Ettore Tiraboschi

Chronic Antidepressants Reduce Depolarization-Evoked Glutamate Release and Protein Interactions Favoring Formation of SNARE Complex in Hippocampus

Glutamate neurotransmission was recently implicated in the action of stress and in antidepressant mechanisms. We report that chronic (not acute) treatment with three antidepressants with different primary mechanisms (fluoxetine, reboxetine, and desipramine) markedly reduced depolarization-evoked release of glutamate, stimulated by 15 or 25 mm KCl, but not release of GABA. Endogenous glutamate and GABA release was measured in superfused synaptosomes, freshly prepared from hippocampus of drug-treated rats. Interestingly, treatment with the three drugs only barely changed the release of glutamate (and of GABA) induced by ionomycin. In synaptic membranes of chronically treated rats we found a marked reduction in the protein-protein interaction between syntaxin 1 and Thr286-phosphorylated αCaM kinase II (α-calcium/calmodulin-dependent protein kinase II) (an interaction previously proposed to promote neurotransmitter release) and a marked increase in the interaction between syntaxin 1 and Munc-18 (an interaction proposed to reduce neurotransmitter release). Furthermore, we found a selective reduction in the expression level of the three proteins forming the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. These findings suggest that antidepressants work by stabilizing glutamate neurotransmission in the hippocampus and that they may represent a useful tool for the study of relationship between functional and molecular processes in nerve terminals.

Fear Erasure in Mice Requires Synergy Between Antidepressant Drugs and Extinction Training

Long-term loss of fearful memories can be achieved through a combination of antidepressant drugs and exposure therapy. Antidepressant drugs and psychotherapy combined are more effective in treating mood disorders than either treatment alone, but the neurobiological basis of this interaction is unknown. To investigate how antidepressants influence the response of mood-related systems to behavioral experience, we used a fear-conditioning and extinction paradigm in mice. Combining extinction training with chronic fluoxetine, but neither treatment alone, induced an enduring loss of conditioned fear memory in adult animals. Fluoxetine treatment increased synaptic plasticity, converted the fear memory circuitry to a more immature state, and acted through local brain-derived neurotrophic factor. Fluoxetine-induced plasticity may allow fear erasure by extinction-guided remodeling of the memory circuitry. Thus, the pharmacological effects of antidepressants need to be combined with psychological rehabilitation to reorganize networks rendered more plastic by the drug treatment.

Selective phosphorylation of nuclear CREB by fluoxetine is linked to activation of CaM kinase IV and MAP kinase cascades

Regulation of gene expression is purported as a major component in the long-term action of antidepressants. The transcription factor cAMP-response element-binding protein (CREB) is activated by chronic antidepressant treatments, although a number of studies reported different effects on CREB, depending on drug types used and brain areas investigated. Furthermore, little is known as to what signaling cascades are responsible for CREB activation, although cAMP-protein kinase A (PKA) cascade was suggested to be a central player. We investigated how different drugs (fluoxetine (FLX), desipramine (DMI), reboxetine (RBX)) affect CREB expression and phosphorylation of Ser133 in the hippocampus and prefrontal/frontal cortex (PFCX). Acute treatments did not induce changes in these mechanisms. Chronic FLX increased nuclear phospho-CREB (pCREB) far more markedly than pronoradrenergic drugs, particularly in PFCX. We investigated the function of the main signaling cascades that were shown to phosphorylate and regulate CREB. PKA did not seem to account for the selective increase of pCREB induced by FLX. All drug treatments markedly increased the enzymatic activity of nuclear Ca2+/calmodulin (CaM) kinase IV (CaMKIV), a major neuronal CREB kinase, in PFCX. Activation of this kinase was due to increased phosphorylation of the activatory residue Thr196, with no major changes in the expression levels of α- and β-CaM kinase kinase, enzymes that phosphorylate CaMKIV. Again in PFCX, FLX selectively increased the expression level of MAP kinases Erk1/2, without affecting their phosphorylation. Our results show that FLX exerts a more marked effect on CREB phosphorylation and suggest that CaMKIV and MAP kinase cascades are involved in this effect.

