Interrogating the Role of Cocaine-Generated Silent Synapses in the Regulation of Cocaine-Associated Memory Dynamics

Drug addiction is an acquired behavioral state that develops progressively through repeated drug experience and is characterized by maladaptive and compulsive behavior associated with drug seeking and taking. Cravings and subsequent drug seeking are often precipitated by the reactivation of memories associated with drug use, which are formed between various external stimuli, or cues, and the rewarding and pleasurable experience of taking the drug. As such, drug addiction is often conceptualized as a pathological form of memory that drives maladaptive behavior. This has spurred intensive investigation into the neural substrates underlying drug-associated memories, with the ultimate goal of targeting these substrates to disrupt drug seeking behaviors. To explore the synaptic underpinnings of cocaine-associated memories, we studied AMPA receptor (AMPAR)-silent excitatory synapses, which are generated in the nucleus accumbens (NAc) by cocaine experience. These synapses functionally mature during withdrawal through the recruitment of AMPARs and contribute to subsequent cocaine seeking behavior, indicating these synapses contribute to the encoding of cocaine-associated memories and behaviors. In this dissertation, we have further investigated the role of cocaine-generated silent synapses in the encoding of cocaine-associated memories by examining their role in regulating the natural dynamics of cocaine-associated memories. Our results demonstrate that dynamic changes in the functional state of cocaine-generated synapses contributes to the natural destabilization and reconsolidation of cocaine-associated memories following memory retrieval, and that disrupting these synaptic dynamics impairs subsequent cocaine seeking behaviors. In addition, we also demonstrate that cocaine-generated synapses contribute to the recruitment and activation of neurons within the NAc associated with cocaine seeking behavior during withdrawal, suggesting they may contribute to the encoding of cocaine-associated memories at the circuit level. Collectively, these findings provide further support to the hypothesis that cocaine-generated synapses serve as discrete synaptic substrates underlying aspects of cocaine-associated memories and behaviors

The Role of System XC- in Cognition: The Importance of Neuron-Astrocyte Signaling

The biological basis of human intelligence is largely a mystery, but likely required evolutionary adaptations to achieve the information processing capacity needed to expand the complexity of cognition among species. The link between evolutionary expansion of signaling complexity in the brain and cognition has largely focused on neuronal mechanisms, in part because information processing has historically been attributed to these cells. However, astrocytes are emerging as a second type of brain cell that is capable of processing information due to their capacity to release glutamate and, thereby, regulate neural circuits. Hence, a modern question is whether astrocytes contributed to the signaling complexity required for sophisticated forms of cognition. The glutamate release mechanism system xc- (Sxc) is the ideal mechanism to investigate this question because it is evolutionarily novel to vertebrate species and it is expressed by astrocytes. The central hypothesis tested herein is that Sxc increased the complexity of glutamate signaling and is required for behavior requiring complex cognition. To test, a genetically modified rat with Sxc activity eliminated was generated (MSxc rats). Phenotyping revealed that loss of Sxc activity produced changes in behavior that reflect diminished cognition or top-down processing including impaired reversal learning, set-shifting, and attentional allocation. Remarkably, loss of Sxc did not impact central regulation of metabolism, Pavlovian conditioning, instrumental conditioning, locomotor activity, and novel-object recognition. Additionally, Sxc is integral to the regulation of neural networks. In the nucleus accumbens, we found that a loss of Sxc altered synaptic strength in a circuit specific manner. Further, we found that Sxc-mediated glutamate release is regulated by presynaptic (the neuropeptide PACAP), postsynaptic (endocannabinoid) and hormonal (glucocorticoids) signaling mechanisms. Further interrogation of Sxc regulation by PACAP revealed that this neuropeptide acts on both neurons and astrocytes to facilitate bidirectional neuron-astrocyte signaling between Sxc and extrasynaptic NMDA receptors. The in vivo relevance of this mechanism is established by our findings that PACAP microinjected into the nucleus accumbens attenuates cocaine-primed reinstatement, and the regulation of this behavior requires both Sxc and NMDA receptors. These findings support the possibility that future therapeutics could restore cognition by targeting astrocytes

