Chronic treatment with desipramine blocks the time-dependent effects of acute stress on working memory

S44

Behavioural and systems

received rifaximin, a non-absorbable antibiotic. At the end of the CVS, open field behavior and sugar preference tests were performed. Composition of the gut microbiome was analyzed in colon samples by real time quantitative PCR based on 16S rRNA. Plasma corticosterone level was measured by radioimmune assay and expressions of selected genes were analyzed from colon tissue by TaqMan PCR. In the open field behavior test, stressed mice display increased anxiety and locomotor activity. Chronically stressed mice had decreased sucrose preference, which indicates anhedonia. Plasma corticosterone level was elevated in the stressed groups. Amount of the Bacteroides, γ-Proteobacteria, Bifidobacteria and Clostridia were increased in colon microbiome of the stressed mice, however rifaximin treatment normalized the number of Clostridia only. Moreover, tight junction proteins and occludin mRNA expression were elevated in response to antibiotic treatment, suggesting decreased gut permeability. Finally, expression of different proinflammatory cytokines and chemokines remained unchanged in the colon of chronically stressed and/or rifaximin treated animals. Two way ANOVA was used for assessment (n = 5/group, p < 0.05). In conclusion, chronic stress changed the gut microbiome and behavior. Although rifaximin treatment reduced the level of Clostridia and induced occludin and tight junction protein expression in colon tissue, antibiotic treatment did not affect stress-induced anxiety behavior and anhedonia. Reference(s) [1] Forsythe, P., Sudo, N., Dinan, T., Taylor, V.H., Bienenstock, J., 2010. Mood and gut feelings. Brain, Behavior and Immunity 24, 9–16. [2] Mayer, E.A., Knight, R., Mazmanian, S.K., Cryan, J.F., Tillisch, K., 2014. Gut microbes and the brain: Paradigm shift in neuroscience. The Journal of Neuroscience 34(46), 15490–15496. P.2.023 Chronic treatment with desipramine blocks the time-dependent effects of acute stress on working memory N. Sala1*, L. Musazzi1, P. Tornese1, C. Bazzini1, M. Popoli1. 1University of Milan, Scienze farmacologiche e biomolecolari, Milano, Italy Background: Stress can induce rapid functional and morphological changes in excitatory synapses and transmission, thus representing a risk factor for the development of neuropsychiatric disorders. In recent studies, we demonstrated that acute inescapable footshock (FS)-stress

induces in prefrontal and frontal cortex (PFC/FC) a rapid and sustained increase (up to 24 h) of both depolarizationevoked glutamate release, and readily releasable pool of glutamate presynaptic vesicles [1]. Moreover, we found that the same protocol of acute stress exerts fast and longlasting architectural alterations in PFC/FC. FS-stress induced an early increase in the number of vesicles docked to the presynaptic membrane in perforated synapses and a dramatic sprout of the total number of non-perforated synapses (+42%) [2,3]. Moreover, the density of synaptic spines in PFC/FC was elevated 24 h after stress and returned to basal level 7 days later, while apical dendrites arborization was significantly reduced 24 h after stress, and up to 2 weeks later. Remarkably, the antidepressant desipramine resulted to prevent at least in part the effects induced by acute FS-stress [4]. Aims: Here we aimed at assessing the time dependent behavioural outcome of acute FS-stress, by evaluating alterations of working memory performance, an executive function mostly regulated by the PFC. Moreover we wanted to verify whether desipramine was able to modify the stressinduced effects on cognitive ability. Methods: Half of the animals were subjected to chronic treatment (14 days) with DMI (10 mg/kg) delivered in drinking water. Twenty-four hours after the last drug or vehicle administration, half of the animals from each group were subjected to a single session of acute FS-stress [2]. Working memory performance was evaluated using the delayed alternation T-maze test, performed 2 h and 24 h after FS-stress. The T-maze task consisted in 12 random trials with 0, 30 and 60 s delays between information and test runs [5]. Data were analyzed by means of two-way ANOVA followed by Bonferroni post-hoc test; statistical significance was assumed at p < 0.05. Results: 2 h after the stress protocol, ANOVA showed a significant effect of FS-stress in trials with 0 and 30 s delays ( p < 0.05 and p < 0.01, respectively), and in 30 s delay trials Bonferroni post-hoc test revealed a significant increase of cognitive performance in stressed animals treated with vehicle vs. controls ( p < 0.05). 24 h after the FS-stress, the effect of treatment was significant when no delay was applied ( p < 0.05), while stress exerted a statistically significant effect when the delay time was 30 and 60 s ( p < 0.05). Acute FS-stress decreased the cognitive performance only in stressed animals treated with vehicle (0 s delay, p < 0.05 vs. stressed animals treated with desipramine; 30 s delay, p < 0.05 vs. controls). Conclusions: Acute FS-stress exerted time-dependent and bi-phasic alterations on working memory, with an early improvement of cognitive performance, followed by a cognitive impairment the day after the protocol. Previous chronic treatment with desipramine was able to prevent