Regulation of Editing and Expression of Glutamate α-Amino-Propionic-Acid (AMPA)/Kainate Receptors by Antidepressant Drugs

BACKGROUND Several reports have shown that the glutamatergic system is involved in both the pathogenesis of affective and stress-related disorders and in the action of antidepressant drugs. In particular, antidepressant treatment was shown to modulate expression and function of ionotropic glutamate receptors, to inhibit glutamate release and to restore synaptic plasticity impaired by stress. METHODS We analyzed the mRNA expression and RNA editing of alpha-amino-propionic-acid (AMPA) and kainate (KA) receptor subunits, in the pre-frontal/frontal cortex (P/FC) and hippocampus (HI) of rats chronically treated with three different drugs: the selective serotonin (5-HT) reuptake inhibitor fluoxetine, the selective noradrenaline (NA) reuptake inhibitor reboxetine and the tricyclic antidepressant desipramine. RESULTS Our data showed that fluoxetine and desipramine exerted moderate but selective effects on glutamate receptor expression and editing, while reboxetine appeared to be the drug that affects glutamate receptors (GluR) most. The most consistent effect, observed with pronoradrenergic drugs (desipramine and reboxetine), was a decrease of GluR3 expression both in P/FC and HI. Interestingly, in HI, the same drugs also decreased the editing levels of either the flip (desipramine) or flop (reboxetine) form of GluR3. CONCLUSIONS Overall, these results point to specific and regionally discrete changes in the expression and editing level of glutamate receptors and, in particular, to a selective reduction of conductance for GluR3-containing receptors following treatment with antidepressant drugs. These data support the hypothesis that changes in glutamate neurotransmission are involved in the therapeutic effects induced by these drugs.

Serotonin triggers a transient epigenetic mechanism that reinstates adult visual cortex plasticity in rats

Cortical circuitries are highly sensitive to experience during early life but this phase of heightened plasticity decreases with development. We recently demonstrated that fluoxetine reinstates a juvenile‐like form of plasticity in the adult visual system. Here we explored cellular and molecular mechanisms that underlie the occurrence of these plastic phenomena. Adult rats were intracortically treated with serotonin (5‐HT) whereas long‐term fluoxetine‐treated rats were infused with the 5‐HT1A‐receptor antagonist WAY‐100635, brain‐derived neurotrophic factor (BDNF) scavenger trkB‐IgG or the mitogen‐activated protein kinase inhibitor U0126. Plasticity was assessed as variations of visual cortex responsiveness after unilateral eyelid suture and reverse occlusion by using an electrophysiological approach. Real‐time PCR and chromatin immunoprecipitation analysis were then used to explore alterations in gene expression and modifications of chromatin structure associated with the plastic outcome caused by fluoxetine in the visual system. Local infusion of 5‐HT into visual cortex restored susceptibility to monocular deprivation in adulthood whereas infusion of WAY‐100635, trkB‐IgG or U0126 prevented the process of plasticity reactivation in fluoxetine‐treated animals. Long‐term fluoxetine treatment promoted a transient increase of Bdnf expression in the visual cortex, which was paralleled by an increased histone acetylation status at Bdnf promoter regions and by decreased expression of Hdac5. Accordingly, enhancing histone acetylation levels by systemic treatment with Trichostatin‐A reactivated plasticity in the adult while WAY‐100635‐infusion prevented epigenetic modifications in Bdnf promoter areas. The data suggest a key role for 5‐HT1A receptor and BDNF‐trkB signalling in driving a transitory epigenetic remodelling of chromatin structure that underlies the reactivation of plasticity in the visual system.

Chronic Antidepressants Induce Redistribution and Differential Activation of αCaM Kinase II between Presynaptic Compartments

Changes in synaptic plasticity are involved in pathophysiology of depression and in the mechanism of antidepressants. Ca2+/calmodulin (CaM) kinase II, a protein kinase involved in synaptic plasticity, has been previously shown to be a target of antidepressants. We previously found that antidepressants activate the kinase in hippocampal neuronal cell bodies by increasing phosphorylation at Thr286, reduce the kinase phosphorylation in synaptic membranes, and in turn its phosphorylation-dependent interaction with syntaxin-1 and the release of glutamate from hippocampal synaptosomes. Here, we investigated the chronic effect of different antidepressants (fluoxetine, desipramine, and reboxetine) on the expression and function of the kinase in distinct subcellular compartments in order to dissect the different kinase pools affected. Acute treatments did not induce any change in the kinase. In total tissue extracts chronic drug treatments induced activation of the kinase; in hippocampus (HC), but not in prefrontal/frontal cortex, this was partially accounted for by increased Thr286 phosphorylation, suggesting the involvement of different mechanisms of activation. In synaptosomes, all drugs reduced the kinase phosphorylation, particularly in HC where, upon fractionation of the synaptosomal particulate into synaptic vesicles and membranes, we found that the drugs induced a redistribution and differential activation of the kinase between membranes and vesicles. Furthermore, a large decrease in the level and phosphorylation of synapsin I located at synaptic membranes was consistent with the observed decrease of CaM kinase II. Overall, antidepressants induce a complex pattern of modifications in distinct subcellular compartments; at presynaptic level, these changes are in line with a dampening of glutamate release.

