3D analysis of synaptic vesicle density and distribution after acute foot-shock stress by using serial section transmission electron microscopy.

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.

Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.

Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect.

Pharmacological characterization of BDNF promoters I, II and IV reveals that serotonin and norepinephrine input is sufficient for transcription activation.

Compelling evidence has shown that the effects of antidepressants, increasing extracellular serotonin and noradrenaline as a primary mechanism of action, involve neuroplastic and neurotrophic mechanisms. Brain-derived neurotrophic factor (BDNF) has been shown to play a key role in neuroplasticity and synaptic function, as well as in the pathophysiology of neuropsychiatric disorders and the mechanism of action of antidepressants. The expression of BDNF is mediated by the transcription of different mRNAs derived by the splicing of one of the eight 5' non-coding exons with the 3' coding exon (in rats). The transcription of each non-coding exon is driven by unique and different promoters. We generated a gene reporter system based on hippocampal and cortical neuronal cultures, in which the transcription of luciferase is regulated by BDNF promoters I, II, IV or by cAMP response element (CRE), to investigate the activation of selected promoters induced by monoaminergic antidepressants and by serotonin or noradrenaline agonists. We found that incubation with fluoxetine or reboxetine failed to induce any activation of BDNF promoters or CRE. On the other hand, the incubation of cultures with selective agonists of serotonin or noradrenaline receptors induced a specific and distinct profile of activation of BDNF promoters I, II, IV and CRE, suggesting that the monoaminergic input, absent in dissociated cultures, is essential for the modulation of BDNF expression. In summary, we applied a rapidly detectable and highly sensitive reporter gene assay to characterize the selective activation profile of BDNF and CRE promoters, through specific and different pharmacological stimuli.

Emotional memory impairments in a genetic rat model of depression: involvement of 5-HT/MEK/Arc signaling in restoration.

Cognitive dysfunctions are common in major depressive disorder, but have been difficult to recapitulate in animal models. This study shows that Flinders sensitive line (FSL) rats, a genetic rat model of depression, display a pronounced impairment of emotional memory function in the passive avoidance (PA) task, accompanied by reduced transcription of Arc in prefrontal cortex and hippocampus. At the cellular level, FSL rats have selective reductions in levels of NMDA receptor subunits, serotonin 5-HT(1A) receptors and MEK activity. Treatment with chronic escitalopram, but not with an antidepressant regimen of nortriptyline, restored memory performance and increased Arc transcription in FSL rats. Multiple pharmacological manipulations demonstrated that procognitive effects could also be achieved by either disinhibition of 5-HT(1A)R/MEK/Arc or stimulation of 5-HT4R/MEK/Arc signaling cascades. Taken together, studies of FSL rats in the PA task revealed reversible deficits in emotional memory processing, providing a potential model with predictive and construct validity for assessments of procognitive actions of antidepressant drug therapies.

Association between the ionotropic glutamate receptor kainate 3 (GRIK3) ser310ala polymorphism and schizophrenia.

Schizophrenia is a severe psychiatric illness characterised by disturbance of thought, hallucination and delusions.(1) Several studies have suggested that dysfunctions in the glutamatergic transmission are linked to the pathogenesis of schizophrenia, and in particular an excessive activation of glutamate receptors seems to be related to the disruption of neuronal ionic gradients leading to excitotoxicity.(2-7) Numerous findings suggested that the kainate ionotropic glutamate receptors are primarily involved in this mechanism. Recently it has been demonstrated that the GRIK3 gene encoding for the ionotropic glutamate receptor kainate 3 contains a functional polymorphism (T928G) leading to the substitution of a serine with an alanine in position 310 of the protein sequence.(8-11) We performed an association study between the ser310ala GRIK3polymorphism and schizophrenia in a sample of 99 schizophrenic patients and 116 controls. We found a significant difference in the genotype distribution and in particular considering the ala allele as dominant (P = 0.0105, odds ratio (OR) 2.031, 95% confidence interval (CI) 1.177-3.504). This finding suggests a potential role for GRIK3 for susceptibility to schizophrenia.

