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Role of the endocannabinoid system in the altered social behavior observed in the rat valproic acid model of autism
P.1.h. Basic and clinical neuroscience − Animal behaviour cultures of mouse mesencephalic dopaminergic neurons obtained from 12.5 day embryos [3,4]. Hence, we studied the effects of ketamine in this murine cellular model. In addition, we extended these findings to human dopaminergic neurons differentiated from inducible pluripotent stem cells (hiPSC) [5]. hiPSC were generated from healthy donors and differentiated into dopaminergic neurons that were cultured for 80 days to induce a maturation as complete as possible. At this stage tyrosine hydroxylase immunoreactive neurons also contained the dopamine transporter and formed a functional network with GABAergic and glutamatergic neurons. Results: In mouse primary cultures of dopaminergic neurons, ketamine (0.01−1 mM) dose-dependently increased dendritic arborisation and soma size as measured using computer-assisted morphometry. These effects were inhibited by pre-treatment with the AMPA antagonists NBQX (10 mM) and GYKI 52466 (10 mM). In addition, the AMPA positive allosteric modulator CX614 (10 mM) produced similar effects. Intracellular pathways involved in this structural plasticity were studied by western blot and confocal microscopy. Ketamine increased phosphorylation of p-70S6 kinase, located downstream of mTOR, an effect blocked by LY294002 (10 mM), a PI3 kinase inhibitor, and rapamycin (20 nM), a mTORC-1 complex inhibitor. In previous works, cocaine and nicotine promoted structural plasticity of dopaminergic neurons by indirect DA-mediated activation of dopamine D3 receptors (D3R). By analogy, the effects of ketamine, including mTOR recruitment, were blocked by the selective D3 receptor antagonist SB277011-A (50 nM) and were absent in primary cultures of DA D3-KO mice. Immunoneutralization of BDNF also prevented the effects of ketamine suggesting the need for co-activation of TrkB-MEK-ERK and D3R-Akt-mTOR pathways to induce structural plasticity. Similar to mouse dopaminergic neurons, ketamine dose-dependently increased dendritic outgrowth and soma size in hiPSC-derived dopaminergic neurons. These effects were prevented by the AMPA antagonists NBQX and GYKI 52466, consistent with involvement of extracellular glutamate. Ketamine also increased the phosphorylation of p-70S6 kinase, an effect that was prevented by LY294002 and rapamycin. Finally, the D3R antagonist SB277011-A blocked the effects of ketamine. Conclusions: These results show that ketamine induces structural plasticity in DA neurons via a complex pattern of actions involving AMPA receptor activation and engagement of TrkBMEK-ERK and D3R-Akt-mTOR pathways. Furthermore, these observations can be “translated” into humans inasmuch as similar findings as in primary mouse cultures were seen in hiPSC. References [1] Duman, R.S., Li, N., Liu, R.-J., Duric, V., Aghajanian, G., 2012. Signaling pathways underlying the rapid antidepressant actions of ketamine. Neuropharmacology 62, 35−41. [2] Li, N., Lee, B., Liu, R.J., Banasr, M., Dwyer, J.M., Iwata, M., Li, X.Y., Aghajanian, G., Duman, R.S., 2010. mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329, 959–964. [3] Collo, G., Bono, F., Cavalleri, L., Plebani, L., Merlo Pich, E., Millan, M.J., Spano, PF., Missale, C., 2012. Pre-synaptic dopamine D3 receptor mediates cocaine-induced structural plasticity in mesencephalic dopaminergic neurons via ERK and Akt pathways. J Neurochem 120, 765–778. [4] Collo, G., Bono, F., Cavalleri, L., Plebani, L., Mitola, S., Merlo Pich, E., Millan, M.J., Zoli, M., Maskos, U., Spano, PF., Missale, C., 2013. Nicotine-induced structural plasticity in mesencephalic dopaminergic neurons is mediated by dopamine D3 receptors and Akt-mTORC1 signaling. Mol Pharmacol 83, 1176–1189. [5] Kriks, S., Shim, J.W., Piao, J., Ganat, Y.M., Wakeman, D.R., Xie, Z., Carrillo-Reid, L., Auyeung, G., Antonacci, C., Buch, A., Yang, L., Beal, M.F., Surmeier, D.J., Kordower, J.H., Tabar, V., Studer, L., 2011.
