MODULAZIONE DI MECCANISMI DI PLASTICITA' NEURONALE DOPO SOMMINISTRAZIONE ACUTA O CRONICA DI COCAINA

The major aim of this thesis was to assess the role of the neurotrophin BDNF and the imemdiate early gene Arc in the mechanism of action of cocaine. To pursue this purpose we exposed the animals to various drug paradigms. We first injected the animals with different doses of cocaine and sacrificed the animals at different time points. These experiments revealed that acute injection of cocaine produced a finely tuned, time-dependent and regional-selective expression profile of both BDNF and Arc suggesting that they indie partecipate in the rapid action of the psychostimulant. The analysis of repente exposure of the animals to cocaine revealed that precursor (pro-) and mBDNF protein forms were markedly reduced 2 h and 72 h post-injection in the prefrontal cortex. Interestingly, in the striatum we found that repeated cocaine injection increased pro-BDNF levels without altering the mature form of the neurotrophin, thereby suggesting that cocaine differently affects BDNF transcription and translation in a region-selective manner, but might also alter neurotrophin processing. These data further support the notion that the corticostriatal network is highly vulnerable to the effects of cocaine, and suggest that abnormal regulation of BDNF expression could contribute, at least in part, to the functional defects observed in drug abusers. We then decided to evaluate whether repente stress, a major risk factor for cociane relapse, might alter the pattern of BDNF and Arc expression following acute administration of cocaine. we provide evidence that repeated stress prevents cocaine-induced activation of BDNF expression and signaling in rat prefrontal cortex. A single injection of cocaine up-regulates BDNF expression in sham (i.e. unstressed) rats but not in repeatedly stressed rats. Similarly, the expression as well as trafficking of the high affinity BDNF receptor trkB promoted by the psychostimulant is impaired in chronically-stressed rats challenged with cocaine. Moreover, among the different intracellular signaling pathways that can be activated by the neurotrophin, i.e. ERK1/2-, Akt- and PLC\u3b3-pathway, we found that cocaine is able to selectively activate the ERK1/2 pathway in sham animals, but not in rats exposed to repeated stress. These data demonstrate that stress globally interferes with BDNF-mediated signaling responses to cocaine challenge, providing key insights into the molecular basis of stress-cocaine interaction and indicating the critical role of the prefrontal cortex in mediating such interaction. To follow up our studies we decided to employ a new administration paradimg of cocaine in order to separate the direct pharmacological effects of cocaine from those associated with active drug selfadministration. To this end, we employed a yoked control-operant paradigm and sacrificed the animals after a single intravenous (i.v.) cocaine self-administration session. Animals self-administering cocaine (SA, 0.25 mg/0.1 ml saline per infusion, 2-h session) did more active lever-presses than yoked-cocaine (YC) and yoked-vehicle animals (YV). This goal-oriented behaviour was accompanied by a selective increase in Arc mRNA levels in the medial prefrontal cortex. There were no changes in the expression of the other genes in this brain region. mRNA levels of Arc and Zif268 in striatum and Zif268 in the nucleus accumbens markedly increased both in SA and YC animals; but there was no change in the expression of FGF-2 and BDNF. No changes were observed in hippocampus, hypothalamus, frontal cortex, and midbrain in SA and YC animals compared to YV animals in any of the genes. These findings demonstrate that a single session of cocaine i.v. self-administration is sufficient to shape rat behaviour towards goal-directed behaviours and selectively up-regulate Arc expression in mPFC (of SA animals), providing the first evidence that the function of prefrontal cortex is already profoundly influenced by the first voluntary cocaine exposure

A single cocaine administration alters dendritic spine morphology and impairs glutamate receptor synaptic retention in the medial prefrontal cortex of adolescent rats