Prelimbic Cortical Synaptic and Structural Plasticity Following Cocaine Self-administration and Abstinence in Rats: Role of Glutamatergic Pathway Specificity

The primary goal of this dissertation is to further examine the role of the prelimbic (PrL) subdivision of the rodent medial prefrontal cortex in relapse to cocaine seeking following abstinence, and to extend our understanding of pathway-specific adaptations in the PrL cortex projection to the nucleus accumbens (NAc) core that drives relapse. Previous findings indicate that the PrL cortex shows a biphasic response to abstinence from cocaine exposure. Specifically, early withdrawal (two hours after the final self-administration session) results in dephosphorylation of glutamate NMDA receptors and glutamate signaling regulators including extracellular signal-regulated kinase and the downstream transcription factor cAMP response-element binding protein (CREB). One week of abstinence enhances p-CREB and AMPA receptor subunit GluA1 phosphorylation in the PrL cortex, and Synapsin I in the NAc core. Interventions that act to normalize glutamate transmission in the PrL cortex during early withdrawal provide an enduring suppression of drug-seeking by normalizing activity in the PrL-NAc core pathway. Using a combination of biochemical and behavioral pharmacology techniques, we have found that the cocaine-induced activation of STriatal-Enriched protein tyrosine Phosphatase in the PrL cortex during early withdrawal plays a role in subsequent cocaine seeking by dephosphorylating extra-cellular signal-regulated kinase. We also show that chemogenetic-mediated activation of the PrL cortex, or PrL-NAc core neurons, immediately after self-administration transiently reduces drug seeking which is not sustained. Finally, using an array of immunohistochemistry, pathway-specific viral vectors, and high-resolution confocal microscopy techniques, we provide evidence that PrL-NAc core neurons show reduced immunoreactivity of the activity markers Fos and p-CREB, reduced dendritic spine head diameter, and reduced GluA1/2 expression in subsets of dendritic spines during early withdrawal. The opposite effect was found after one week of abstinence. At this timepoint, PrL-NAc core neurons showed heightened nuclear p-CREB, spine head diameter, and GluA1/2 expression in dendritic spines. These findings suggest that the PrL cortex, and specifically PrL-NAc core neurons, undergoes an abstinence duration-dependent transformation in glutamate transmission which may be regulated by the activation of STEP during early withdrawal

Altered dendritic spine plasticity in cocaine-withdrawn rats

金沢大学附属病院神経科精神科Chronic cocaine treatment is associated with changes in dendritic spines in the nucleus accumbens, but it is unknown whether this neuroplasticity alters the effect of a subsequent cocaine injection on spine morphology and protein content. Three weeks after daily cocaine or saline administration, neurons in the accumbens were filled with the lipophilic dye, DiI. Although daily cocaine pretreatment did not alter spine density compared with daily saline, there was a shift from smaller to larger diameter spines. During the first 2 h after an acute cocaine challenge, a bidirectional change in spine head diameter and increase in spine density was measured in daily cocainepretreated animals. In contrast, no change in spine diameter or density was elicited by a cocaine challenge in daily saline animals during the first 2 h after injection. However, spine density was elevated at 6 h after a cocaine challenge in daily saline-pretreated animals. The time-dependent profile of proteins in the postsynaptic density subfraction elicited by a cocaine challenge in daily cocaine-pretreated subjects indicated that the changes in spine diameter and density were associated with a deteriorating actin cytoskeleton and a reduction in glutamate signaling-related proteins. Correspondingly, the amplitude of field potentials in accumbens evoked by stimulating prefrontal cortex was reduced for up to 6 h after acute cocaine in daily cocaine-withdrawn animals. These data indicate that daily cocaine pretreatment dysregulates dendritic spine plasticity elicited by a subsequent cocaine injection. Copyright © 2009 Society for Neuro science