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Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480, 547–551.
P.1.h. Basic and clinical neuroscience − Animal behaviour P.1.h.002 Role of the endocannabinoid system in the altered social behavior observed in the rat valproic acid model of autism M. Servadio1 ° , F. Melancia1 , V. Cartocci1 , V. Pallottini1 , V. Trezza1 1 Roma Tre University, Science, Rome, Italy Purpose: Autism spectrum disorders (ASD) are characterized by impaired social interaction and communication and repetitive patterns of behavior. To date, no effective and specific treatments are yet available for ASD. Given the multifactorial etiology of ASD, the use of animal models is essential to dissect the role of genetic and environmental factors in the pathogenesis of this disease, to unravel the relationships between altered brain function in ASD and behavior and to test new pharmacological options. The endocannabinoid system has been shown to modulate socio-emotional behaviors, and several components of the endocannabinoid system have been found altered in genetic and environmental animal models of ASD. In this study, we investigated the role of the endocannabinoid system in the altered social behavior observed in the rat valproic acid model of ASD. In particular, the present study had a three-fold aim: (1) investigate whether VPA prenatal exposure alters social behavior both in the adolescent and adult rat offspring; (2) study the activation of CB1 receptors in brain areas involved in socio-emotional functioning; (3) test the possibility that pharmacological manipulation of the endocannabinoid system may correct the behavioral deficits found in VPA-exposed animals. Methods: Wistar pregnant rats were treated intraperitoneally with VPA (500 mg/kg/2 ml) [1], or the same volume of saline solution (SAL) at gestational day 12 (GD12). After delivery, the male offspring was tested in the social play behavior test in adolescence (post natal days (PND) 30−35), and in the 3-chamber test both in adolescence and adulthood (PND 70−80). Furthermore, the phosphorylation of CB1 receptors was analyzed both during adolescence and adulthood by western blot in the folloowing brain areas: prefrontal cortex, dorsal striatum, ventral striatum, hippocampus, amygdala and cerebellum. In the last part of the study, the effects of URB597, that inhibits the hydrolysis of the endocannabinoid anandamide, were tested in rats prenatally exposed to either VPA or vehicle. Results: Animals prenatally exposed to VPA responded less to play solicitation compared to SAL-exposed animals (p < 0.05). In the 3 chamber test, both during adolescence and adulthood, VPA-exposed animals spent less time approaching the stimulus compared to SAL-exposed animal (adolescence: p < 0.01; adulthood: p < 0.01). Furthermore, the western blot experiments revealed that the phosphorylation of CB1 receptor was decreased in the hippocampus and increased in dorsal striatum both in adolescent (hippocampus: p < 0.01; dorsal striatum: p < 0.001) and adult (hippocampus: p < 0.001; dorsal striatum: p < 0.001) VPA-exposed animals, and decreased in the amygdala only at adulthood (p < 0.05). Interestingly, URB597 corrected the altered social behavior displayed by VPA-exposed animals at adolescence and adulthood. Conclusion: Altogether, these findings reveal that prenatal exposure to VPA provides a good model for specific aspects of
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ASD. In particular, we observed altered social behavior in VPAexposed rats both during adolescence and adulthood. Furthermore, the altered activation of CB1 receptors in different brain areas involved in socio-emotional functioning, together with the positive effect of pharmacological manipulation of the endocannabinoid system, suggests a possible involvement of the endocannabinoid system in the symptomatology and etiology of ASD. References [1] Schneider, T., Przewlocki, R., 2005. Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism. Neuropsychopharmacology 30(1), 80−9.