Behavioural and systems both the positive and the negative effects induced by acute stress. Taken together, our results showed that the functional and morphological long-term stress-induced alterations found in our previous studies are likely related to time-dependent behavioural alterations. Reference(s) [1] Musazzi, L., Tornese, P., Sala, N., Popoli, M., 2016. Acute stress is not acute: sustained enhancement of glutamate release after acute stress involves readily releasable pool size and synapsin I activation. Mol Psychiatry. 2016 Oct 4. [2] Treccani, G., Musazzi, L., Perego, C., Milanese, M., Nava, N., Bonifacino, T., Lamanna, J., Malgaroli, A., Drago, F., Racagni, G., Nyengaard, J.R., Wegener, G., Bonanno, G., Popoli, M., 2014. Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex. Mol Psychiatry, 19:433–443. [3] Nava, N., Treccani, G., Liebenberg, N., Chen, F., Popoli, M., Wegener, G., Nyengaard, J.R., 2014. Chronic desipramine prevents acute stress-induced reorganization of medial prefrontal cortex architecture by blocking glutamate vesicle accumulation and excitatory synapse increase. Int J Neuropsychopharmacol. 18(3). [4] Nava, N., Treccani, G., Alabsi, A., Kaastrup Mueller, H., Elfving, B., Popoli, M., Wegener, G., Nyengaard, J.R., 2015. Temporal dynamics of acute stressinduced dendritic remodeling in medial prefrontal cortex and the protective effect of desipramine. Cereb Cortex. 2015 Nov 1. pii:bhv254. [Epub ahead of print]. [5] Dudchenko, P.A., 2004. An overview of the tasks used to test working memory in rodents. Neurosci Biobehav Rev. 28(7):699–709. P.2.024 Cross-talk between GABAergic and dopaminergic system: role of GABAA alpha 6 subunit and D3 receptor in ethanol addiction in mice R. Di Marco1*, G.M. Leggio1, S.A. Torrisi1, G. Giurdanella1, A. Fidilio1, F. Caraci2, C. Bucolo1, S. Salomone1, F. Drago1. 1University of Catania, Department of Biomedical and Biotechnological Sciences- Section of Pharmacology - BIOMETEC, Catania, Italy; 2University of Catania, Department of Pharmaceutical Sciences, Catania, Italy Background: The mesolimbic dopamine (DA) pathway mediates the rewarding effects of drugs of abuse [1] including ethanol. However, the role of specific DA receptor subtypes in this control is unclear [2]. DA exerts

S45

its action through five G protein-coupled receptor subtypes (D1–5R); the dopamine D3 receptor (D3R) subtype has an important role in the modulation of the mesolimbic DA pathway and in the control of several DA-related disorders such as addiction and drug-seeking behavior [3]. We previously reported that the D3R gene deletion or the D3R pharmacological blockade inhibits ethanol preference and voluntary intake in mice [4]. We also reported that the genetic deletion or the pharmacological blockade of D3R affect GABAA subunit expression. In particular, D3R deficient mice (D3R −/−) exhibit a 15-fold higher basal alpha 6 GABAA subunit expression in the ventral striatum compared to their wild-type (WT) littermates [5]. Aim: Here, we tested the hypothesis that the GABAA alpha 6 subunit may be involved in ethanol preference and voluntary intake, and the modulation of this subunit could interfere in the control, exhibited by D3R, of ethanol consumption in mice. Materials and Methods: D3R −/− and their WT littermates (males, 8–12 weeks old), treated or not with RO 15–4513 (5 mg/kg, intraperitoneally), an agonist of GABAA receptor containing alpha 6 subunit, were tested in a binge-like ethanol-drinking paradigm, the drinking in the dark (DID). We also analyzed RO 15–4513 effects in WT littermates treated with SB-277011A (10 mg/kg, intraperitoneally), a D3R selective antagonist. The mRNA alpha 6 subunit expression was evaluated by Real Time PCR in the brain regions involved in drug addiction (ventral striatum, prefrontal cortex). Results: The treatment with RO 15-4513 inhibited ethanol intake in WT ( p < 0.05 vs. Vehicle). Conversely, it induced an increase of ethanol consumption in D3R −/− ( p < 0.01 vs. Vehicle). Moreover, the inhibition of the voluntary ethanol intake induced by the pharmacological blockade with SB-277011A (repeated treatment for 7 days) was reverted by the treatment with RO 15-4513 during the drinking in the dark experimental procedure. Furthermore, we reported an increase of GABAA alpha 6 subunit expression and a decrease of D3R expression in WT treated with RO 15-4513 ( p < 0.05 vs. Vehicle). Conclusions: The dopamine D3 receptor-dependent changes in alpha 6 GABAA subunit expression control the rewarding properties of alcohol in mice. Thus, the cross talk between DA and GABA signaling are essential for ethanol-related reward and consumption and it may represent a novel therapeutic target for ethanol addiction. Reference(s) [1] Wise, R.A., Bozarth, M.A., 1987. A psychomotor stimulant theory of addiction. Psychol Rev 94: 469– 492. [2] Bowers, M.S., Chen, B.T., Bonci, A., 2010. AMPA receptor synaptic plasticity induced by