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frontal cortex
P.4.c. Anxiety disorders, OCD, stress related disorders and treatment − Treatment (basic) P.4.c.004 Acute stress selectively affects synaptic vesicle pools in prefrontal cortex glutamatergic terminals: preventing effect of desipramine N. Nava1 ° , M. Popoli2 , L. Musazzi2 , G. Wegener1 , J.R. Nyengaard3 1 Aarhus University, Center for Psychiatric Research, Risskov, Denmark; 2 University of Milan, Laboratory of Neuropsychopharmacology and Functional Neurogenomics, Milan, Italy; 3 Aarhus University, Stereology and Electron Microscopy Laboratory Centre for Stochastic Geometry and Advanced Bioimaging, Aarhus, Denmark Background: Growing evidences have highlighted a deep impairment in stress-related disorders of limbic structures such as amygdala, hippocampus and prefrontal cortex, where glutamatergic transmission is considered to be prevalent [1,2]. A number of studies have shown the cumulative effects of stress and its major mediators, glucocorticoids, on brain volume and dendritic remodeling, in both humans and rodents. Nevertheless, few is still known on the structural changes exerted by behavioral stress on the features of glutamatergic synapses as sites of neuronal communication. Indeed, in excitatory synapses synaptic communication is driven by neurotransmitter which is stored, within the presynaptic terminal, in morphologically distinct pools of vesicles, namely the readily-releasable pool of vesicles (RRP), docked to the active zone and ready for release, and the reserve pool of vesicles. When neurotransmitter is released, exchange of informations takes place through interaction of glutamate with receptors sitting on the post-synaptic density. Alterations of such synaptic ultrastructure might underlie following impairment of glutamatergic release and transmission. Aim: Main goal of the present study was therefore to shade light on the consequences of acute behavioral stress and chronic antidepressant treatment with tricyclic antidepressant desipramine on the features of asymmetric terminals, commonly regarded as excitatory, within medial prefrontal cortex (mPFC). Methods: Rats were treated chronically with either desipramine (DMI) or vehicle (2 weeks); at the end of the treatment animals were subjected to acute Foot-Shock (FS) stress and soon after they were deeply anesthetized and perfusion-fixed. Blood samples were taken through intracardiac puncture for corticosterone measurement. Based on its noticeable citoarchitectural features, mPFC was identified and glutamatergic terminals distribution within mPFC was estimated with glutamate transporter 1 (VGLUT1) immunolabeling [3]. Through serial section electron microscopy, glutamatergic synapses were identified and the number of membranedocked vesicles and total number of vesicles was estimated trough designed-based stereological methods. Extension of post-synaptic density area as well as overall asymmetric synapses distribution were also assessed. Results: Over 90% of mPFC synaptic terminals were found positive to VGLUT1 immunolabeling. Acute behavioral stress selectively induced a significant increase in the number of vesicles docked to the presynaptic membrane (p < 0.001). The stress-induced increase was prevented by previous treatment with chronic DMI (p < 0.001). No effect of either stress or desipramine was observed on total number of vesicles. A strong correlation was found between number of docked vesicles and corticosterone plasma levels in rats subjected to acute stress (Pearson correlation coefficient = 0.8).
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Conclusions: With the present study we showed that acute stress was able to rapidly and selectively induce synaptic vesicles mobilization toward presynaptic active zone, in mPFC glutamatergic temrinals. Chronic treatment with DMI seemed to only partially prevent such stress-induced changes. Identifying the effects of stress on excitatory transmission will provide further knowledge in developing drugs directly targeting the glutamatergic system. References [1] Musazzi, L., Milanese, M., Farisello, P., Zappettini, S., Tardito D., Barbiero, V.S., Bonifacino T., Mallei A., Baldelli P., Racagni G., Raitieri M., Benfenati F., Bonanno G., Popoli M., 2010. Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants. PloS One 5(1):e8566. [2] Ansell E.B., Rando K., Tuit K., Guarnaccia J., Sinha R., 2012. Cumulative adversity and smaller gray matter volume in medial prefrontal, anterior cingulate, and insula regions. Biol Psychiatry 72:57−64. [3] Van Eden C.G., Uylings H.B. 1985. Cytoarchitectonic development of the prefrontal cortex in the rat. J Comp Neurol 241:253−67.
P.4.c.005 Stress and corticosterone increase the readily releasable pool of vesicles in rat synaptosomes of prefrontal/frontal cortex G. Treccani1 ° , L. Musazzi1 , C. Perego1 , M. Milanese2 , T. Bonifacino2 , J. Lamanna3 , A. Malgaroli3 , G. Racagni1 , G. Bonanno2 , M. Popoli1 1 University of Milan, Department of Pharmacological and Biomolecular Sciences, Milan, Italy; 2 University of Genova, Department of Pharmacy − Unit of Pharmacology and Toxicology Centre of Excellence for Biomedical Research, Genova, Italy; 3 Scientific Institute San Raffaele and Universit`a Vita e Salute san Raffaele, Neurobiology of Learning Unit, Milano, Italy Purpose of the study: Stress and its mediators were shown to cause short and long-lasting functional/structural changes in the brain, which may trigger neuropsychatric disorders. In this regard, changes in glutamate release/transmission were shown to play a primary role in stress response [1]. We have previously found that Footshock (FS)-stress induces a marked increase of serum corticosterone (CORT) in serum levels and of depolarization-dependent glutamate release from synaptosomes of prefrontal/frontal cortex (PFC/FC), via activation of glucocorticoid receptor (GR) activation and SNARE complexes accumulation in synaptic membranes. The increase of glutamate release was prevented by chronic antidepressants [2]. Main aim of the present work was to verify if the changes induced by stress on glutamate neurotransmission were mediated by a synaptic (non-genomic) effect of corticosterone (CORT), the main stress hormone, we compared the effects of acute stress ex vivo with that of in vitro application of CORT on purified synaptosomes in PFC/FC. Methods: Rats were subjected to a standard Footshock (FS)stress protocol [2]. Immediately after FS-stress, PFC/FC was dissected and synaptosomes were purified on Percoll gradients; glutamate release was measured in PFC/FC synaptosomes of FSstressed animals or treated with CORT by using the superfusion technique [2]. Changes in vesicles mobilization were measured in PFC/FC synaptosomes treated with CORT by Total Internal Reflection Fluorescence Microscopy (TIRFM). Synapsin I phosphorylation levels were measured in samples of synaptic membranes by SDS-PAGE and Western blot. Electrophysiological experiments were performed on acute medial PFC slices, measuring evoked excitatorypost-synaptic potentials (EPSPs).
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P.4.c. Anxiety disorders, OCD, stress related disorders and treatment − Treatment (basic)
Results: We found that both FS-stress and CORT in vitro increase the size of the readily releasable pool (RRP) of vesicles, measured as glutamate release from PFC/FC synaptosomes in superfusion. This effect of FS-stress and CORT was abolished by selective antagonists for mineralcorticoid receptor (MR), spironolactone (SPIR) or GR, RU486. However, in contrast with what previously reported in hippocampus after acute stress, CORT did not affect glutamate release evoked by depolarization and did not alter excitatory transmission in medial PFC slices, suggesting that, despite the CORT-induced increase of RRP size, the increase of glutamatergic transmission is promoted by other mechanisms. We then confirmed, by using TIRFM, a technique allowing the study of events occurring just below the plasma membrane, that CORT increases vesicle mobilization toward the RRP, via activation of presynaptic MR and GR. Finally we found that both stress and CORT increase the phosphorylation of synapsin I exclusively at site 1 in presynaptic membrane. The increase of phosphorylation of synapsin I was CORT-mediated and blocked by SPIR and RU486, suggesting that this effect is downstream of MR and GR activation. Conclusions: The present results suggest that despite CORT plays a key role in the modulation of glutamatergic neurotransmission induced by acute stress in PFC/FC, its local non-genomic action on synaptic receptor is necessary but not sufficient for increasing glutamate release. References [1] Popoli, M., Yan, Z., McEwen, B.S., Sanacora, G. 2012 The stressed synapse: the impact of stress and glucocorticoids on glutamate transmission. Nat Rev Neurosci, 13, 22−37. [2] Musazzi, L., Milanese, M., Farisello, P., Zappettini, S., Tardito, D., Barbiero, V. S., Bonifacino, T., Mallei, A., Baldelli, P., Racagni, G., Raiteri, M., Benfenati, F., Bonanno, G., Popoli, M. 2010 Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants. PloS ONE 5, e8566.
P.4.c.006 Diagnostic and treatment particularities of early-onset obsessive–compulsive disorder in children A. Simion1 ° , M. Mihaela1 , S. Macovei1 1 Clinical Hospital of Psychiatry “Prof. Al. Dr. Obregia”, Child and Adolescent Departament, Bucharest, Romania Background: Obsessive–Compulsive Disorder (OCD) is a type of anxiety disorder with high debilitating potential due to repetitive thought and behavioral cycles. It usually appears at around 7−12 years. If the symptoms appear and interfere with day-today activities before this age we can talk about an early onset OCD. The recommended treatment for children is cognitivebehavior therapy (CBT) and/or pharmacotherapy. Recommended pharmacotherapy consists of selective serotonin reuptake inhibitor (SSRI) or tricyclic (clomipramine) antidepressants. Studies show that fluvoxamine has a high tolerance rate in children above age 7. Objective: The objective of this presentation is to bring into focus not only the early onset OCD in children and its impact on social interactions but also to evaluate the difficulties in differentiating between it and opposition and conduct disorders. Furthermore it will attempt to objectify improvement in symptomatology and/or the appearance of comorbidities during diazepam and SSRI (fluvoxamine) treatment.
Methods: A 5 year old patient which had presented at the Child and Adolescent Department ‘Prof. Dr. Al. Obregia’ Psychiatry Hospital in Bucharest on 27.03.2012 underwent physiological (EEG), mental and psychological (STONY questionnaire) tests that supported the OCD diagnosis. The patient has been observed for a period of one year. Results: A satisfactory improvement of symptoms was observed following diazepam 1 mg and fluvoxamine 25 mg treatment. When discharged, the patient received a 6 month prescription for Fevarine. After 6 months, a significant improvement of daily rituals was observed without the appearance of any comorbidities. The pursued concerns were separation anxiety (especially from the mother), fear of danger and behavior with other children and her family members (the patient was trying to control everybody and if they didn’t do what she wants then she would scream). Conclusions: The results strengthen the importance of therapy in early onset OCD [3]. Most of the children with OCD exhibit a relatively low insight into their illness and a low awareness of their aberrant behaviors. Children at this age (age 5) don’t have an insight so they are predisposed to developing relationship problems. In this case, the differential diagnosis included conduct disorder and opposition disorder which, at this age, are particularly difficult to differentiate from OCD due to their shared symptoms: anger outbursts and inadequate social behavior due to the child’s inability to understand his illness and to control his thoughts. The importance of diagnostic OCD in early childhood is to prevent or to keep under observation the appearence of comorbidities such as tic disorders, depression, bipolar disorder, schizophrenia in adolescents and later on adults. References [1] Geller DA, Biederman J, Jones J, Shapiro S, Schwartz S, Park KS: Obsessive–compulsive disorder in children and adolescents: a review. Harv Rev Psychiatry 1998; 5: 260–273. [2] Leonardo F. Fontenelle, Mauro V. Mendlowicz, Carla Marquesa, Marcio Versiania: Early- and late-onset obsessive–compulsive disorder in adult patients: an exploratory clinical and therapeutic study. Journal of Psychiatric Research Volume 37, Issue 2, March–April 2003. [3] Rapoport JL, Inoff-Germain G. Treatment of obsessive–compulsive disorder in children and adolescents. J Child Psychol Psychiatry. 2000; 41: 419–431.
P.4.c.007 Acetyl-l-carnitine prevents the stressinduced chromatin remodeling within the glutamatergic synapse C. Nasca1 ° , T. Rubin2 , J. Gray2 , B. Bigio3 , B.S. McEwen2 University of Rome-The Rockefeller University New York, Physiology and Pharmacology “V. Erspamer”-Laboratory of Neuroendocrinology, Rome-New York, Italy; 2 The Rockefeller University New York, Laboratory of Neuroendocrinology, New York, USA; 3 The Rockefeller University New York, Center for Clinical & Translational Science, New York, USA
1 Sapienza
Stressful events trigger an epigenetic program that can predispose individuals to a variety of mental disorders, such as depression, anxiety and post-traumatic stress disorders. Understanding how epigenetic changes, induced by psychological stressors, alter brain plasticity, shaping neuronal vulnerability to stress in ways that resemble those observed in depressed patients, is crucial to uncovering both pharmaceutical medications that will ameliorate mood and more efficient antidepressants with faster onset of action.
S521
Conclusions: With the present study we showed that acute stress was able to rapidly and selectively induce synaptic vesicles mobilization toward presynaptic active zone, in mPFC glutamatergic temrinals. Chronic treatment with DMI seemed to only partially prevent such stress-induced changes. Identifying the effects of stress on excitatory transmission will provide further knowledge in developing drugs directly targeting the glutamatergic system. References [1] Musazzi, L., Milanese, M., Farisello, P., Zappettini, S., Tardito D., Barbiero, V.S., Bonifacino T., Mallei A., Baldelli P., Racagni G., Raitieri M., Benfenati F., Bonanno G., Popoli M., 2010. Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants. PloS One 5(1):e8566. [2] Ansell E.B., Rando K., Tuit K., Guarnaccia J., Sinha R., 2012. Cumulative adversity and smaller gray matter volume in medial prefrontal, anterior cingulate, and insula regions. Biol Psychiatry 72:57−64. [3] Van Eden C.G., Uylings H.B. 1985. Cytoarchitectonic development of the prefrontal cortex in the rat. J Comp Neurol 241:253−67.
P.4.c.005 Stress and corticosterone increase the readily releasable pool of vesicles in rat synaptosomes of prefrontal/frontal cortex G. Treccani1 ° , L. Musazzi1 , C. Perego1 , M. Milanese2 , T. Bonifacino2 , J. Lamanna3 , A. Malgaroli3 , G. Racagni1 , G. Bonanno2 , M. Popoli1 1 University of Milan, Department of Pharmacological and Biomolecular Sciences, Milan, Italy; 2 University of Genova, Department of Pharmacy − Unit of Pharmacology and Toxicology Centre of Excellence for Biomedical Research, Genova, Italy; 3 Scientific Institute San Raffaele and Universit`a Vita e Salute san Raffaele, Neurobiology of Learning Unit, Milano, Italy Purpose of the study: Stress and its mediators were shown to cause short and long-lasting functional/structural changes in the brain, which may trigger neuropsychatric disorders. In this regard, changes in glutamate release/transmission were shown to play a primary role in stress response [1]. We have previously found that Footshock (FS)-stress induces a marked increase of serum corticosterone (CORT) in serum levels and of depolarization-dependent glutamate release from synaptosomes of prefrontal/frontal cortex (PFC/FC), via activation of glucocorticoid receptor (GR) activation and SNARE complexes accumulation in synaptic membranes. The increase of glutamate release was prevented by chronic antidepressants [2]. Main aim of the present work was to verify if the changes induced by stress on glutamate neurotransmission were mediated by a synaptic (non-genomic) effect of corticosterone (CORT), the main stress hormone, we compared the effects of acute stress ex vivo with that of in vitro application of CORT on purified synaptosomes in PFC/FC. Methods: Rats were subjected to a standard Footshock (FS)stress protocol [2]. Immediately after FS-stress, PFC/FC was dissected and synaptosomes were purified on Percoll gradients; glutamate release was measured in PFC/FC synaptosomes of FSstressed animals or treated with CORT by using the superfusion technique [2]. Changes in vesicles mobilization were measured in PFC/FC synaptosomes treated with CORT by Total Internal Reflection Fluorescence Microscopy (TIRFM). Synapsin I phosphorylation levels were measured in samples of synaptic membranes by SDS-PAGE and Western blot. Electrophysiological experiments were performed on acute medial PFC slices, measuring evoked excitatorypost-synaptic potentials (EPSPs).
S522
P.4.c. Anxiety disorders, OCD, stress related disorders and treatment − Treatment (basic)
Results: We found that both FS-stress and CORT in vitro increase the size of the readily releasable pool (RRP) of vesicles, measured as glutamate release from PFC/FC synaptosomes in superfusion. This effect of FS-stress and CORT was abolished by selective antagonists for mineralcorticoid receptor (MR), spironolactone (SPIR) or GR, RU486. However, in contrast with what previously reported in hippocampus after acute stress, CORT did not affect glutamate release evoked by depolarization and did not alter excitatory transmission in medial PFC slices, suggesting that, despite the CORT-induced increase of RRP size, the increase of glutamatergic transmission is promoted by other mechanisms. We then confirmed, by using TIRFM, a technique allowing the study of events occurring just below the plasma membrane, that CORT increases vesicle mobilization toward the RRP, via activation of presynaptic MR and GR. Finally we found that both stress and CORT increase the phosphorylation of synapsin I exclusively at site 1 in presynaptic membrane. The increase of phosphorylation of synapsin I was CORT-mediated and blocked by SPIR and RU486, suggesting that this effect is downstream of MR and GR activation. Conclusions: The present results suggest that despite CORT plays a key role in the modulation of glutamatergic neurotransmission induced by acute stress in PFC/FC, its local non-genomic action on synaptic receptor is necessary but not sufficient for increasing glutamate release. References [1] Popoli, M., Yan, Z., McEwen, B.S., Sanacora, G. 2012 The stressed synapse: the impact of stress and glucocorticoids on glutamate transmission. Nat Rev Neurosci, 13, 22−37. [2] Musazzi, L., Milanese, M., Farisello, P., Zappettini, S., Tardito, D., Barbiero, V. S., Bonifacino, T., Mallei, A., Baldelli, P., Racagni, G., Raiteri, M., Benfenati, F., Bonanno, G., Popoli, M. 2010 Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the dampening action of antidepressants. PloS ONE 5, e8566.
P.4.c.006 Diagnostic and treatment particularities of early-onset obsessive–compulsive disorder in children A. Simion1 ° , M. Mihaela1 , S. Macovei1 1 Clinical Hospital of Psychiatry “Prof. Al. Dr. Obregia”, Child and Adolescent Departament, Bucharest, Romania Background: Obsessive–Compulsive Disorder (OCD) is a type of anxiety disorder with high debilitating potential due to repetitive thought and behavioral cycles. It usually appears at around 7−12 years. If the symptoms appear and interfere with day-today activities before this age we can talk about an early onset OCD. The recommended treatment for children is cognitivebehavior therapy (CBT) and/or pharmacotherapy. Recommended pharmacotherapy consists of selective serotonin reuptake inhibitor (SSRI) or tricyclic (clomipramine) antidepressants. Studies show that fluvoxamine has a high tolerance rate in children above age 7. Objective: The objective of this presentation is to bring into focus not only the early onset OCD in children and its impact on social interactions but also to evaluate the difficulties in differentiating between it and opposition and conduct disorders. Furthermore it will attempt to objectify improvement in symptomatology and/or the appearance of comorbidities during diazepam and SSRI (fluvoxamine) treatment.
Methods: A 5 year old patient which had presented at the Child and Adolescent Department ‘Prof. Dr. Al. Obregia’ Psychiatry Hospital in Bucharest on 27.03.2012 underwent physiological (EEG), mental and psychological (STONY questionnaire) tests that supported the OCD diagnosis. The patient has been observed for a period of one year. Results: A satisfactory improvement of symptoms was observed following diazepam 1 mg and fluvoxamine 25 mg treatment. When discharged, the patient received a 6 month prescription for Fevarine. After 6 months, a significant improvement of daily rituals was observed without the appearance of any comorbidities. The pursued concerns were separation anxiety (especially from the mother), fear of danger and behavior with other children and her family members (the patient was trying to control everybody and if they didn’t do what she wants then she would scream). Conclusions: The results strengthen the importance of therapy in early onset OCD [3]. Most of the children with OCD exhibit a relatively low insight into their illness and a low awareness of their aberrant behaviors. Children at this age (age 5) don’t have an insight so they are predisposed to developing relationship problems. In this case, the differential diagnosis included conduct disorder and opposition disorder which, at this age, are particularly difficult to differentiate from OCD due to their shared symptoms: anger outbursts and inadequate social behavior due to the child’s inability to understand his illness and to control his thoughts. The importance of diagnostic OCD in early childhood is to prevent or to keep under observation the appearence of comorbidities such as tic disorders, depression, bipolar disorder, schizophrenia in adolescents and later on adults. References [1] Geller DA, Biederman J, Jones J, Shapiro S, Schwartz S, Park KS: Obsessive–compulsive disorder in children and adolescents: a review. Harv Rev Psychiatry 1998; 5: 260–273. [2] Leonardo F. Fontenelle, Mauro V. Mendlowicz, Carla Marquesa, Marcio Versiania: Early- and late-onset obsessive–compulsive disorder in adult patients: an exploratory clinical and therapeutic study. Journal of Psychiatric Research Volume 37, Issue 2, March–April 2003. [3] Rapoport JL, Inoff-Germain G. Treatment of obsessive–compulsive disorder in children and adolescents. J Child Psychol Psychiatry. 2000; 41: 419–431.
P.4.c.007 Acetyl-l-carnitine prevents the stressinduced chromatin remodeling within the glutamatergic synapse C. Nasca1 ° , T. Rubin2 , J. Gray2 , B. Bigio3 , B.S. McEwen2 University of Rome-The Rockefeller University New York, Physiology and Pharmacology “V. Erspamer”-Laboratory of Neuroendocrinology, Rome-New York, Italy; 2 The Rockefeller University New York, Laboratory of Neuroendocrinology, New York, USA; 3 The Rockefeller University New York, Center for Clinical & Translational Science, New York, USA
1 Sapienza
Stressful events trigger an epigenetic program that can predispose individuals to a variety of mental disorders, such as depression, anxiety and post-traumatic stress disorders. Understanding how epigenetic changes, induced by psychological stressors, alter brain plasticity, shaping neuronal vulnerability to stress in ways that resemble those observed in depressed patients, is crucial to uncovering both pharmaceutical medications that will ameliorate mood and more efficient antidepressants with faster onset of action.