P.4.b.003 Tryptophan hydroxylase and serotonin transporter gene polymorphism does not affect the diagnosis and treatment outcome of panic disorder

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P.4.b. Anxiety disorders and anxiolytics − Anxiety disorders (basic)

P.4.b.002 New hippocampal gene expression in a rat model of PTSD B.C. Lee1 ° , J.S. Kim2 , J.E. Park3 , I.G. Choi4 1 Hangang Sacred Heart Hospital, Department of Psychiatry, Seoul, South-Korea; 2 Hangang Sacred Heart Hospital, Burn Institute, Seoul, SouthKorea; 3 Keyo Hospital, Psychiatry, Uiwang-si, South-Korea; 4 Hangang Sacred Heart Hospital, Psychiatry, Seoul, South-Korea Purpose of the study: Post-traumatic stress disorder is a longlasting psychiatric disease following the traumatic experience of severe fatal stress with the consequence of hippocampal atrophy. Accumulating evidence from animal and human studies suggests that the etiology of PTSD may be a reduction in the inhibition of memory circuits for fear extinction due to an exaggerated amygdala response that in turn occurs due to a hypofunctioning medial prefrontal cortex and hippocampus. This study identified novel potential molecular and cellular targets for therapeutic intervention in PTSD. Method used: We used 7-week-old male Wistar rats for a PTSD model using a shuttle-box task (active avoidance/escape) [1]; the PTSD group was selected based on the changes in rats’ behaviour and levels of glucocorticoids two weeks after inescapable shock stress. RNA collected from the hippocampus was subjected to DNA microarray analysis and quantitative RT-PCR. Gene expression profiling was performed using Illumina Rat Ref-12 Expression BeadChips containing 21,666 probes. Q-PCR assays were used to verify the results of the gene expression changes measured using microarrays. Tnfrsf11b, Cldn5, Map2, or Map1b of these genes were selected and reconfirmed by IHC or western blotting. Summary of results: Contrary to expectation, cortisol levels were significantly increased in both PTSD and PTSD-RS groups compared with control, though they were increased more in the PTSD-RS group [2]. Fold-changes greater than 1.5 and a P < 0.05 were arbitrarily selected (296 genes) for further analysis; 27 genes were up-regulated and 269 genes were downregulated in the PTSD/PTSD-Restress groups. Many of the downregulated genes, such as NMDA, AMPA, or cannabinoids, have already been observed in neurodegeneration as well as PTSD. In addition, almost all of the genes related ‘Ionotropic glutamate receptors’, ‘endogenous cannabinoid system’, ‘cytokine signaling and inflammatory pathway’, ‘ribosomal proteins’, ‘Spliceosome’, ‘Neuron projection’, ‘Mitochondrion’, and ‘Regulation of synaptic plasticity’ had also been described already in other microarray or molecular studies. In fact, our study not only confirmed previous studies but also provides new candidates for a key role in the pathogenesis of PTSD, although their function is still to be determined. We selected genes that exhibited high fold changes and had not yet been predicted. The genes for Tnfrsf11b, Cldn5, Prom1, and Abcg2 were significantly up-regulated and Snap91, Map2, Myo5a, Map1b, and Pgrmc1 were down-regulated in PTSD/ PTSD-Restress rat hippocampus compared to controls and correlated well with the microarray results. Of these genes, Tnfrsf11b, Cldn5, Map2 and Map1b were selected and reconfirmed by IHC or western blotting. We observed that protein expression of Tnfrsf11b or Cldn5 was increased and that of Map2 or Map1b was decreased in the rat hippocampus. Conclusion: In this study, we showed that 296 genes were upor down-regulated in PTSD/PTSD-RS, and studied their molecular attribution. The study provides new evidence of hippocampal

change at the molecular level in PTSD, and suggests novel potential molecular and cellular targets for therapeutic intervention in PTSD. References [1] Wakizono T, Sawamura T, Shimizu K, Nibuya M, Suzuki G, Toda H, et al. (2007). Stress vulnerabilities in an animal model of post-traumatic stress disorder. Physiol Behav 90(4): 687–695. [2] Yehuda R (2006). Advances in understanding neuroendocrine alterations in PTSD and their therapeutic implications. Ann N Y Acad Sci 1071: 137–166.

P.4.b.003 Tryptophan hydroxylase and serotonin transporter gene polymorphism does not affect the diagnosis and treatment outcome of panic disorder W. Kim1 ° , J.M. Woo1 1 Seoul Paik Hospital, Department of Psychiatry, Seoul, South-Korea Objectives: An increasing body of evidence suggests that serotonergic neurotransmission may be linked with the modulation of anxiety via serotonin metabolism, as well as alterations in serotonin reuptake and activity, etc, in the brain, under the influence of a variety of external and internal stimuli. Therefore, genetic variants of the serotonergic system would be reasonable candidate for study of panic disorder. Tryptophan hydroxylase (TPH) is a rate-limiting enzyme in serotonin synthesis, and two common polymorphisms (A218C, A779C) have been reported in intron 7 of the TPH gene. The serotonin transporter promoter polymorphism, 5-HTTLPR, is the functional polymorphism of the serotonin transporter, and can be designated by the long (L) or short (S) allele. Several studies have reported an association between the S allele of the 5-HTTLPR and anxiety-related traits. This study investigated the association between the tryptophan hydroxylase (TPH) gene and a serotonin transporter gene promoter polymorphism (5-HTTLPR), with panic disorder in a Korean population. Method: Two hundred and forty four Korean panic disorder patients and the 227 controls were genotyped by a polymerase chain reaction-based method. The severity of panic disorders was assessed by number of panic attacks during the previous one month, as well as scores for anticipatory anxiety, panic distress, and agoraphobic distress, as determined by a visual analogue scale (VAS). All the subjects completed the assessment measures including Spielberger State-Trait Anxiety Inventory-State (STAI-S), Spielberger State-Trait Anxiety Inventory-Trait (STAI-T), Beck Depression Inventory (BDI), Symptom Checklist-90-Revised (SCL90-R), Revised Anxiety Sensitivity Index (ASI-R), Clinical Global Impression scale − Severity of Illness (CGI-S), Panic Disorder Severity Scale (PDSS), and the Hamilton Depression Rating Scale (HAMD). Responder analyses were conducted on the basis of changes in CGI-I scores after 10 weeks of treatment. Genotype distributions in each polymorphism were compared via Chisquared tests. Monte Carlo methods were applied in the assessment of the contingency table, for small cell counts. Continuous variables were evaluated via Mann–Whitney and Kruskal– Wallis tests between the comparisons, where appropriate, as the Kolmogorov-Smirnov test indicated a skew of the data from normality. Results: We found no significant differences in the genotype and allele frequencies in TPH A218C and 5-HTTLPR polymor-

P.4.b. Anxiety disorders and anxiolytics − Anxiety disorders (basic) phisms between the panic patients and the control group. Subgroup analyses in terms of comorbidities and other primary clinical variables, indicated no differences in these polymorphisms. Patients’ responses to a 10-week treatment regimen of paroxetine also did not significantly differ according to genotypes. Compared to other studies that reported different antidepressants responses according to genotype of TPH A218C and 5-HTTLPR in cases of major depressive disorder, our results suggest that the primary therapeutic mechanisms exploited by antidepressants in cases of panic disorder may be different from those in cases of major depressive disorder. Conclusion: Our findings suggest that the TPH A218C polymorphism and 5-HTTLPR play no significant roles in the pathogenesis and clinical symptomatologies, at least in a Korean population. References [1] Perna, G., Favaron, E., Di Bella, D., Bussi, R., Bellodi, L., 2005. Antipanic efficacy of paroxetine and polymorphism within the promoter of the serotonin transporter gene. Neuropsychopharmacology. 12, 2230– 2235. [2] Serretti, A., Zanardi, R., Rossini, D., Cusin, C., Lilli, R., Smeraldi, E., 2001. Influence of trypophan hydroxylase and serotonin transporter genes on fluvoxamine antidepressant activity. Mol Psychiatry. 6, 586– 592.

P.4.b.004 Repeated dorsal periaqueductal gray injection of corticotrophin-releasing factor facilitates the expression of a panic response T. Oliveira Sergio1 ° , H. Zangrossi1 Pharmachology, S˜ao Paulo, Brazil

