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Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter-containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot-shock stress and (2) rats with sham stress as control group. Two-dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in-section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three-dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one-dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot-shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.

Aims: Several studies suggest a critical role of glutamatergicneurotransmission in the stress response. We compared the effects of acutebehavioural stress with that of in vitroapplication of corticosterone (CORT) to prefrontal/frontal cortex (PFC/FC) synaptosomes,to verify if stress effects on glutamate neurotransmission were mediated by asynaptic action of CORT. Methods: Rats were subjected to acute Footshock (FS)-stress. Synaptosomeswere purified from PFC/FC; glutamate release was measured by superfusion.Electrophysiological experiments were performed on acute PFC slices. Changes invesicle mobilization were measured by Total Internal Reflection FluorescenceMicroscopy (TIRFM). Synapsin I phosphorylation was measured by Western blot. Results: We found that both acute stress and CORT in vitro increase the size of thereadily releasable pool (RRP) of vesicles in PFC/FC synaptosomes. This effectwas abolished by selective mineralocorticoid or glucocorticoid receptor (MR/GR)antagonists. However, CORT did not affect glutamate release evoked bydepolarization and did not alter excitatory transmission in medial PFC slices.We then confirmed, by using TIRFM, that CORT increases vesicle mobilizationtoward the RRP acting on synaptic GR/MR. Finally, we found that both stress andCORT increase the phosphorylation of synapsin I at site 1 in presynapticmembrane. This increase of synapsin I phosphorylation was also found to bedependent on GR/MR and necessary for the increase of RRP. Conclusions: These results suggest that, despite the rapid increaseof RRP size whereby CORT primes synaptic terminals in PFC/FC, the synapticnon-genomic action of CORT is necessary but not sufficient to enhance glutamaterelease.

Glutamate-mediated excitotoxicity plays a major role in the degeneration of motor neurons in amyotrophic lateral sclerosis and reduced astrocytary glutamate transport, which in turn increases the synaptic availability of the amino acid neurotransmitter, was suggested as a cause. Alternatively, here we report our studies on the exocytotic release of glutamate as a possible source of excessive glutamate transmission. The basal glutamate efflux from spinal cord nerve terminals of mice-expressing human soluble superoxide dismutase (SOD1) with the G93A mutation [SOD1/G93A(+)], a transgenic model of amyotrophic lateral sclerosis, was elevated when compared with transgenic mice expressing the wild-type human SOD1 or to non-transgenic controls. Exposure to 15 mM KCl or 0.3 μM ionomycin provoked Ca(2+)-dependent glutamate release that was dramatically increased in late symptomatic and in pre-symptomatic SOD1/G93A(+) mice. Increased Ca(2+) levels were detected in SOD1/G93A(+) mouse spinal cord nerve terminals, accompanied by increased activation of Ca(2+)/calmodulin-dependent kinase II and increased phosphorylation of synapsin I. In line with these findings, release experiments suggested that the glutamate release augmentation involves the readily releasable pool of vesicles and a greater capability of these vesicles to fuse upon stimulation in SOD1/G93A(+) mice.