P.1.h.004 A new model of impulsivity, social interactions and preference of alcohol in mice living in groups L. Szumiec1 ° , J. Rodriguez-Parkitna1 1 Institute of Pharmacology- Polish Academy of Sciences, Molecular Pharmacology, Krak´ow, Poland Background: Impulsive behaviour is an important component of clinical symptoms in several psychiatric disorders. Inability to tolerate a delay of grafitication is one important factor which might lead to aggression, frustration or violence [2,3]. The goal of our project is identification of neuronal mechanisms responsible for economic decision making. We have developed a set of new models that assess the effects of delay on choice behavior. The rationale for the development of novel models was to test behaviors under minimal environmental constraints, as similar to natural conditions as possible [1]. Methods: We have worked on model of testing delay discounting in mice living in groups. A cohort of 10−16 mice was implanted subcutaneously with radiofrequency identification chips and placed into a cage equipped with sensors for automatic tracking and 4 corners with 2 drinking bottles for each accessed through a small compartment (Intellicage Plus). Mice can be individually monitored and restricted to model effects of delay discounting. Results: Two models were tested: choice between saccharin and water and choice among two concentrations of saccharin (0.01%, 0.1%) and water. The former is simpler, while the latter allows for comparison of the same reward with different magnitudes. In both models mice showed initially high preference for 0.1% sachharin and shifted preference towards water or 0.01% saccharin when the delay reached more than 8s. To validate the model we have tested the effects of an irreversible inhibitor of the monoamine oxidase − tranylcypromine (3 mg/kg ip., single injection) on delay discounting. In line with expectations the drug induced increase in monoamine levels caused acceleration of delay discounting (t-test with Bonferroni, p(14s.) = 0.04; p(17s.) = 0.02; p(20s) = 0.006). Also we have tested cloccinamox (10 mg/kg ip.), an opioid receptor antagonist but it hadn’t effect on delay discounting (t-test with Bonferroni, p = 0.35) We have tested also the preference of saccharin and the most important parameter called the indifference point, which represents the value of the delayed outcome. It is exactly when the preference between values is equal (in our case is about 17s of delay on the greater one). Furthermore, we have tested an example of social interactions with following (5 pairs of mice: leader/follower). Mice would get a reward only if had entered to corner after suitable leader. Followers could follow their leaders to get a reward (preference even 50%). Moreover, we have
tested the preference of drinking alcohol (4, 8, 12%), as alleged the preference decreased steeply from 0.70 (4%), 0.46 (8%) and 0.11 (12%). Conclusions: The main advantages of the new model are the ability to test behaviour in the home cage in group housed mice, during natural activity cycles, no interaction with the experimenter and without pharmacological treatments or genetic modifications on delay discounting. The further research will be based on social interactions and ADE (model of alcoholism). References [1] Parkitna, J.R., Sikora, M., Gołda, S., Gołembiowska, K., Bystrowska, B., Engblom, D., Bilbao, A., Przewlocki, R., 2013. Novelty-seeking behaviors and the escalation of alcohol drinking after abstinence in mice are controlled by metabotropic glutamate receptor 5 on neurons expressing dopamine d1 receptors. Biological Psychiatry 73(3), 263−27. [2] Yates, J.R., Batten, S.R., Bardo, M.T., Beckmann, J.S., 2015. Role of ionotropic glutamate receptors in delay and probability discounting in the rat. Psychopharmacology 232(7), 1187–119. [3] Evenden, J.L., Ryan, C.N., 1996. The pharmacology of impulsive behaviour in rats: the effects of drugs on response choice with varying delays of reinforcement. Psychopharmacology 128(2), 161–170.