IGF-1 Restores Visual Cortex Plasticity in Adult Life by Reducing Local GABA Levels

The central nervous system architecture is markedly modified by sensory experience during early life, but a decline of plasticity occurs with age. Recent studies have challenged this dogma providing evidence that both pharmacological treatments and paradigms based on the manipulation of environmental stimulation levels can be successfully employed as strategies for enhancing plasticity in the adult nervous system. Insulin-like growth factor 1 (IGF-1) is a peptide implicated in prenatal and postnatal phases of brain development such as neurogenesis, neuronal differentiation, synaptogenesis, and experience-dependent plasticity. Here, using the visual system as a paradigmatic model, we report that IGF-1 reactivates neural plasticity in the adult brain. Exogenous administration of IGF-1 in the adult visual cortex, indeed, restores the susceptibility of cortical neurons to monocular deprivation and promotes the recovery of normal visual functions in adult amblyopic animals. These effects were accompanied by a marked reduction of intracortical GABA levels. Moreover, we show that a transitory increase of IGF-1 expression is associated to the plasticity reinstatement induced by environmental enrichment (EE) and that blocking IGF-1 action by means of the IGF-1 receptor antagonist JB1 prevents EE effects on plasticity processes.

Experience‐dependent expression of NPAS4 regulates plasticity in adult visual cortex

•  Transcription factors at the basis of plasticity in the adult visual system are unknown. •  Enhanced levels of NPAS4 transcription factor parallel visual cortical plasticity in adult life. •  Overexpression of NPAS4 restores plasticity in the adult visual cortex. •  NPAS4 down‐regulation prevents the plastic outcome caused by fluoxetine (FLX) in adulthood. •  NPAS4 regulates the expression of plasticity genes in the adult visual cortex.

Early induction of CREB activation and CREB-regulating signalling by antidepressants

Converging evidence points to adaptive changes in neuroplasticity and gene expression as mediators of therapeutic action of antidepressants. Activation of cAMP response-element binding protein (CREB) and CREB-regulating signalling are considered main effectors in these mechanisms. We analysed the temporal profile of intracellular changes induced by antidepressants, by measuring activation of major CREB-regulating signalling cascades and activation (Ser133 phosphorylation) of CREB. The main aims of the study were to investigate how these different variables are modulated with time, whether stronger activation of signalling cascades corresponds to stronger activation of CREB, and whether these changes are different in distinct brain areas. Rat groups were treated for 1, 2 or 3 wk with the antidepressants fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Activation of CREB and major effectors in signalling cascades were analysed by Western blot analysis with phospho-antibodies, in nuclear and cytosolic fractions from hippocampus and prefrontal/frontal cortex (P/FC). Surprisingly, CREB activation was already maximal after 1-wk treatment. In hippocampus early and stronger CREB activation was consistent with early and stronger activation of signalling. For both drugs, the profile of activation in P/FC was different from that observed in hippocampus. The results also showed that, contrary to the activatory role of MAP-ERKs and CaM kinase IV, nuclear alphaCaM kinase II was inactivated in parallel with activation of CREB.

Selective regulation of presynaptic calcium/calmodulin-dependent protein kinase II by psychotropic drugs

BACKGROUND Changes in neuroplasticity have been involved in the pathogenesis of psychiatric disorders as well as in psychotropic drug action. Calcium/calmodulin-dependent protein kinase II (CaM kinase II), an enzyme with a pivotal role in synaptic plasticity and cognitive functions, has been implicated in the action of anticonvulsants, benzodiazepines, and antidepressants, but little is known as to its role in the action of different drugs employed for treatment of psychiatric disorders. METHODS We studied the function and expression of CaM kinase II following chronic treatment of rats with two antidepressants, fluvoxamine and desipramine, a typical antipsychotic drug, haloperidol, and the typical medication for manic-depressive disorder, lithium. RESULTS Antidepressants significantly increased the kinase activity in presynaptic vesicles of frontal/prefrontal cortex. Haloperidol induced no change, whereas lithium significantly decreased the activity. Kinase activation by antidepressants was further demonstrated by increased phosphorylation of exogenously added recombinant synaptotagmin. Immunoreactivity of vesicular kinase (alpha-isoform) was significantly increased by reuptake blockers but not by the two other drugs. Kinetic analysis showed that limiting value of enzymatic velocity (Vmax) of the kinase for substrate was also increased by reuptake blockers and decreased by lithium; however, neither messenger ribonucleic acid nor protein expression level of the kinase was increased in frontal/prefrontal cortex homogenates of antidepressant-treated rats, suggesting the involvement of local synaptic mechanisms. CONCLUSIONS These findings show that functional regulation of presynaptic CaM kinase II is selectively affected by different psychotropic drugs, and suggest local synaptic mechanisms for pharmacological regulation of the kinase.