Long-term treatment with S-adenosylmethionine induces changes in presynaptic CaM kinase II and synapsin I.

BACKGROUND: According to current hypotheses, antidepressant drug action is the result of adaptive changes in neuronal signaling mechanisms rather than a primary effect on neurotransmitter transporters, receptors, or metabolic enzymes. Among the signaling mechanisms involved, protein kinases and phosphorylation have been shown to be modified by drug treatment. Presynaptic signaling (calcium/calmodulin-dependent protein kinase II [CaMKII]) and the protein machinery regulating transmitter release have been implicated in the action of these drugs. METHODS: We investigated the effect of S-adenosylmethionine (SAM), a compound with putative antidepressant activity, on presynaptic CaMKII and its synaptic vesicle substrate synapsin I. The activity of CaMKII was assayed in synaptic subcellular fractions prepared from hippocampus (HI), frontal cortex (FCX), striatum (STR), and parieto-temporal cortex. RESULTS: The kinase activity was increased after SAM treatment in the synaptic vesicle fraction of HI (31.7%), FCX (35.9%), and STR (18.4%). The protein level of CaMKII was also increased in synaptic vesicles of HI (40.4%). The synapsin I level was unchanged in synaptic vesicles but markedly increased in synaptic cytosol of HI (75.8%) and FCX (163.0%). No changes for both CaMKII and synapsin I level were found in homogenates, suggesting that synaptic protein changes are not explained by an increase in total level of proteins, but rather by translocation to nerve terminals. CONCLUSIONS: Similar to typical antidepressant drugs, SAM induces changes in CaMKII activity and increases synapsin I level in HI and FCX nerve terminals, suggesting a modulatory action on transmitter release.

Serine/threonine kinases as molecular targets of antidepressants: implications for pharmacological treatment and pathophysiology of affective disorders.

It is currently a widely accepted opinion that adaptive, plastic changes in the molecular and cellular components of neuronal signaling systems correlate with the effects on mood and cognition observed after long-term treatment with antidepressant drugs. Protein phosphorylation represents a key step for most signaling systems, and it is involved in the regulation of virtually all cellular functions. Two serine/threonine kinases, Ca2+ /calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, have been shown to be activated in the brain following antidepressant treatment. The changes in kinase activity are mirrored by changes in the phosphorylation of selected protein substrates in subcellular compartments (presynaptic terminals and microtubules), which, in turn, may contribute to the modulation of synaptic transmission observed with antidepressants. The molecular consequences of protein kinase activation may account for some of the alterations in neural function induced by antidepressants, and may suggest novel possible strategies of pharmacological intervention.

Effect of reboxetine treatment on brain cAMP- and calcium/calmodulin-dependent protein kinases.

Previous studies showed that the type II Ca(2+)/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA) are affected by long-term antidepressant treatment in presynaptic and somatodendritic compartments, respectively. This study describes the long-term effects of the selective noradrenaline reuptake inhibitor reboxetine on PKA and CaMKII, in both the microtubule and subsynaptosomal fractions of rat brain. Unlike other antidepressants, chronic reboxetine induced in the cerebrocortical soluble and microtubule fractions a decrease in the [(32)P]cAMP binding to the type II PKA regulatory subunit. No change in the cAMP-dependent endogenous phosphorylation of the protein substrate, microtubule-associated protein 2 was observed. In the hippocampal subsynaptosomal fractions (synaptic vesicles and synaptosomal membranes) reboxetine induced a robust increase in the activity but not in the expression of CaMKII. An increase in the calcium/calmodulin-dependent phosphorylation of presynaptic substrates was also detected. These findings showed that reboxetine modulates post-receptor signal transduction systems in rat brain.