Regulator of G Protein Signaling-12 (RGS12) in dopaminergic and kappa opioid receptor-dependent signaling and behavior

Dopaminergic neurotransmission is critically involved in the etiology and treatment of many psychiatric and neurological disorders. One modulator of dopaminergic neurotransmission is the kappa opioid receptor (KOR) -- a G protein-coupled receptor (GPCR) that is densely expressed within dopaminergic neurons and circuits. GPCRs are tightly regulated by a variety of intracellular signaling molecules, including Regulator of G Protein Signaling (RGS) proteins. Canonically, RGS proteins act as GTPase accelerating proteins (GAPs) on GTP-bound Ga subunits following GPCR activation, thereby hastening the rate at which GPCR-mediated G protein signaling is terminated. However, some RGS proteins exhibit more complex mechanisms of action on cellular signaling. One such example is RGS12, which harbors the capacity to regulate both G protein-dependent and -independent signaling cascades. RGS12 is widely expressed across the developing and adult brain; we show that expression levels are notably high within dopamine- and KOR-enriched regions. We also observed that Rgs12 mRNA exhibits marked expression overlap with Oprk1 mRNA encoding KOR, which potently regulates dopaminergic neurotransmission following environmental stress and pharmacological challenge with psychostimulants. We revealed that genetic ablation of Rgs12 in mice upregulates dopamine transporter (DAT) function and attenuates behavioral responses to psychostimulants via KOR-dependent mechanisms. Moreover, we showed that RGS12 differentially regulates G protein-dependent versus G protein-independent signaling and behavior downstream of KOR activation. We also demonstrated that RGS12 displays region-specific effects in the striatum, and that RGS12 selectivity modulates KOR over other opioid receptors. Further studies are required to more completely elucidate the complex interaction between RGS12 and KOR, as well as to identify precisely the neuronal cell populations and brain regions mediating the effect of RGS12 on dopaminergic and KOR-dependent signal transduction and behavior

Investigations into the role of the metabotropic glutamate receptor, mGluR5, in incentive learning and some behavioural and neurobiological effects of cocaine

The metabotropic glutamate receptor, mGluR5, is densely expressed in brain regions involved in incentive learning processes. There is considerable evidence to suggest that following exposure to addictive drugs such as cocaine, adaptations in these brain areas may underlie the development and maintenance of behavioural responses related to addictive processes. The present thesis examines the role of mGluR5 in both incentive learning processes and some behavioural and neurobiological effects of cocaine. First, using a novel mutant mouse line in which mGluR5 is selectively knocked down in cells that express dopamine D1 receptors (D1R), I argue that this mGluR5 population is critically important for specific incentive learning processes. By blocking mGluR5 in wild-type mice with a selective antagonist, I then propose mGluR5 as necessary for the acquisition, but not the expression of an incentive association. Next, I present data showing that mGluR5 on dopaminoceptive neurons are not necessary for the „conditioned rewarding‟ properties of cocaine, measured in the conditioned place preference model, but do contribute to the psychomotor activating effects of cocaine. Finally, I present an immunohistochemistry study that examines cocaine-induced activation of the extracellular-signal related kinase (ERK) pathway. In the mGluR5 knock-down mice, activation of the ERK pathway in the striatum is disrupted following an acute injection of cocaine. Given the importance of the ERK pathway in establishing and maintaining long term memories, I propose that disruption of this pathway could contribute, in part, to some findings reported in the present thesis. Taken together, this thesis will argue that signalling through mGluR5 on D1R expressing neurons is important for the formation of incentive associations, and may contribute to neural adaptations necessary for the development and maintenance of behavioural responses related to addictive processes