1 University

of Sao Pulo,

Introduction: Corticotrophin-releasing factor (CRF) is a peptidergic neurotransmitter that plays a critical role in the endocrine, autonomic and behavioral responses to a variety of stressors. Recent evidence suggests that dysregulation of CRF signaling system may contribute to the etiology of anxiety disorders [1]. In experimental animals, repetitive administration of sub-anxiogenic doses of CRF in limbic structures such amygdala and bed nucleus of the stria terminals (BNST) results in a chronic anxiety-like state, a phenomenon known as priming [2]. The dorsal periaqueductal gray matter (DPAG) in the midbrain contains substantial levels of CRF1 and CRF2 receptors, and has been considered a key region involved in the mediation of defensive behaviors related to both anxiety and panic. It has been consistently shown that acute administration of CRF in this structure enhances anxiety in different animal models [3]. The aim of this study was to investigate the effect of the repetitive intra-DPAG injection of a sub-anxiogenic dose of CRF in rats exposed to the elevated T-maze. This animal model measures, in the same animal, avoidance and escape responses that have been related to anxiety and panic responses, respectively. Methods: Male Wistar rats were implanted with a guidecannula aimed at the DPAG. In experiment 1, the animals were injected (0.2ul) in the DPAG with ovine CRF (250, 500, 1000 ng) or saline, and ten minutes later, they were tested in elevated T-maze. In experiment 2, the rats were intra-DPAG microinjected with ovine CRF (62.5 pg) or saline for 5 consecutive days. One group of animals was injected with saline for 4 days and on the fifth day they were injected with CRF (62.5 pg; acute group). Ten minutes after the last injection, the animals were tested in the elevated T-maze.

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Results: In experiment 1, intra-DPAG injection of CRF significantly (p < 0.05) facilitated escape expression [escape (mean±EPM): saline = 14.4±2.1 s, CRF-250 = 7.67±0.78 s, CRF-500 = 7.33±1.15 s, CRF-1000 = 7.67±1.17 s], without affecting inhibitory avoidance acquisition. In experiment 2, repeated administration of a sub-effective dose of CRF also potentiated escape expression [escape (mean±EPM): saline = 13.5±2.8, CRF-acute = 7.17±1.89 s, CRF-5days = 4.64±1.4] and this effect was not followed by change in inhibitory avoidance acquisition. No drug treatment affected the total distance traveled in the open field test, suggesting that the results observed in the elevated T-maze were not due to non-specific alterations in motor function. Conclusions: Intra-DPAG injection of CRF selectively facilitates a defensive response associated with panic attacks. As in other fear/anxiety-related areas such as the amygdala and BNST, repetitive stimulation of CRF receptors in the DPAG resulted in priming, a phenomenon that may be of importance in the pathophysiology of panic disorder. References [1] Risbrough, V.B., Stein, M.B., 2006. Role of corticotrophin releasing factor in anxiety disorders: a translational research perspective. Horm Behav 50, 550–561. [2] Younglim, L., Fitz, S., Johnson P.L., Shekar, A. 2008. Repeated stimulation of CRF receptors in the BNST of rats selectively induces social but not panic-like anxiety. Neuropsychopharmacology 11, 2586–2594. [3] Martins A.P., Marras R.A., Guimar˜aes F.S. 1997. Anxiogenis effect of corticotrophin-releasing hormone in the dorsal periaqueductal grey. Neuroreport. 16, 3601–3604.

P.4.b.005 Activation of 5-HT1A receptors in the prelimbic cortex changes anxiety-related, but not panic-related, defensive responses P.S.M. Yamashita1 ° , H. Zangrossi Jr.1 1 University of S˜ao Paulo − School of Medicine of Ribeirao Preto, Department of Pharmacology, Ribeirao Preto, Brazil Introduction: The dorsal raphe nucleus, the main source of serotonergic (5-HT) projections to limbic areas involved in the regulation/genesis of anxiety and fear, is densely innervated by the medial prefrontal cortex. The glutamatergic projection linking the medial prefrontal cortex to the dorsal raphe nucleus is shown to indirectly inhibit 5-HT cell firing, via activation of local GABAergic interneurons. It has been also shown that 5-HT receptors in the medial prefrontal cortex can exert a postsynaptic control on the activity of 5-HT neurons in the dorsal raphe nucleus. Electrophysiological studies revealed that local application of 5-HT1A receptor agonists into medial prefrontal cortex decreases 5-HT cell firing in the dorsal raphe nucleus [1,2]. In the present study, we investigated the behavioral effects caused by the activation of 5-HT1A receptors in the prelimbic cortex of animals exposed to the elevated T-maze. This animal model, derived from the elevated plus-maze, allows the measurement, in the same rat, of an anxiety- and a panic-related defensive response, respectively, inhibitory avoidance and escape. Methods: Male Wistar rats weighing 270–290 g were anaesthetized and fixed in a stereotaxic frame. Guides cannulae were bilaterally implanted in the forebrain aimed at the prelimbic cortex. Seven days after surgery, the animals were injected (0.2 mL) into the prelimbic cortex with the 5-HT1A agonist 8-OHDPAT (2, 4, 8 nmol) or saline and tested in the elevated T-maze. The test apparatus consists of three elevated arms − one enclosed