S.04.04 Stress-induced alterations in hippocampal and amygdalar microRNAs: effects of lithium

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forward genetic strategy employing a panel of mouse chromosome substitution strains (CSSs) based on the A/J (donor) and C57BL/6J (host) strain. Febrile seizures were induced by warm-air (50ºC) induced hyperthermia on postnatal day 14. Video/EEG monitoring showed that the onset of tonic-clonic seizures (measured as febrile seizure latency: FSL) highly correlates with the onset of spike wave discharges. Therefore FSL was used as phenotypic parameter to measure FS susceptibility in mice. CSS1 mice were less susceptible than C57BL/6J mice [1]. Behavioural phenotyping and genetic mapping of the CSS1 F2progeny resulted in a significant QTL with a LOD-score of 7.6. This QTL harbors 32 known brain expressed genes. Only FS1 was differentially expressed between CSS1 and C57BL/6J brain. The ultimate proof that FS1 is a mouse FS susceptibility gene came from functional interference studies in vivo. Reducing FS1 expression by microinjecting FS1 antisense oligonucleotides into the ventrical of C57BL/6J mouse resulted in a significant reduction in FS susceptibility. FS1 expression was significantly higher in hippocampus of TLE patients with FS than without. FS1 (in combination with other susceptibility genes) may be a valuable marker to assess risk factors for complex FS and TLE in children. References [1] Hessel EV, van Gassen KL, Wolterink-Donselaar IG, Stienen PJ, Fernandes C, Brakkee JH, Kas MJ, de Graan PN. Phenotyping mouse chromosome substitution strains reveal multiple QTLs for febrile seizure susceptibility. Genes Brain Behav. 2009 Mar;8(2):248−55.

S.04.02 Lack of central serotonin synthesis: effects on development of GABAergic subpopulations J. Waider1 ° , L. Gutknecht1 , K.P. Lesch1 . 1 University of W¨urzburg, Molecular and Clinical Psychobiology Laboratory for Translational Neurobiology Clinic and Policlinic for Psychiatry Psychosomatic and Psychotherapy, W¨urzburg, Germany Alterations in the serotonergic system have been associated with a wide spectrum of psychiatric disorders and various processes of early development. [1] Tryptophan hydroxylase 2 (TPH2) is the key enzyme in central serotonin (5-HT) synthesis. Recently, Tph2 knockout (−/−) mice were generated displaying lack of 5-HT synthesis in the brain. However, survival, migration and projection pattern of serotonergic neurons do not appear to be impaired in these mice. [2] In this study, GABAergic neurons were identified by in-situ hybridization (ISH) or double immunohistochemistry (dIHC) in hippocampus and cortex of adult Tph2 (−/−), Tph2 (−/+) and Tph2 (+/+) mice. Different layers of both brain structures were delineated with contours in 4−5 animals per group and mean cell densities of 3−4 equivalent sections per animal were calculated. Furthermore, neurotransmitter levels of several brain regions were assessed by HPLC. Analysis of the dorsal hippocampus with dIHC and ISH against Gad65/67 revealed no significant difference in mean cell densities of GABAergic interneurons among genotypes. However, by separating different hippocampal layers the density of Gad65/67 immunoreactive neurons in stratum lacunosum moleculare was reduced in Tph2 (−/−) mice. A similar trend was detected in stratum lacunosum moleculare by ISH. Thus, decreased 5-HT levels may influence the inhibitory GABAergic control in the dorsal hippocampus by affecting the development of a specific subtype of GABAergic interneurons. However, due to possible bias caused by counting design and group size, further

studies are required to confirm this finding and to identify the affected interneuron subpopulation. References [1] Vitalis T, Cases O, Passemard S, Callebert J, Parnavelas JG. Embryonic depletion of serotonin affects cortical development. Eur J Neurosci. 2007 Jul;26(2):331−44. [2] Gutknecht L, Waider J, Kraft S, Kriegebaum C, Holtmann B, Reif A, Schmitt A, Lesch KP. Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice. J Neural Transm. 2008 Aug;115(8):1127−32. Epub 2008 Jul 30.