Modifications in brain CaM kinase II after long-term treatment with desmethylimipramine.

The present study investigated the effect of long-term (15 mg/kg for 15 days) and acute (15 mg/kg, single administration) treatment with desmethylimipramine, a tricyclic antidepressant drug, on calcium/calmodulin-dependent protein kinase II (CaMKII), a kinase implicated in the mechanism of antidepressant drug action. Similar to selective and non-selective serotonin reuptake inhibitors, long-term, but not acute, treatment with desmethylimipramine markedly increased the activity of CaMKII in the hippocampal synaptic vesicle fraction (+51.9%). The kinase activity was also increased in the same fraction of frontal cortex (+24.2%) and in the striatum (+45.9%), although in this last area the mechanism appeared to be different because the protein level of the kinase was also markedly increased (+43.7%). However, the effect of treatment was not restricted to the presynaptic kinase, because CaMKII activity was also increased in the total cellular cytosol in cortical areas. The autonomous (calcium-independent) activity of CaMKII was assayed for the first time after antidepressant treatment, and found to be increased in synaptic vesicles of all three areas. These results confirmed the involvement of CaMKII in antidepressant drug action and suggested that modulation of transmitter release is a primary component in the action of psychotropic drugs.

Modification of presynaptic CaM kinase II affinity for ATP in hippocampus after long term blockade of serotonin reuptake.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is markedly enriched at synapses, where it is involved in the control of synaptic transmission, transmitter release and synaptic plasticity. CaMKII has also been found to be involved in the long-term action of antidepressants on post-receptor signaling mechanisms, because monoamine reuptake inhibitors induced an increase in autophosphorylation and activity of the kinase in nerve terminals of hippocampus. To study whether changes in the amount of enzyme or kinetic changes, due to posttranslational modifications, are responsible for kinase activation in nerve terminals, alpha-CaMKII level and kinetic constants of the autophosphorylation reaction as a function of ATP concentration were measured in presynaptic cytosol from hippocampus. Treatment with two serotonin reuptake inhibitors did not change the level of presynaptic kinase or the Vmax of autophosphorylation reaction. Instead the Km of the kinase for ATP was decreased 2.8-fold with fluvoxamine and 3.5-fold with paroxetine, implying an increase in the affinity for ATP. This result represents the first finding of changes in kinetic constants of a major brain enzyme after treatment with antidepressant drugs.

Second messenger-regulated protein kinases in the brain: their functional role and the action of antidepressant drugs.

Depression has been treated pharmacologically for over three decades, but the views regarding the mechanism of action of antidepressant drugs have registered recently a major change. It was increasingly appreciated that adaptive changes in postreceptor signaling pathways, rather than primary action of drugs on monoamine transporters, metabolic enzymes, and receptors, are connected to therapeutic effect. For some of the various signaling pathways affected by antidepressant treatment, it was shown that protein phosphorylation, which represents an obligate step for most pathways, is markedly affected by long-term treatment. Changes were reported to be induced in the function of protein kinase C, cyclic AMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase. For two of these kinases (cyclic AMP- and calcium/calmodulin-dependent), the changes have been studied in isolated neuronal compartments (microtubules and presynaptic terminals). Antidepressant treatment activates the two kinases and increases the endogenous phosphorylation of selected substrates (microtubule-associated protein 2 and synaptotagmin). These modifications may be partly responsible for the changes induced by antidepressants in neurotransmission. The changes in protein phosphorylation induced by long-term antidepressant treatment may contribute to explain the therapeutic action of antidepressants and suggest new strategies of pharmacological intervention.

Changes of synaptotagmin interaction with t-SNARE proteins in vitro after calcium/calmodulin-dependent phosphorylation.

The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein-protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP-25, and VAMP-synaptobrevin. Other proteins interacting with the core of the SNARE complex, such as voltage-activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation-dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin and SNAP-25 in native complexes was not found to be calcium-dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin-syntaxin interaction. Stimulation of Ca2+/calmodulin-dependent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP-25 with synaptotagmin (particularly when measured in the presence of calcium), as well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin-t-SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation-dephosphorylation balance in regulation of the synaptotagmin-t-SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.

Modifications in brain cAMP- and calcium/calmodulin-dependent protein kinases induced by treatment with S-adenosylmethionine.

Several lines of evidence suggest that the mechanism of action of antidepressant drugs (AD) involves adaptive changes occurring in intraneuronal post-receptor signal transduction cascades. Protein phosphorylation has a key role in signal transduction and was previously found to be a target in the action of AD (5-HT and/or NA reuptake blockers). Several studies showed that cAMP- and type II Ca2+/calmodulin-dependent protein kinases (PKA and CaMKII) are markedly affected by typical AD in two different and complementary cellular districts, respectively microtubules (a somatodendritic compartment) and synaptic vesicles (a presynaptic terminal compartment). In order to investigate whether the effect on protein kinases may be involved in the therapeutic action of drugs it is interesting to compare the effect of atypical AD with that of typical drugs. In this study the effect of the atypical AD S-adenosylmethionine (SAMe) was tested. Repeated (12 days) SAMe treatment induced in cerebrocortical microtubules an increase in the binding of cAMP to the RII PKA regulatory subunit and an increase in the endogenous phosphorylation of microtubule-associated protein 2, an effect resembling that of typical AD. In synaptic terminals the treatment induced an increase in the activity of CaMKII and in the endogenous phosphorylation of vesicular substrates. However, this modification was found in the cerebral cortex rather than in the hippocampus, where typical AD affect CaMKII. In addition the synapsin I level was decreased in the hippocampus and increased in the cerebral cortex, an effect not detected with typical AD.

cAMP-dependent phosphorylation system after short and long-term administration of moclobemide.

Accumulating evidence suggested that signal transduction cascade including protein phosphorylation is implicated in the neurochemical action of antidepressant agents. Clinical data indicated that moclobemide, a short acting and reversible inhibitor of monoamino oxidase type. A, is an effective antidepressant medication. However, little is known about the intracellular effects of this compound. Thus, in the present study we assessed the binding of cAMP to cAMP-dependent protein kinase (PKA) in rat cerebral cortex following short and long-term administration of moclobemide. The results showed that 21 days of treatment with moclobemide significantly increased the specific [32P]-cAMP covalent binding into the soluble 52-54 kDa cAMP-receptor. This effect was not seen following 1, 5 and 12 days of treatment. These findings suggest that PKA could be implicated in the biochemical effects of moclobemide.

Long-term blockade of serotonin reuptake affects synaptotagmin phosphorylation in the hippocampus.

Synaptic vesicle trafficking and transmitter release from presynaptic terminals are precisely regulated by a complex array of protein/protein interactions. Several of these proteins are substrates of endogenous protein kinases present in presynaptic terminals. The activity of Ca2+/calmodulin-dependent protein kinase II(CaMKII), one of the kinases involved in the modulation of transmitter release, was previously shown to increase in the hippocampus after long-term blockade of 5-hydroxytryptamine (5-HT) reuptake (a treatment known to elicit an increase in 5-HT release in this area). To investigate the changes induced in presynaptic protein phosphorylation by 5-HT reuptake blockade and concomitant CaMKII up-regulation, we analyzed two major CaMKII presynaptic substrates (synapsin I and synaptotagmin). All 5-HT reuptake blockers that we used, which induce an increase in CaMKII activity and autophosphorylation, also caused a large (2-3-fold) increase in the Ca2+/calmodulin-dependent post hoc phosphorylation of synaptotagmin. Conversely, the phosphorylation of synapsin I is much less affected. The change in synaptotagmin phosphorylation, as determined through immunoprecipitation and quantitative immunoblot analysis after fluvoxamine treatment, is due exclusively to increased phosphate incorporation (presumably caused by the increased kinase activity) and not to a change in the level of substrate protein after the treatment. Thus, drugs known to induce an increase in 5-HT release simultaneously induce an increase in the activity of presynaptic CaMKII and in the phosphate incorporation (post hoc) by a major CaMKII substrate in synaptic vesicles (synaptotagmin). This finding establishes a link between the facilitation of transmitter release induced by antidepressant drugs and the phosphorylation of synaptotagmin by CaMKII.

