatum after 24 weeks. After 8 and 24 weeks DA turnover was significantly raised. The increase in the DA level was also seen in the SN after 8 weeks of treatment but no changes in DA turnover were detected. No deficits of DA in the SN and frontal cortex as well as of N A and 5-HT in any of the examined structures were observed. Binding of [SH]GBR12.935 to DAT was decreased after 4 and 8 weeks of treatment, after 12 weeks it was normalized and after 24 weeks it was again reduced by 11% and 33% in the dorsal and ventral striatum,
respectively. Paraquat elicited also decreases in binding of the ligand to DAT in the SN after 4, 8 and 12 weeks of injections. These data show three succeeding stages of paraquat toxicity: (i) a period of enhanced dopaminergic transmission and metabolism, with reduced binding to DAT, (ii) reversion to control DA levels, (iii) a stage of decreased dopaminergic transmission and binding to DAT. We postulate that long-lasting exposure of rats to paraquat leads to the loss of striatal dopaminergic transmission due to degeneration of nigral neurons which is preceded by a compensatory activation of this system. Acknowledgements: This study was supported by the State Committee for Scientific Research, project PB2-MIN-001/PO5/18 which is a part of a research project PB2-MIN-001/PO5/2002 entitled: Polish-German solicited research projects in the field of neurological sciences.
IP.1.241A transmembrane calcium flux is required for circadian clock gene rhythmicity in mammalian pacemaker neurons G. Lundkvist, Y. Kwak, E. Davis, H. Tel, G. Block.
Department of Neuroscience, Karolinska Instituter, Stockholm, Sweden Calcium (Ca 2+) homeostasis have in numerous studies been suggested to play a role in the pathophysiology of human major depression, and Ca 2+ channel antagonists have been ascribed antidepressant effects (Paul and Skolnick, 2003). One feature of major depression is sleep disturbances and disruption of the sleep-wake cycle (Paul and Skolnick, 2003), possibly via brain circuits involved in the generation of circadian rhythms, daily rhythms in physiological and behavioral processes with a period of approximately 24 h, which in mammals are primarily driven by the hypothalamic suprachiasm atic nucleus (SCN; Hastings and Maywood, 2000). The SCN expresses circadian rhyttnns in spontaneous electrical impulse activity and neuropeptide release. Rhythm generation within the SCN is a cell-autonomous property. It is generally believed that circadian rhythms are driven by molecular feedback loops of rhythmically expressed 'clock genes' and their protein products (Hastings and Maywood, 2000). Membrane phenomena have not been generally recognized as part of the core clock mechanism but rather as pathways by which information reaches the clock for light synchronization and by which the clock regulates tissue and organ targets. This view has been challenged recently in Drosophila, suggesting that membrane potential and Ca 2+ flux may play a more central role in circadian rhythm generation (Nitabach et al., 2002).
Molecular Neuropsychopharmacology Purpose of the study: The aim of this study was to address the question if membrane potential and Ca 2+ flux can impact the expression of clock genes in mammalian pacemaker neurons, and if Ca 2+ flux is acritical component of the circadian timing mechanism. Methods: The rhythmic expression of mPerl was monitored in vitro by continuously recording light emission in cultured SCN tissue from transgenic rats and mice, in which a reporter for luciferase has been linked to the mouse Perl promoter region. In addition, an roPer2L~ knock-in mouse was used to monitor expression of PER2. The SCN was cultured in media containing different concentrations of K + and Ca 2+ concentrations. Patch clamp recordings were performed to estimate membrane potentials. Results: Low [K+] hyperpolarized the membrane potential of SCN neurons and immediately reduced the amplitude or stopped the rhythmic expression of mPerl. Low [Ca 2+] similarly abolished the rhythm in mPerland mPER2 expression but depolarized the membrane potential. Per rhythms were restored after washout with control medium. The amplitude of the Perl expression was markedly decreased by voltage-gated Ca 2+ channel blockers. Conclusion: These results strongly suggest that a transmembrane Ca 2+ flux is necessary for sustained molecular rhythmicity in the SCN, and that Ca 2+ influx, due to circadian variations in membrane potential, is a critical process for circadian pacemaker function.
References [l] Hastings M, Maywood ES (2000). Circadian clocks in the mammalian brain. 13ioEssays 22: 23-31. [2] Nitabach MN, Blau J, Holmes TC (2002). Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock. Cell 109: 485-95. [3] Paul IA, Skolnick P (2003). Glutamate and depression: clinical and preclinical studies. Ann N Y Acad Sci 1003: 250-72.
IP.1.251 Galantamine increases firing activity of dopaminergic neurons in the ventral tegmental area 13. Schilstr6m, V. Ivanov, C. Wiker, T.H. Svensson. Department of Physiology and Pharmacology, Section of Neuropsyehopharmaeology, Karolinska Instituter, Stockholm, Sweden. Galantamine is currently used in the treatment of Alzheimer's disease. Recent clinical studies suggest that galantamine may also be useful as adjunct therapy in
$21
schizophrenia, treating negative and cognitive symptoms (Rosse and Deutch, 2002). Galantarnine has two separate mechanisms of action which both enhance cholinergic neurotransmission in the brain. At low doses it binds allosterically to nicotinic acetylcholine receptors (nAChRs) and potentiates their function and at higher doses it also becomes an inhibitor of aeetylcholine esterase (ACHE; Maelicke et al., 2000). Given that several lines of evidence suggest a role for ~7 nAChRs in schizophrenia and that the selective ACHE-inhibitor donepezil has not proven effective in schizophrenia, galantamine's beneficial effect may be mediated, largely, through potentiation of nAChRs. Cognitive deficits in schizophrenia, such as impairments in working memory, are thought to be linked to an impaired doparninergic neurotransmission in the prefrontal cortex. Nicotine, which is known to enhance working memory, activates dopaminergic neurons by stimulating nAChRs in the cell body region (Schilstr6m et al., 2003). Thus, we hypothesized that the beneficial effects of galantamine on cognition in schizophrenia might be due to potentiation of those somatodendritic nAChRs. To test this hypothesis, the effects of galantamine (0.011.0 mg/kg s.c.) on doparnine cell firing were tested, using in vivo extracellular single cell recordings in anaesthetized rats. Galantamine increased both firing rate and burst firing of dopaminergic cells in the ventral tegmental area. The effect was observed already at a low dose, unlikely to result in significant ACHE-inhibitiort Moreover, the effect of galantamine was not mimicked by the selective ACHE-inhibitor donepezil (1.0 mg/kg s.c.) and it was blocked by the nAChR antagonist mecamylamine (1.0 mg/kg s.c.) but not the muscarinic receptor antagonist scopolamine (0.1 mg/kg s.c.). Thus, our data indicate that galantamine enhances dopaminergic activity through potentiation of nAChRs and supports the notion that allosteric potentiation of nAChRs may improve cognitive function in schizophrenia. Supported by Janssen-Cilag A13, Sweden, the Lundbeck Foundation, Denmark, the Swedish Research Council and the Karolinska Institutet.
References [1] Maelicke, A., Schrattenholz, A., Samochocki, M., Radina, Iv[ and Albuquerque E.X., 2000. Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer's disease. 13char. Brain Res 113, 199-206. [2] Rosse, tL13., and Deutsch, S.I., 2002. Adjuvant galantamine administration improves negative symptoms in a patient with treatment-refractory schizophrenia. Clin Neuropharmacol. 25, 272-275. [3] Schilstr6m, 13., Rawal, N., Mameli-Engvall, M., Nomikos, G.G., and Svensson, T.H., 2003. Dual