S.04.03 Inositol monophosphatase: drug target or false alarm? N. Singh1 ° , S. Vasudevan1 , J. Thomas1 , O. Kuznetsova1 , M. Knight1 , P. Aley1 , T. Sharp1 , G. Churchill1 . 1 University of Oxford, Dept. of Pharmacology, Oxford, United Kingdom Purpose of study: Inositol monophosphatase (IMPase) has been a subject of scrutiny since Berridge et. al., 1989 [1] , proposed that it may play a central role in the treatment of bipolar disorder (BD). Lithium, which is used therapeutically in BD [2] , is a known inhibitor of IMPase with an IC50 of 0.8 mM in vivo. However, it has not been possible to correlate the therapeutic effect of lithium with IMPase inhibition, since lithium inhibits several other enzymes and ion transporters. Methods: Taking a step towards direct testing of the inositol depletion hypothesis, we used the Open Eye Scientific Software to search for potential inhibitors of IMPase. A ligandbased virtual screen was used to identify potential inhibitors using inositol-1 phosphate as a template. The ZINC database of drug-like molecules was used to screen, and the compounds were ranked according to shape similarity. The top 20 hits were purchased and tested for inhibitory activity in vitro. In an alternate approach, we used a library of FDA-approved compounds, with proven safety, to screen for IMPase inhibition. Results: We found hits from both approaches. The best one has an IC50 of 2 mM, n = 5, and comes from the FDA library. This drug is being tested in different behavioural rodent models and shows some lithium-like activity in preliminary tests. Conclusion: After further analysis in animal models, this drug can be used in clinical trials in bipolar patients, since it already has proven safety in humans. References [1] Berridge, M. J., Downes, C.P., Hanley, M.R., 1989 Neural and Developmental Actions of Lithium: A Unifying Hypothesis. Cell 59: 411– 419. [2] Cade, J.F., 1949 Lithium salts in the treatment of psychotic excitement. Medical Journal of Australia 36, 349−52.

S.04.04 Stress-induced alterations in hippocampal and amygdalar microRNAs: effects of lithium R.M. O’ Connor1 ° , O.F. O’ Leary1 , A. Dinan2 , A. Gokul1 , T.G. Dinan3 , J.F. Cryan3 . 1 University College Cork, School of Pharmacy Dept. of Pharmacology & Therapeutics, Cork, Ireland; 2 University College Cork, Alimentary Pharmabiotic Centre, Cork, Ireland; 3 University College Cork, School of Pharmacy Dept. of Pharmacology & Therapeutics Alimentary Pharmabiotic Centre, Cork, Ireland Our understanding of the molecular basis of Bipolar disorder (BD) is severely lacking. The most widely used pharmaceutical

S.04. Young Scientists Award symposium 1 treatment for BD is the mood stabiliser lithium. Many of the cellular targets that mediate the therapeutic benefit of lithium remain unknown. miRNAs are short non-coding RNA species which regulate gene expression by repressing mRNA translation [1]. We studied the impact of chronic immobilisation stress and chronic lithium treatment on specific miRNAs in the murine hippocampus and amygdala. BALB/cOLaHsd mice received a 0.2% lithium chloride diet or regular diet for 21 days, for the final 10 days mice underwent the immobilisation stress for 3 hours daily or remained in their homecage. miRNA levels were measured using RT-PCR. Interestingly, in the stress-sensitive BALB/c mice, RTPCR revealed no significant change due to lithium treatment or chronic stress to let-7b or miR-34c levels the amygdala or hippocampus. miR-34a was unchanged in the amygdala while in the hippocampus miR-34a levels were unaffected by lithium treatment, however stress significantly downregulated miR-34a levels with lithium treatment partially normalising this down-regulation (t-test, p < 0.005). Chronic stress increased hippocampal miR-15a levels, however, lithium had no effect alone or in conjunction with the stress treatment (2 way ANOVA, p < 0.005). miR-15a levels in the amygdala were reduced due to stress (2 way ANOVA, p < 0.005) with lithium partially normalising this upregulation. This work adds to the current knowledge on the molecular changes that may occur in psychiatric disorders and how lithium may provide its therapeutic benefit. References [1] Filipowicz, W., S. N. Bhattacharyya, et al. (2008). “Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?” Nat Rev Genet 9 (2): 102–114.

S.04.05 Interactions of oligomeric and fibrillar b-amyloid with a7 neuronal nicotinic receptors and synaptic targets in Alzheimer’s disease A. Lilja1 ° , O. Porras1 , E. Storelli1 , A. Nordberg1 , A. Marutle1 . 1 Karolinska Institutet, Division of Alzheimer Neurobiology Department of Neurobiology Care Sciences and Society, Stockholm, Sweden Amyloid (Ab) is considered to be one of the major pathological hallmarks in Alzheimer’s disease (AD). Ab peptides formed through proteolytical cleavage of the amyloid precursor protein (APP) aggregate into different species. Specific Ab assemblies are suggested to affect and trigger pathological events such as the activation of inflammatory processes, altered protein kinase signaling and downregulation of growth factors, leading to impaired cholinergic neurotransmission and cell death. A potential link between Ab toxicity and cholinergic dysfunction in AD involves an interaction between A b and a7 neuronal nicotinic receptors (a7 nAChRs), suggesting that A b−a7 nAChR complexes are formed through the binding of Ab to a7 nAChR located on neurons. These complexes are then internalized, leading to intraneuronal accumulation of A b and impaired synaptic plasticity [1]. However, the interaction between different Ab species and specific a7 nAChRs are yet unknown. Herein, we investigated how various aggregation forms of Ab interact with a 7 nAChR using the novel specific agonists varenicline and JN403. a7 nAChR activation protected neuronal cells against neurotoxicity caused by fibrillar Ab 1−40 , suggesting an antagonistic effect of fibrillar Ab. On the other hand, nanomolar concentrations of oligomeric but not fibrillar Ab 1−40 induced an a7 nAChR dependent increase in

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[Ca2+ ]i in neuronal cells, demonstrating a receptor activation. In conclusion, these findings indicate that A b aggregation is critical for the functional interaction with cellular targets such as a 7 nAChRs, and warrant for further studies to elucidate the relation between amyloid and neuroprotective mechanisms. References [1] Snyder E. M., Nong Y., Almeida C. G., Paul S., Moran T., Choi E. Y., Nairn A. C., Salter M. W., Lombroso P. J., Gouras G. K., Greengard P., 2005 Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8, 1051–1058.

S.04.06 Altered aggressive behaviour following genetic and pharmacological manipulation of serotonin autoinhibition E. Audero1 ° , B. Mlinar2 , Z. Skachokova1 , R. Corradetti2 , C. Gross1 . 1 EMBL, Mouse Biology Unit, Monterotondo − Roma, Italy; 2 University of Firenze, Dep. of Preclinical and Clinical Pharmacology, Firenze, Italy In humans, nonhuman primates and other mammals, preclinical and clinical studies have identified the brain neurotransmitter serotonin system as the major modulator of impulsiveness and aggression [1]. Numerous pharmacological and neurochemical investigations have provided evidence of a negative correlation existing between serotonin levels and aggression [2]. Release of serotonin in the brain is actively regulated by serotonergic neurons firing activity in the raphe nuclei of the brainstem. In turn, firing activity of serotonergic neurons is modulated by negative feedback inhibition via somato-dentritic serotonin 1A autoreceptors (Htr1a) [3]. To better understand the contribution of serotonergic firing activity in setting the level of aggressive behaviour and to dissect the developmental programming effects, we developed two transgenic lines in which serotonergic neurons activity can be specifically and transiently modulated. First, we used conditional over-expression of Htr1a in serotonergic neurons of the brainstem to selectively increase serotonergic auto-inhibition and reduce serotonin firing in mice, demonstrating that chronic reduction in serotonin neuron firing is associated with decreased serotonin metabolite and heightened aggression. We also showed that over-expression of Htr1a autoreceptor in adulthood was both necessary and sufficient for altered aggression behaviour excluding a role for serotonin in the developmental programming of the circuits that control aggression. Second, we generated a transgenic line where Htr1a expression is confined to serotonergic neurons of the brainstem and we used this transgenic line as a pharmacogenetic tool to demonstrate that acute activation of Htr1a results in inhibition of serotonin neuron activity and increased aggressive response. References [1] Nelson, R.J. and Trainor, B.C. (2007). Neural mechanisms of aggression. Nature Review Neuroscience 8, 536–546. [2] Ferrari, P.F., Palanza, P., Parmigiani, S., de Almeida, R.M., and Miczek, K.A. (2005). Serotonin and aggressive behavior in rodents and nonhuman primates: predispositions and plasticity. European Journal of Pharmacology 526, 259–273. [3] Blier, P., Pineyro, G., el Mansari, M., Bergeron, R., and de Montigny, C. (1998). Role of somatodendritic 5-HT autoreceptors in modulating 5-HT neurotransmission. Annals of the New York Academy of Sciences 861, 204–216.