Purpose and Methods: Genetic studies found no evidence of classic Mendelian inheritance for human depression, while stress factors, such as early-life adverse events, have been shown to interact with a variable background of genetic vulnerability. Indeed, the experience of stressful events in childhood was found to increase the risk for the development of mood disorders in adult life [1]. Moreover, stress can profoundly affect cognitive functions and alter hippocampal synaptic plasticity. Antidepressants have been shown to induce various effects beyond neurotransmitters receptors, such as adaptive changes in gene expression and neuroplasticity. These drugs were also shown to affect long-term potentiation (LTP), a synaptic mechanism underlying learning and memory [2]. We employed an innovative experimental design, attempting at reproducing the combination of environmental adverse events and genetic susceptibility. We used the Flinders Sensitive Line (FSL) rats, a well-validated model of depression carrying genetic vulnerability associated to distinct features of pathology [3]. To reproduce early life stress events the FSL rats and their controls, the Flinders Resistant Line (FRL) rats, were subjected to a standard maternal separation protocol. Moreover, FSL and FRL rats, with or without early-life stress, were treated with escitalopram. LTP was induced in vivo in the hippocampus of FSL/FRL rats by means of high frequency stimulation (HFS) protocol. Furthermore, we purified synaptosomes from the hippocampus of FSL/FRL (not subjected to HFS) and analyzed protein-protein interactions pre- and postsynaptically, as well as changes in synaptic signaling, in order to identify molecular correlates of early-life stress and response to escitalopram treatment. Results and Conclusions: In vivo LTP was significantly lower in basal FSL rats. LTP was reduced in both FRL and FSL escitalopram-treated rats. Maternal separation did not induce significant LTP changes; however, there was a tendency toward increased LTP in FSL after maternal separation. LTP was not further changed by escitalopram in FRL maternally separated, while it was reduced, although not significantly, in FSL maternally separated treated with the antidepressant. Basal expression level of NMDA-NR1 subunit in synaptosomes of FSL was lower and early life stress upregulated NR1 levels. Interaction between CaMKII and NMDA-NR2A/B subunit was reduced in FSL rats compared with FRL, consistent with a lower synaptic NMDA receptor content. Furthermore, we found basal differences between FSL and FRL in phosphorylation levels of CaMKII and of Synapsin-1 (CaMKII site) and in the interaction between CaMKII and syntaxin-1. These results combined suggest a dysfunction of glutamate neurotransmission in FSL. Finally, basal phosphorylation levels of the stress-sensitive kinases ERK1/2 in synaptosomes of FSL were higher and early life stress failed to further increase kinases activation, contrary to the results obtained in FRL. Our experimental design superimposing early environmental adverse events on a genetic background of vulnerability allowed for functional and molecular studies in an animal model more thoroughly reproducing pathology. Our results may contribute to characterize the molecular effectors of plasticity that mediate vulnerability to stress and response to antidepressant action. References [1] Caspi A, Sugden K, Moffitt TE, et al., 2003, Influence of life stress on depression: moderation by a polymorphism in the 5-HTT gene. Science 301, 386– 389. [2] Shakesby AC, Anwyl R, Rowan MJ, 2002, Overcoming the effects of stress on synaptic plasticity in the intact hippocampus: rapid actions of serotonergic and antidepressant agents. J Neurosci 22, 3638–3644. [3] Overstreet DH, Friedman E, Math AA, et al., 2005, The Flinders Sensitive Line rat: a selectively bred putative animal model of depression. Neurosci Biobehav Rev 29, 739–759.

Introduction: Agomelatine is a potent melatonergic receptor (MT1/MT2) agonist and 5HT2C receptor antagonist with proven antidepressant efficacy in both animal models of depression and in depressed patients. In previous studies we had found that following a 2 week-chronic treatment with different antidepressants [1] depolarization-evoked release of glutamate in the hippocampus but not GABA was markedly reduced, suggesting that antidepressants may change the balance between excitatory and inhibitory neurotransmission. The functional changes in glutamatergic neurotransmission were accounted for by alterations of neurotransmitter release regulating protein-protein interactions: in particular, we found a reduction of syntaxin-1/aCalcium-CaM dependent Kinase II (aCaMKII) interaction, suggested to promote the formation of exocytotic SNARE complex, and an increase of syntaxin-1/Munc-18 interaction, that reduces formation of SNARE complex and neurotransmitter release [1,2]. Purpose: In order to determine whether also a chronic treatment with agomelatine would induce similar synaptic functional and molecular changes, we measured depolarization-evoked release of glutamate and selected presynaptic molecular mechanisms involved in the regulation of glutamatergic transmission in the hippocampus of chronically treated rats. Methods: Rats were intraperitoneally treated with agomelatine (40 mg/kg), the reference antidepressant venlafaxine (10 mg/kg) or vehicle at 5 pm (2 h before the start of the 12 h-dark-phase), for 21 days. 17 hrs after the final administrations, rat hippocampi were dissected and synaptosomes and synaptic membranes were prepared by centrifugation on Percoll gradients followed by differential centrifugation and ultracentrifugation. Glutamate release was measured in freshly purified synaptosomes by using superfusion technique [1]. aCaMKII and Munc-18 were independently immunoprecipitated with specific antibodies and coimmunoprecipitated syntaxin-1 was measured by SDS-PAGE and Western blot. SDS-resistant SNARE complex was measured by SDS-PAGE and Western blot. Results: Chronic treatment with agomelatine significantly (p