Role of Palmitoylation of Postsynaptic Proteins in Promoting Synaptic Plasticity

Many postsynaptic proteins undergo palmitoylation, the reversible attachment of the fatty acid palmitate to cysteine residues, which influences trafficking, localization, and protein interaction dynamics. Both palmitoylation by palmitoyl acyl transferases (PAT) and depalmitoylation by palmitoyl-protein thioesterases (PPT) is regulated in an activity-dependent, localized fashion. Recently, palmitoylation has received attention for its pivotal contribution to various forms of synaptic plasticity, the dynamic modulation of synaptic strength in response to neuronal activity. For instance, palmitoylation and depalmitoylation of the central postsynaptic scaffold protein postsynaptic density-95 (PSD-95) is important for synaptic plasticity. Here, we provide a comprehensive review of studies linking palmitoylation of postsynaptic proteins to synaptic plasticity

Efeitos do metilfenidato : uma abordagem experimental

Embora o uso e o abuso de metilfenidato estejam aumentando na infância e na adolescência, pouco se sabe sobre as consequências de sua utilização em longo prazo sobre o cérebro em desenvolvimento. O objetivo do presente estudo foi investigar os efeitos neuroquímicos hipocampais e comportamentais do tratamento crônico com metilfenidato em ratos jovens. Também avaliamos se o tratamento com metilfenidato (1μM) influencia a via Akt-mTOR em células de feocromocitoma de ratos (PC12), um modelo celular bem caracterizado. Ratos Wistar receberam injeções intraperitoneais de metilfenidato (2,0 mg/kg) ou volume equivalente de solução salina (controles), uma vez por dia, do 15º ao 45º dia de idade. O tratamento com metilfenidato alterou o perfil de aminoácidos, diminuindo os níveis de glutamina, bem como a captação de glutamato e a atividade da Na+,K+-ATPase. Não foram observadas alterações no imunoconteúdo dos transportadores de glutamato (GLAST e GLT-1), no imunoconteúdo das subunidades catalíticas da Na+,K+-ATPase (α1, α2 e α3) e nos parâmetros de equlíbrio redox. Os níveis de ATP foram diminuídos pelo metilfenidato e as atividades da citrato sintase, succinato desidrogenase e complexos da cadeia respiratória (II, II-III e IV), bem como a massa e o potencial de membrana mitocondrial não foram alterados pelo metilfenidato. Hipofosforilação da GFAP e a redução do seu imunoconteúdo foram observadas em ratos tratados com metilfenidato. Os neurofilamentos de médio e alto peso molecular também foram hipofosforilados, porém seus imunoconteúdos não foram alterados. O imunoconteúdo da proteína fosfatase 1 e 2A foram aumentados. O conteúdo total de gangliosídeos e fosfolipídeos, assim como os principais gangliosídeos (GM1, GD1a e GD1b) e fosfolipídeos cerebrais foram reduzidos pelo tratamento com metilfenidato. O colesterol total também foi reduzido no hipocampo desses animais. Além disso, resultados mostraram que a administração crônica de metilfenidato causou uma perda de astrócitos e neurônios no hipocampo de ratos jovens. O imunoconteúdo de BDNF e pTrkB, bem como os níveis de NGF foram reduzidos, enquanto que os níveis de TNF-α e IL-6 e os imunoconteúdos de Iba-1 e caspase 3 foram aumentados. A razão pERK/ERK e o imunoconteúdo de PKCaMII foram reduzidos, porém a razão pAkt/Akt e pGSK-3β/GSK-3β não foram alterados. O imunoconteúdo de SNAP-25 foi diminuído e GAP-43 e sinaptofisina não foram alterados. A atividade exploratória e a memória de reconhecimento de objetos foram prejudicadas pelo metilfenidato. Em relação às células, o tratamento com metilfenidato de curta duração diminuiu as razões pAkt/Akt, pmTOR/mTOR e pS6K/S6K, bem como o imunoconteúdo de pFoxO1. Por outro lado, o tratamento em longo prazo aumentou a razão de pAkt/Akt, pmTOR/mTOR e pGSK-3β/GSK-3β. Os níveis de fosforilação de 4E-BP1 foram diminuídos aos 15 e 30 minutos e aumentados