P.1.h.005 Anxiolytic-like effect of gallic acid and quercetin in young/healthy rats A. Georgieva1 ° , I. Belcheva2 , S. Belcheva3 , R. Tashev4 , S. Valcheva-Kuzmanova1 1 Medical University “Prof. Dr. Paraskev Stoyanov”, Preclinical and Clinical Pharmacology, Varna, Bulgaria; 2 Institute of Neurobiology- Bulgarian Academy of Sciences- Sofia- Bulgaria, Behavioral neurobiology, Sofia, Bulgaria; 3 Sofia University, Department of Pre-school and Primary School Education, Sofia, Bulgaria; 4 Medical universitySofia, Pathophysiology, Sofia, Bulgaria Introduction: Some of the most widespread polyphenols in natural food sources are phenolic acids such as gallic acid and flavonoids such as quercetin. An anxiolytic-like effect of gallic acid was observed after a single intraperitoneal administration [1]. There are some data suggesting an anxiolytic-like effect of quercetin given as a single dose [2], and repeatedly at a high dose [3]. Aim: The aim of the present study was to investigate the effect of gallic acid and quercetin administered subchronically at equal low doses on anxiety in young/healthy rats. Methods: Male Wistar rats (200–250 g) were used in the experiments. The treatment lasted for 7, 14, 21 or 30 days. For each treatment period, there were three groups receiving daily orally one of the following treatments: saline 10 ml/kg (controls), gallic acid (20 mg/kg as a 10 ml/kg solution) or quercetin (20 mg/kg as a 10 ml/kg solution). After each treatment period, animals were tested in the elevated plus-maze which is a common test for anxiety behavior in rodents. Its concept lies in the conflict between the natural fear of the animals from open areas and their curiosity to a novel environment [4]. The test was carried out on 12 different animal groups (3 kinds of treatment, 4 treatment durations) consisting of 10 rats each. The following indices were recorded during the 5-min test period: the number of entries into open arms and the time spent there, the number of entries into closed arms of the maze and the time spent there, the total number of arm entries, and the ratio of the number of entries into the open arms vs. total number of entries. The statistical analyses were performed by one-way ANOVA using GraphPad Prism statistical software. Results: Applied for 7 days, gallic acid and quercetin had no significant effects on the recorded indices. The two kinds
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Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease. Nature 480, 547–551.
P.1.h. Basic and clinical neuroscience − Animal behaviour P.1.h.002 Role of the endocannabinoid system in the altered social behavior observed in the rat valproic acid model of autism M. Servadio1 ° , F. Melancia1 , V. Cartocci1 , V. Pallottini1 , V. Trezza1 1 Roma Tre University, Science, Rome, Italy Purpose: Autism spectrum disorders (ASD) are characterized by impaired social interaction and communication and repetitive patterns of behavior. To date, no effective and specific treatments are yet available for ASD. Given the multifactorial etiology of ASD, the use of animal models is essential to dissect the role of genetic and environmental factors in the pathogenesis of this disease, to unravel the relationships between altered brain function in ASD and behavior and to test new pharmacological options. The endocannabinoid system has been shown to modulate socio-emotional behaviors, and several components of the endocannabinoid system have been found altered in genetic and environmental animal models of ASD. In this study, we investigated the role of the endocannabinoid system in the altered social behavior observed in the rat valproic acid model of ASD. In particular, the present study had a three-fold aim: (1) investigate whether VPA prenatal exposure alters social behavior both in the adolescent and adult rat offspring; (2) study the activation of CB1 receptors in brain areas involved in socio-emotional functioning; (3) test the possibility that pharmacological manipulation of the endocannabinoid system may correct the behavioral deficits found in VPA-exposed animals. Methods: Wistar pregnant rats were treated intraperitoneally with VPA (500 mg/kg/2 ml) [1], or the same volume of saline solution (SAL) at gestational day 12 (GD12). After delivery, the