The brain is still maturing during adolescence and interfering with such a vulnerable period may lead to structural and functional consequences. We investigated the effect of a single cocaine exposure on dendritic spine structure and glutamate dynamics in the medial prefrontal cortex (mPFC) of adolescent rats 7 days after a single cocaine administration. We found a reduced number of dendritic spines, suggesting that cocaine lowers the density of dendritic spines in the mPFC of adolescent rats. Since dendritic spines are postsynaptic glutamatergic protrusions, we investigated the main determinants of glutamate postsynaptic responsiveness. In the postsynaptic density, cocaine reduced the expression of the NMDA receptor subunits GluN1, GluN2A and GluN2B as well as of the AMPA GluA1 and GluA2 subunits. Cocaine also impaired their synaptic stability since the expression of the scaffolding proteins SAP102 and SAP97, critical for the anchoring of such receptors at the postsynaptic membrane, was reduced as well. The expression of PSD-95 and Arc/Arg3.1, which play structural and functional roles in glutamate neurons, was also similarly reduced. Such changes were not found in the whole homogenate, ruling out a translational effect of cocaine and implying, rather, an impaired synaptic retention at the active zones of the synapse. Notably, neither these critical glutamate determinants nor the density and morphology of the dendritic spines were altered in the mPFC of adult animals, suggesting that a single cocaine exposure selectively impairs the developmental trajectory of the glutamate synapse. These results indicate a dynamic impairment of mPFC glutamate homeostasis during a critical developmental window that persists for at least one week after a single cocaine administration. Our results identify dysfunctional glutamate synapse as a major contributor to the mechanisms that distinguish adolescent vs. adult outcomes of a single cocaine exposure

Repeated cocaine exposure during adolescence impairs recognition memory in early adulthood: A role for BDNF signaling in the perirhinal cortex

The perirhinal cortex (PrhC) is critical for object recognition memory; however, information regarding the molecular mechanisms underlying this type of memory following repeated exposure to drugs of abuse during adolescence is unknown. To this end, adolescent or adult rats were exposed to cocaine from postnatal day (PND) 28 to PND 42 or PND 63 to PND 77, respectively. Two weeks later, rats were subjected to the cognitive test named Novel Object Recognition (NOR) test. We found that adolescent, but not adult, cocaine exposure caused a significant impairment in the NOR test, independently from changes in the stress response system. In adolescent saline-treated rats, NOR test up-regulated BDNF and its downstream signaling whereas a downregulation of the same pathway was observed in cocaine-treated rats together with a reduction of Arc/Arg3.1 and PSD95 expression, indicating reduced pro-cognitive structural adaptations in the PrhC. Of note, cocaine-treated adult rats correctly performed in the NOR test indicating intact recognition memory mechanisms, despite a significant cocaine-induced reduction of BDNF levels in the PrhC, suggesting that recognition memory is heavily dependent on BDNF during adolescence whereas during adulthood other mechanisms come into pla

Short-term withdrawal from repeated exposure to cocaine during adolescence modulates dynorphin mRNA levels and BDNF signaling in the rat nucleus accumbens

BACKGROUND: Early-life stressful events affect the neurobiological maturation of cerebral circuitries including the endogenous opioid system and the effects elicited by adolescent cocaine exposure on this system have been poorly investigated. Here, we evaluated whether cocaine exposure during adolescence causes short- or long-term alterations in mRNAs codifying for selected elements belonging to the opioid system. Moreover, since brain-derived neurotrophic factor (BDNF) may undergo simultaneous alterations with the opioid peptide dynorphin, we also evaluated its signaling pathway as well. METHODS: Adolescent male rats were exposed to cocaine (20\u2009mg/kg/day) from post-natal day (PND) 28 to PND42, approximately corresponding to human adolescence. After short- (PND45) or long-term (PND90) abstinence, prodynorphin-\u3ba-opioid receptor (pDYN-KOP) and pronociceptin-nociceptin receptor (pN/OFQ-NOP) gene expression were evaluated in the nucleus accumbens (NAc) and hippocampus (Hip) together with the analysis of BDNF signaling pathways. RESULTS: In the NAc of PND45 rats, pDYN mRNA levels were up-regulated, an effect paralled by increased BDNF signaling. Differently from NAc, pDYN mRNA levels were down-regulated in the Hip of PND45 rats without significant changes of BDNF pathway. At variance from PND45 rats, we did not find any significant alteration of the investigated parameters either in NAc and Hip of PND90 rats. CONCLUSIONS: Our results indicate that the short-term withdrawal from adolescent cocaine exposure is characterized by a parallel pDYN mRNA and BDNF signaling increase in the NAc. Given the depressive-like state experienced during short abstinence in humans, we hypothesize that such changes may contribute to promote the risk of cocaine abuse escalation and relapse

Increased cocaine self-administration in rats lacking the serotonin transporter : a role for glutamatergic signaling in the habenula

Serotonin (5-HT) and the habenula (Hb) contribute to motivational and emotional states such as depression and drug abuse. The dorsal raphe nucleus, where 5-HT neurons originate, and the Hb are anatomically and reciprocally interconnected. Evidence exists that 5-HT influences Hb glutamatergic transmission. Using serotonin transporter knockout (SERT-/- ) rats, which show depression-like behavior and increased cocaine intake, we investigated the effect of SERT reduction on expression of genes involved in glutamate neurotransmission under both baseline conditions as well as after short-access or long-access cocaine (ShA and LgA, respectively) intake. In cocaine-na\uefve animals, SERT removal led to reduced baseline Hb mRNA levels of critical determinants of glutamate transmission, such as SLC1A2, the main glutamate transporter and N-methyl-D-aspartate (Grin1, Grin2A and Grin2B) as well as \u3b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (Gria1 and Gria2) receptor subunits, with no changes in the scaffolding protein Dlg4. In response to ShA and LgA cocaine intake, SLC1A2 and Grin1 mRNA levels decreased in SERT+/+ rats to levels equal of those of SERT-/- rats. Our data reveal that increased extracellular levels of 5-HT modulate glutamate neurotransmission in the Hb, serving as critical neurobiological substrate for vulnerability to cocaine addiction

Regulation of Schwann cells oncotransformation by changes in Nf2/merlin expression, Hippo/YAP signaling and DNA methylation

Schwann cell (SC) express the Neurofibromin type 2 gene (Nf2), encoding the tumor suppressor protein merlin, a cytoskeleton-associated protein regulating cell proliferation and survival. Nf2/merlin inactivation causes protein loss and leads to SC transformation into a form of benign tumor called schwannoma. Moreover, Nf2/merlin is mutated in an autosomal dominant multiple syndrome, called neurofibromatosis type 2. In line with observation that physio/mechanical cues, such as environmental challenges, may be pathogenetically relevant for SC oncotransformation, we recently showed that the exposure to electromagnetic fields (EMFs) causes changes in SC Nf2/merlin expression, cell migration, chemotactic responsivity and cytoskeleton reorganization. We showed a downstream MAPK/Erk activation, involved in SC proliferation, as well as activation of Hippo/YAP signalling commonly altered during tumorigenesis. We also showed that some genes, known to be upstream or downstream mediators of Hippo (Amotl2, Dchs, Fat, Wnt1) were changed. Further studies on rat SC oncotransformation following acute EMF exposure (0.1 T, 50 Hz, 10 min) demonstrated that the number of cells in G1 phase was increased. Focus forming analysis, after repeated exposures, showed an increase in 3D SC growth. EMF affects also the SC epigenome, as total DNA methylation, de novo DNMT and HDAC were reduced. Furthermore, RT2-profile assay evidenced that genes crucial for SCs are upregulated in EMF exposed cells. Overall, we identified some mechanisms responsible of environmental-induced SC changes toward a proliferative/migrating state, which may be pathologically relevant for nerve tumor development

The metaplastic effects of NMDA receptors blockade on reactivation of instrumental memories in rats

Metaplasticity, defined as the plasticity of synaptic plasticity, could affect learning and memory at different neural levels. It was hypothesized that metaplasticity changes on glutamate receptors may affect memory destabilization, promoting or preventing reconsolidation. We investigated the metaplastic effect of NMDA channel blocker MK-801 on sucrose instrumental memory reconsolidation in a behavioural rat model associated to the assessment of molecular markers of metaplasticity, memory retrieval, destabilization and reconsolidation. Following instrumental conditioning and forced abstinence, rats were intraperitoneally treated with MK-801 or vehicle 24\u202fh before the exposure to memory retrieval or not-retrieval. Separate groups were tested for in-vivo extinction of responding (24\u202fh and 7\u202fd after reactivation) or ex-vivo assessment of transcription factor Zif268 and ribosomal protein rpS6 phosphorylation in nucleus accumbens (NAc) and amygdala (Amy). MK-801 significantly inhibited instrumental responding at extinction test, suggesting reconsolidation blockade of instrumental memory. The decrease of Zif268 and phosphorylated-rpS6 levels in NAc and Amy in MK-801/Retrieval vs. Vehicle/Retrieval group supported the behavioural findings. An increase of GluN2B, GluA1 and mGluR5 in NAc, and GluN2B in Amy, 24\u202fh after MK-801 indicated the trigger of associated metaplastic changes. Our findings show that metaplastic changes induced by NMDA receptors blockade affected sucrose instrumental memory retrieval as shown by both behavioural and molecular changes. We hypothesize that these findings however suggested a switch to extinction rather than a reconsolidation

Short-term withdrawal from developmental exposure to cocaine activates the glucocorticoid receptor and alters spine dynamics

Although glucocorticoid receptors (GRs) contribute to the action of cocaine, their role following developmental exposure to the psychostimulant is still unknown. To address this issue, we exposed adolescent male rats to cocaine (20. mg/kg/day) from post-natal day (PND) 28 to PND 42 and sacrificed them at PND 45 or 90. We studied the medial prefrontal cortex (mPFC), a brain region that is still developing during adolescence. In PND 45 rats we found enhanced GR transcription and translation as well as increased trafficking toward the nucleus of the receptor, with no alteration in plasma corticosterone levels. We also showed reduced expression of the GR co-chaperone FKBP51, that normally keeps the receptor in the cytoplasm, and increased expression of Src1, which cooperates in the activation of GR transcriptional activity, revealing that short withdrawal alters the finely tuned mechanisms regulating GR action. Since activation of GRs regulate dendritic spine morphology, we next investigated spine dynamics in cocaine-withdrawn rats. We found that PSD95, cofilin and F-actin, molecules regulating spine actin network, are reduced in the mPFC of PND 45 rats suggesting reduced spine density, confirmed by confocal imaging. Further, formation of filopodia, i.e. the inactive spines, is enhanced suggesting the formation of non-functional spines. Of note, no changes were found in molecules related to GR machinery or spine dynamics following long-term abstinence, i.e. in adult rats (PND 90). These findings demonstrate that short withdrawal promotes plastic changes in the developing brain via the dysregulation of the GR system and alterations in the spine network

Ketamine Self-Administration Reduces the Homeostasis of the Glutamate Synapse in the Rat Brain

Ketamine is a non-competitive antagonist of the NMDA glutamate receptor with psychotomimetic and reinforcing properties, although recent work has pointed out its antidepressant action following acute exposure. Our aim was to investigate the expression of crucial components of the glutamate synapse following chronic ketamine self-administration (S/A), focusing our attention on medial prefrontal cortex (mPFC) and hippocampus (Hip), two brain regions involved in compulsive drug-seeking and drug-related cognitive disorders. Rats self-administered ketamine at a sub-anesthetic dose for 5\u20136 weeks and were sacrificed 24 h after the last drug exposure. We found a general downregulation of glutamate receptor expression that was brain region-dependent. In fact, in the mPFC, we found reduced expression of NMDA receptor subunits, whereas AMPA receptor protein levels were reduced in Hip; of note, specific scaffolding proteins of NMDA and AMPA receptors were also reduced in mPFC and Hip, respectively. Moreover, the metabotropic mGluR5 receptor was similarly downregulated in these brain regions. These findings reveal a dynamic impairment of glutamate homeostasis in the mPFC and Hip that may represent a signature of long-term exposure to ketamine S/A. Further, this decrement, similarly observed in humans and animal models of schizophrenia may represent a specific feature of the human disease endophenotype

Schwann Cell Autocrine and Paracrine Regulatory Mechanisms, Mediated by Allopregnanolone and BDNF, Modulate PKCε in Peripheral Sensory Neurons

Protein kinase type C-\u3b5 (PKC\u3b5) plays important roles in the sensitization of primary afferent nociceptors, such as ion channel phosphorylation, that in turn promotes mechanical hyperalgesia and pain chronification. In these neurons, PKC\u3b5 is modulated through the local release of mediators by the surrounding Schwann cells (SCs). The progesterone metabolite allopregnanolone (ALLO) is endogenously synthesized by SCs, whereas it has proven to be a crucial mediator of neuron-glia interaction in peripheral nerve fibers. Biomolecular and pharmacological studies on rat primary SCs and dorsal root ganglia (DRG) neuronal cultures were aimed at investigating the hypothesis that ALLO modulates neuronal PKC\u3b5, playing a role in peripheral nociception. We found that SCs tonically release ALLO, which, in turn, autocrinally upregulated the synthesis of the growth factor brain-derived neurotrophic factor (BDNF). Subsequently, glial BDNF paracrinally activates PKC\u3b5 via trkB in DRG sensory neurons. Herein, we report a novel mechanism of SCs-neuron cross-talk in the peripheral nervous system, highlighting a key role of ALLO and BDNF in nociceptor sensitization. These findings emphasize promising targets for inhibiting the development and chronification of neuropathic pain