Ca2+/phospholipid-binding and syntaxin-binding of native synaptotagmin I.

Synaptotagmin, a synaptic vesicle protein endowed with multiple properties, is the putative calcium sensor in neuroexocytosis. Ca2+/phospholipid binding and syntaxin binding activity of synaptotagmin were previously investigated using recombinant fusion proteins. In phospholipid binding the EC50 for calcium obtained was different when fusion proteins containing one (C2A) or both (C2A+C2B) binding domains were used. It was alternatively proposed that one or both synaptotagmin binding domains are important for calcium-sensing and triggering of transmitter release. In this study the binding activity of native full-length synaptotagmin, immobilized on beads, was investigated. We found the kinetic parameters of Ca2+/phospholipid binding to be compatible with the role of calcium sensor for synaptotagmin (EC50 for calcium = 72 +/- 7 microM), with the two C2 domains supporting separate and complementary calcium sensing properties. The binding of native syntaxin to synaptotagmin was measurable in the absence of calcium, but was markedly stimulated (2.2-fold) in the presence of mM calcium. It may be speculated that the two domains have a synergistic action in fast synchronous transmitter release, whereas C2B domain alone may support slow asynchronous release, working as a high affinity calcium sensor.

Inhibitory effect of lithium on cAMP dependent phosphorylation system.

The aim of the present study was to assess the direct effect of lithium on cAMP dependent phosphorylation. The results show that lithium, but not rubidium, at therapeutic and high concentrations significantly decreases the cAMP stimulated MAP2 endogenous phosphorylation in microtubule fraction. An inhibitory effect of lithium has also been found using purified heat stable microtubule proteins phosphorylated by the catalytic subunit of PKA. These data suggest a direct effect of lithium on the cAMP dependent protein kinase.

Presynaptic Ca2+/calmodulin-dependent protein kinase II: autophosphorylation and activity increase in the hippocampus after long-term blockade of serotonin reuptake.

It is known that long-term treatment with antidepressants induces an enhancement of neurotransmission in the pathway projecting from raphe nuclei to the hippocampus. In the case of selective serotonin (5-HT) reuptake inhibitors, this enhancement is due to a desensitization of presynaptic 5-HT autoreceptors and a concomitant increase in 5-HT release in terminal areas. To investigate whether this effect is accompanied by adaptive changes in the molecular machinery regulating transmitter release at serotonergic terminals, autophosphorylation and activity of Ca2+/calmodulin-dependent protein kinase II were measured in subsynaptosomal fractions from hippocampus and total cortex. Long-term treatment with two selective serotonin reuptake inhibitors (paroxetine and fluvoxamine) and with a nonselective reuptake inhibitor (venlafaxine) induces a large increase of kinase autophosphorylation in synaptic vesicles and synaptic cytosol in the hippocampus but not in synaptosomal membranes. No significant change was detected in total cortex. The change is not reproduced by the direct addition of the drugs to the phosphorylation system and is not elicited by acute treatment of the animals. The increase in autophosphorylation is not accounted for by neosynthesis or translocation of the kinase to synaptic terminals. The change is restricted to the kinase located inside the terminals and is not detected in synaptosomal membranes, containing predominantly postsynaptic kinase, suggesting that only presynaptic kinase is affected. In the same fractions, the kinase activity is increased. These results are in agreement with reports suggesting a presynaptic effect for the SSRIs and disclose a new putative site of action for psychotropic drugs.