Background and aims: The simultaneous and wide-scale analysis of gene and protein expression provides a powerful strategy for the exploration at molecular level of complex pathophysiological mechanisms, such as the response to treatment with psychotropic agents. GENDEP is an Integrated Project that combines large-scale clinical pharmacogenomic studies on depressed patients with preclinical investigations on animal models, focusing on treatment with proserotonergic and pronoradrenergic antidepressants. Methods: Two different, complementary rat models of depression were used: the flinders sensitive line (FSL) and the learned helplessness. FSL rats were additionally subjected to a maternal separation protocol to investigate the impact of gene-environment interaction. Animals were chronically treated with escitalopram or nortriptyline, with behavioural studies and transcriptomic and proteomic profiling, in order to identify novel genes and gene products differentially expressed in the different paradigms. Transcriptome analyses were performed from prefrontal cortex and hippocampus using Affymetrix GeneChips, whilst for proteomics two-dimensional electrophoresis with mass spectrometry was used. Further proteomic studies, including functional investigations focusing on the detection of protein phosphorylation changes, were performed on both total extracts and purified synaptic terminals. Finally, modifications of synaptic plasticity (LTP) in hippocampus were investigated by electrophysiology with parallel measurements of molecular correlates. Results and conclusions: Preliminary results on proteome and transcriptome analysis of escitalopram treated rats allowed the identification of molecular and functional correlates of vulnerability to the disease, the effect of early-life stress and the response to pharmacological treatment.

The wide-scale analysis of protein expression provides a powerful strategy for the exploration at a molecular level of complex pathophysiological mechanisms, such as the response to treatment with psychotropic agents. GENDEP is an Integrated Project that combines large-scale clinical pharmacogenomic studies on depressed patients with preclinical investigations on animal models of disease, focusing on treatment with antidepressants. In this work, the Flinders Sensitive Line (FSL), a genetically selected rat model of depression displaying good face, predictive and construct validity (Overstreet 2005) was investigated by a two dimensional proteomic approach. As a control, analyses were carried out on the corresponding Flinders Resistant Line (FRL), which does not show the depressive-like behaviour. To evaluate gene–environment interactions, the FSL and FRL animals were subjected to maternal separation (MS), since early life trauma is considered to be an important antecedent of major depression. Following weaning, FSL and FRL rats were treated with escitalopram (ES), a selective serotonin reuptake inhibitor. FSL and FRL rats were subjected to 180 min. maternal separation from postnatal day 2 to 14. Control groups were not maternally separated. Both stressed and control animals were split into groups receiving ES admixed to food pellets (25 mg/kg/day) or vehicle for 30 days. One week before the end of treatments, the animals were subjected to the Porsolt swim test in order to assess the antidepressant effect of ES. After sacrifice, extracts were prepared from frontal cortex and hippocampus and proteins were separated by two-dimensional electrophoresis. Spots were detected by staining with a fluorescent dye. Image analysis was carried out on maps with PDQuest software to compare protein levels in different gels. Protein level comparisons were carried out with both univariate (Student’s t test) and multivariate (Partial Least Squares) statistical analysis methods. Spots displaying statistically significant differences were cut and proteins were identified using peptide fingerprinting mass spectrometry. Comparisons were carried out between groups in different electrophoretic runs to detect changes induced by genetic background, stress exposure and pharmacological treatment in each brain region. Modified proteins were identified. By the comparison FRL vs FSL, information was gathered about genetic differences between the lines, which are probably involved in the depressive-like behaviour. In the comparison (FSL+noMS+vehicle) vs (FSL+no MS+ES) vs (FSL+MS+ES) the different effect of pharmacological treatment was revealed on control and maternally separated FSL. By the comparison (FSL+noMS+vehicle) vs (FSL+MS+vehicle) vs (FSL+MS+ES), information was collected about the effect of the pharmacological treatment on the modifications induced by maternal separation. The same designs were also analysed on the FRL. Proteins involved in the different pharmacological response in different genetic backgrounds were revealed in the comparison (FRL+MS+ES) vs (FSL+MS+ ES). Results on proteome analysis of ES treated rats allowed the identification of molecular correlates of vulnerability to the disease, the effect of early life stress and the response to pharmacological treatment.