em 1 e 6 h pelo metilfenidato. A razão pCREB/CREB foi diminuída. Esses resultados fornecem evidências adicionais de que a exposição crônica precoce ao metilfenidato pode ter efeitos complexos, bem como fornece novas bases para a compreensão das consequências neuroquímicas e comportamentais associadas ao tratamento com esse psicoestimulante. Além disso, fornece uma nova base para a compreensão dos mecanismos associados ao tratamento com metilfenidato, que pode conduzir estudos futuros.Although the use, and misuse, of methylphenidate is increasing in childhood and adolescence, there is little information about the consequences of this psychostimulant chronic use on brain and behavior during development. The aim of the present study was to investigate neurochemical, histochemical, and behavioral effects of chronic methylphenidate treatment to juvenile rats. We also attempted to determine whether the treatment with methylphenidate (1μM) influences Akt-mTOR signaling pathways in rat pheochromocytoma cells (PC12), a well characterized cellular model. Wistar rats received intraperitoneal injections of methylphenidate (2.0 mg/kg) or an equivalent volume of 0.9 % saline solution (controls), once a day, from the 15th to the 45th day of age. Results showed that chronic methylphenidate altered amino acid profile in hippocampus, decreasing the levels of glutamine. Glutamate uptake and Na+,K+-ATPase activity were decreased after treatment in rat hippocampus. No changes were observed in the glutamate transporters (GLAST and GLT-1), immunocontent of catalytic subunits of Na+,K+-ATPase (α1, α2 and α3), and redox status. Methylphenidate provoked a decrease in ATP levels, while citrate synthase, succinate dehydrogenase, respiratory chain complexes activities (II, II-III and IV), as well as mitochondrial mass and mitochondrial membrane potential were not altered. Methylphenidate also provoked hypophosphorylation of GFAP and reduced its immunocontent. Middle and high molecular weight neurofilament subunits (NF-M, NF-H) were hypophosphorylated by methylphenidate on KSP repeat tail domains, while their immunocontents were not altered. Methylphenidate increased protein phosphatase 1 and 2A immunocontents. Methylphenidate decreased the total content of ganglioside and phospholipid, as well as the main brain gangliosides (GM1, GD1a, and GD1b) and the major brain phospholipids in rat hippocampus. Total cholesterol content was also reduced in the hippocampi of juvenile rats by methylphenidate. In addition, methylphenidate caused loss of astrocytes and neurons in the hippocampus of juvenile rats. BDNF and pTrkB immunocontents and NGF levels were decreased, while TNF-α and IL-6 levels, as well as Iba-1 and caspase 3 cleaved immunocontents (microglia marker and active apoptosis marker, respectively) were increased. ERK and PKCaMII signaling pathways, but not Akt and GSK-3β, were decreased. SNAP-25 was decreased after methylphenidate treatment, while GAP-43 and synaptophysin were not altered. Both exploratory activity and object recognition memory were impaired. In relation to PC12 cells, short term methylphenidate treatment decreased the pAkt/Akt, pmTOR/mTOR and pS6K/S6K ratios, as well as pFoxO1 immunocontent. On the other hand, long term treatment increased pAkt/Akt, pmTOR/mTOR and pGSK-3β/GSK-3β ratios. Phosphorylation levels of 4E-BP1 were decreased at 15 and 30 minutes and increased at 1 and 6 h by methylphenidate. pCREB/CREB ratio was decreased. These findings provide additional evidence that early-life exposure to methylphenidate can have complex effects during central nervous system development, as well as provide new basis for understanding of the biochemical and behavioral effects associated with methylphenidate treatment

Modulation of Brain Chemistry with Small Molecule Probes: From Opioid to Growth Factor Signaling Systems