male offspring was tested in the social play behavior test in adolescence (post natal days (PND) 30−35), and in the 3-chamber test both in adolescence and adulthood (PND 70−80). Furthermore, the phosphorylation of CB1 receptors was analyzed both during adolescence and adulthood by western blot in the folloowing brain areas: prefrontal cortex, dorsal striatum, ventral striatum, hippocampus, amygdala and cerebellum. In the last part of the study, the effects of URB597, that inhibits the hydrolysis of the endocannabinoid anandamide, were tested in rats prenatally exposed to either VPA or vehicle. Results: Animals prenatally exposed to VPA responded less to play solicitation compared to SAL-exposed animals (p < 0.05). In the 3 chamber test, both during adolescence and adulthood, VPA-exposed animals spent less time approaching the stimulus compared to SAL-exposed animal (adolescence: p < 0.01; adulthood: p < 0.01). Furthermore, the western blot experiments revealed that the phosphorylation of CB1 receptor was decreased in the hippocampus and increased in dorsal striatum both in adolescent (hippocampus: p < 0.01; dorsal striatum: p < 0.001) and adult (hippocampus: p < 0.001; dorsal striatum: p < 0.001) VPA-exposed animals, and decreased in the amygdala only at adulthood (p < 0.05). Interestingly, URB597 corrected the altered social behavior displayed by VPA-exposed animals at adolescence and adulthood. Conclusion: Altogether, these findings reveal that prenatal exposure to VPA provides a good model for specific aspects of
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ASD. In particular, we observed altered social behavior in VPAexposed rats both during adolescence and adulthood. Furthermore, the altered activation of CB1 receptors in different brain areas involved in socio-emotional functioning, together with the positive effect of pharmacological manipulation of the endocannabinoid system, suggests a possible involvement of the endocannabinoid system in the symptomatology and etiology of ASD. References [1] Schneider, T., Przewlocki, R., 2005. Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism. Neuropsychopharmacology 30(1), 80−9.
P.1.h.004 A new model of impulsivity, social interactions and preference of alcohol in mice living in groups L. Szumiec1 ° , J. Rodriguez-Parkitna1 1 Institute of Pharmacology- Polish Academy of Sciences, Molecular Pharmacology, Krak´ow, Poland Background: Impulsive behaviour is an important component of clinical symptoms in several psychiatric disorders. Inability to tolerate a delay of grafitication is one important factor which might lead to aggression, frustration or violence [2,3]. The goal of our project is identification of neuronal mechanisms responsible for economic decision making. We have developed a set of new models that assess the effects of delay on choice behavior. The rationale for the development of novel models was to test behaviors under minimal environmental constraints, as similar to natural conditions as possible [1]. Methods: We have worked on model of testing delay discounting in mice living in groups. A cohort of 10−16 mice was implanted subcutaneously with radiofrequency identification chips and placed into a cage equipped with sensors for automatic tracking and 4 corners with 2 drinking bottles for each accessed through a small compartment (Intellicage Plus). Mice can be individually monitored and restricted to model effects of delay discounting. Results: Two models were tested: choice between saccharin and water and choice among two concentrations of saccharin (0.01%, 0.1%) and water. The former is simpler, while the latter allows for comparison of the same reward with different magnitudes. In both models mice showed initially high preference for 0.1% sachharin and shifted preference towards water or 0.01% saccharin when the delay reached more than 8s. To validate the model we have tested the effects of an irreversible inhibitor of the monoamine oxidase − tranylcypromine (3 mg/kg ip., single injection) on delay discounting. In line with expectations the drug induced increase in monoamine levels caused acceleration of delay discounting (t-test with Bonferroni, p(14s.) = 0.04; p(17s.) = 0.02; p(20s) = 0.006). Also we have tested cloccinamox (10 mg/kg ip.), an opioid receptor antagonist but it hadn’t effect on delay discounting (t-test with Bonferroni, p = 0.35) We have tested also the preference of saccharin and the most important parameter called the indifference point, which represents the value of the delayed outcome. It is exactly when the preference between values is equal (in our case is about 17s of delay on the greater one). Furthermore, we have tested an example of social interactions with following (5 pairs of mice: leader/follower). Mice would get a reward only if had entered to corner after suitable leader. Followers could follow their leaders to get a reward (preference even 50%). Moreover, we have
tested the preference of drinking alcohol (4, 8, 12%), as alleged the preference decreased steeply from 0.70 (4%), 0.46 (8%) and 0.11 (12%). Conclusions: The main advantages of the new model are the ability to test behaviour in the home cage in group housed mice, during natural activity cycles, no interaction with the experimenter and without pharmacological treatments or genetic modifications on delay discounting. The further research will be based on social interactions and ADE (model of alcoholism). References [1] Parkitna, J.R., Sikora, M., Gołda, S., Gołembiowska, K., Bystrowska, B., Engblom, D., Bilbao, A., Przewlocki, R., 2013. Novelty-seeking behaviors and the escalation of alcohol drinking after abstinence in mice are controlled by metabotropic glutamate receptor 5 on neurons expressing dopamine d1 receptors. Biological Psychiatry 73(3), 263−27. [2] Yates, J.R., Batten, S.R., Bardo, M.T., Beckmann, J.S., 2015. Role of ionotropic glutamate receptors in delay and probability discounting in the rat. Psychopharmacology 232(7), 1187–119. [3] Evenden, J.L., Ryan, C.N., 1996. The pharmacology of impulsive behaviour in rats: the effects of drugs on response choice with varying delays of reinforcement. Psychopharmacology 128(2), 161–170.
P.1.h.005 Anxiolytic-like effect of gallic acid and quercetin in young/healthy rats A. Georgieva1 ° , I. Belcheva2 , S. Belcheva3 , R. Tashev4 , S. Valcheva-Kuzmanova1 1 Medical University “Prof. Dr. Paraskev Stoyanov”, Preclinical and Clinical Pharmacology, Varna, Bulgaria; 2 Institute of Neurobiology- Bulgarian Academy of Sciences- Sofia- Bulgaria, Behavioral neurobiology, Sofia, Bulgaria; 3 Sofia University, Department of Pre-school and Primary School Education, Sofia, Bulgaria; 4 Medical universitySofia, Pathophysiology, Sofia, Bulgaria Introduction: Some of the most widespread polyphenols in natural food sources are phenolic acids such as gallic acid and flavonoids such as quercetin. An anxiolytic-like effect of gallic acid was observed after a single intraperitoneal administration [1]. There are some data suggesting an anxiolytic-like effect of quercetin given as a single dose [2], and repeatedly at a high dose [3]. Aim: The aim of the present study was to investigate the effect of gallic acid and quercetin administered subchronically at equal low doses on anxiety in young/healthy rats. Methods: Male Wistar rats (200–250 g) were used in the experiments. The treatment lasted for 7, 14, 21 or 30 days. For each treatment period, there were three groups receiving daily orally one of the following treatments: saline 10 ml/kg (controls), gallic acid (20 mg/kg as a 10 ml/kg solution) or quercetin (20 mg/kg as a 10 ml/kg solution). After each treatment period, animals were tested in the elevated plus-maze which is a common test for anxiety behavior in rodents. Its concept lies in the conflict between the natural fear of the animals from open areas and their curiosity to a novel environment [4]. The test was carried out on 12 different animal groups (3 kinds of treatment, 4 treatment durations) consisting of 10 rats each. The following indices were recorded during the 5-min test period: the number of entries into open arms and the time spent there, the number of entries into closed arms of the maze and the time spent there, the total number of arm entries, and the ratio of the number of entries into the open arms vs. total number of entries. The statistical analyses were performed by one-way ANOVA using GraphPad Prism statistical software. Results: Applied for 7 days, gallic acid and quercetin had no significant effects on the recorded indices. The two kinds