This report describes the use of small molecule probes in the modulation of brain chemistry with the ultimate goal of developing novel therapeutics for the treatment of mood disorders. With an increasing number of people suffering from depression, there is a need to explore more diverse mechanisms of these diseases to better understand their cause and therefore provide insight into their treatment. Chapter 1 serves as an introduction and describes the current understanding of depression mechanisms, as well as a history of antidepressant therapeutics. The chapter then goes on to discuss, in depth, the mechanisms of G Protein-Coupled Receptor (GPCR) function and the implications of biased signaling. There is also an introductory overview of basic pharmacological terms. The chapter finishes with a summary of current technology available to measure GPCR function, including those utilized in the rest of this report. The remainder of the report is broken up into two parts. In the first part, I will describe my work to understand the opioid receptor system in the context of mood disorders. In Chapter 2, the atypical antidepressant tianeptine is discovered to act through the mu-opioid receptor (MOR), and a biochemical exploration is reported including an exploration of its unique properties in the context of G protein-dependent and -independent signaling, as well as preliminary in vivo and structure activity relationship studies into the mechanism of action. In Chapter 3, I will describe the biological characterization of the Mitragyna speciosa alkaloids at the opioid receptors. In particular, the major alkaloids mitragynine and 7-OH mitragynine are found to be partial agonists at the MOR and antagonists at the kappa-opioid receptor (KOR) with apparent G protein bias. In Chapter 4, alkaloids inspired by those found in Tabernanthe iboga, such as ibogaine, are synthesized and characterized at the opioid receptors. Through a novel 12- hydroxy-oxaibogamine scaffold, opioid activity is uncovered that is greatly increased in comparison to the ibogaine metabolite noribogaine. Analogs tested have varying degrees of potency and efficacy at all three opioid receptors, and one analog in particular is found to be a selective G protein biased partial KOR agonist. In Chapter 5, I will conclude the opioid section by taking a critical examination of commonly used assays for measuring arrestin recruitment by dissecting assay components and analyzing what is necessary to determine accurate calculations of bias within a cellular system. The alleged G protein bias of KOR agonist dynorphin is studied at great length, and a discussion on the future of understanding ligand bias is presented. In the second part of this report, I move away from opioids and instead focus on the growth factor signaling system as a second approach to uncovering novel therapeutics for depression. In Chapter 6, I describe a second potential mechanism of action of the natural product ibogaine in the context of glial cell line-derived neurotrophic factor (GDNF) signaling. The deconstructed iboga analog XL-008 is studied that is a superior releaser of GDNF and potentiates the signaling of a second growth factor, fibroblast growth factor 2 (FGF2). In the final Chapter 7, I look to the FGF family, both receptor and growth factor, as a novel target for depression. In order to identify small molecule modulators of the FGF receptor 1 (FGFR1), cell- based assays are developed and validated in a pilot screen. The strength of these assays are assessed, and the initial results from a full high throughput screen are presented

LRRK2 effect on dopamine receptor trafficking: implication in Parkinson’s disease

Parkinson disease is the second most common neurodegenerative disorder affecting 4 million people worldwide. It is characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc) and by the presence of cytoplasmic inclusion bodies (Lewy bodies). Cell death leads to a profound depletion of dopamine neurotransmitter involved mainly in the control of the movement. Mutations in LRRK2 (leucine-rich repeat kinase 2) gene (PARK8; OMIM 609007) are responsible for one of the autosomal-dominant forms of Parkinson’s disease. Up to date, the LRRK2 biological function is largely unknown. LRRK2 has been found in different subcellular districts that play a crucial role in the control of vesicular trafficking: ER, Golgi apparatus and associated vesicles, cytoskeleton, lipid raft and lysosomes. The results of this work indicates that PD-associated mutant G2019S LRRK2 impairs dopamine receptor D1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect dopamine receptor D2 turnover by decreasing the rate of the receptor trafficking from the Golgi complex to the cell membrane. Collectively, these findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking