Nucleus Accumbens Tissue Research Articles

Decreased Caffeine-Induced Locomotor Activity via Microinjection of CART Peptide into the Nucleus Accumbens Is Linked to Inhibition of the pCaMKIIa-D3R Interaction.

The purpose of this study was to characterize the inhibitory modulation of cocaine- and amphetamine-regulated transcript (CART) peptides, particularly with respect to the function of the D3 dopamine receptor (D3R), which is activated by its interaction with phosphorylated CaMKIIα (pCaMKIIα) in the nucleus accumbens (NAc). After repeated oral administration of caffeine (30 mg/kg) for five days, microinjection of CART peptide (0.08 μM/0.5 μl/hemisphere) into the NAc affected locomotor behavior. The pCaMKIIα-D3R interaction, D3R phosphorylation and cAMP/PKA/phosphorylated CREB (pCREB) signaling pathway activity were measured in NAc tissues, and Ca2+ influx and pCaMKIIα levels were measured in cultured NAc neurons. We found that CART attenuated the caffeine-mediated enhancement of depolarization-induced Ca2+ influx and CaMKIIα phosphorylation in cultured NAc neurons. Repeated microinjection of CART peptides into the NAc decreased the caffeine-induced enhancement of Ca2+ channels activity, pCaMKIIα levels, the pCaMKIIα-D3R interaction, D3R phosphorylation, cAMP levels, PKA activity and pCREB levels in the NAc. Furthermore, behavioral sensitization was observed in rats that received five-day administration of caffeine following microinjection of saline but not in rats that were treated with caffeine following microinjection of CART peptide. These results suggest that caffeine-induced CREB phosphorylation in the NAc was ameliorated by CART peptide due to its inhibition of D3R phosphorylation. These effects of CART peptides may play a compensatory role by inhibiting locomotor behavior in rats.

Nucleus Accumbens AMPA Receptors Are Necessary for Morphine-Withdrawal-Induced Negative-Affective States in Rats.

Dependence is a hallmark feature of opiate addiction and is defined by the emergence of somatic and affective withdrawal signs. The nucleus accumbens (NAc) integrates dopaminergic and glutamatergic inputs to mediate rewarding and aversive properties of opiates. Evidence suggests that AMPA glutamate-receptor-dependent synaptic plasticity within the NAc underlies aspects of addiction. However, the degree to which NAc AMPA receptors (AMPARs) contribute to somatic and affective signs of opiate withdrawal is not fully understood. Here, we show that microinjection of the AMPAR antagonist NBQX into the NAc shell of morphine-dependent rats prevented naloxone-induced conditioned place aversions and decreases in sensitivity to brain stimulation reward, but had no effect on somatic withdrawal signs. Using a protein cross-linking approach, we found that the surface/intracellular ratio of NAc GluA1, but not GluA2, increased with morphine treatment, suggesting postsynaptic insertion of GluA2-lacking AMPARs. Consistent with this, 1-naphthylacetyl spermine trihydrochloride (NASPM), an antagonist of GluA2-lacking AMPARs, attenuated naloxone-induced decreases in sensitivity to brain stimulation reward. Naloxone decreased the surface/intracellular ratio and synaptosomal membrane levels of NAc GluA1 in morphine-dependent rats, suggesting a compensatory removal of AMPARs from synaptic zones. Together, these findings indicate that chronic morphine increases synaptic availability of GluA1-containing AMPARs in the NAc, which is necessary for triggering negative-affective states in response to naloxone. This is broadly consistent with the hypothesis that activation of NAc neurons produces acute aversive states and raises the possibility that inhibiting AMPA transmission selectively in the NAc may have therapeutic value in the treatment of addiction. Morphine dependence and withdrawal result in profound negative-affective states that play a major role in the maintenance of addiction. However, the underlying neurobiological mechanisms are not fully understood. We use a rat model of morphine dependence to show that GluA1 subunits of AMPA glutamate receptors in the nucleus accumbens (NAc), a brain region critical for modulating affective states, are necessary for aversive effects of morphine withdrawal. Using biochemical methods in NAc tissue, we show that morphine dependence increases cell surface expression of GluA1, suggesting that neurons in this area are primed for increased AMPA receptor activation upon withdrawal. This work is important because it suggests that targeting AMPA receptor trafficking and activation could provide novel targets for addiction treatment.

Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration

It is well established that cocaine-induced changes in glutamate receptor expression in the nucleus accumbens (NAc) play a significant role in animal models of cocaine addiction. Far less is known about cocaine-induced changes in GABA transmission, despite its importance in regulating NAc output via local interneurons and medium spiny neuron (MSN) axon collaterals (GABA ‘microcircuit’). Here we investigated whether GABAA receptor surface or total expression is altered following an extended-access cocaine self-administration regimen that produces a time-dependent intensification (incubation) of cue-induced cocaine craving in association with strengthening of AMPA receptor (AMPAR) transmission onto MSN. Rats self-administered cocaine or saline (control condition) 6h/day for 10 days. NAc tissue was obtained and surface proteins biotinylated on three withdrawal days (WD) chosen to span incubation of craving and associated AMPAR plasticity: WD2, WD25 and WD48. Immunoblotting was used to measure total and surface expression of three GABAA receptor subunits (α1, α2, and α4) that are strongly expressed in the NAc. We found a transient increase in surface, but not total, expression of the α2 subunit on WD2 from cocaine self-administration, an effect that was no longer observed by WD25. The expression of α1 and α4 subunits was not altered at these withdrawal times. On WD48, when AMPAR transmission is significantly potentiated, we did not find any alteration in GABAA receptor surface or total expression. Our findings suggest that the strengthening of AMPAR-mediated glutamate transmission in the NAc is not accompanied by compensatory strengthening of GABAergic transmission through insertion of additional GABAA receptors.

An Acute Methamphetamine Injection Downregulates the Expression of Several Histone Deacetylases (HDACs) in the Mouse Nucleus Accumbens: Potential Regulatory Role of HDAC2 Expression

Methamphetamine (METH) administration alters gene expression in the nucleus accumbens (NAc). We recently demonstrated that an acute METH injection produced prolonged increases in the expression of immediate early genes in the NAc of HDAC2-deficient mice, suggesting that HDAC2 might be an important regulator of gene expression in the rodent brain. Here, we tested the possibility that HDAC2 deletion might also impact METH-induced changes in the expression of various HDAC classes in the NAc. Wild-type (WT) and HDAC2 knockout (KO) mice were given a METH (20mg/kg) injection, and NAc tissue was collected at 1, 2, and 8h post treatment. We found that METH decreased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA expression but increased HDAC6 mRNA levels in the NAc of WT mice. In contrast, the METH injection increased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA levels in HDAC2KO mice. These observations suggest that METH may induce large-scale transcriptional changes in the NAc by regulating the expression of several HDACs, in part, via HDAC2-dependent mechanisms since some of the HDACs showed differential responses between the two genotypes. Our findings further implicate HDACs as potential novel therapeutic targets for neurotoxic complications associated with the abuse of certain psychostimulants.

Differential miRNA expression in inherently high- and low-active inbred mice.

Despite established health benefits of regular exercise, the majority of Americans do not meet the recommended levels of physical activity. While it is known that voluntary activity levels are largely heritable, the genetic mechanisms that regulate activity are not well understood. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit transcription by binding to a target gene, inhibiting protein production. The purpose of this study was to investigate differential miRNA expression between inherently high- (C57L/J) and low- (C3H/HeJ) active inbred mice in soleus, extensor digitorum longus (EDL), and nucleus accumbens tissues. Expression was initially determined by miRNA microarray analysis, and selected miRNAs were validated by qRT-PCR. Expression of 13 miRNAs varied between strains in the nucleus accumbens, 20 in soleus, and eight in EDL, by microarray analysis. Two miRNAs were validated by qRT-PCR in the nucleus accumbens; miR-466 was downregulated (∼4 fold; P < 0.0004), and miR-342-5p was upregulated (∼115 fold; P < 0.0001) in high-active mice. MiR-466 was downregulated (∼5 fold; P < 0.0001) in the soleus of high-active mice as well. Interestingly, miR-466 is one of several miRNA families with sequence located in intron 10 of Sfmbt2; miRNAs at this locus are thought to drive imprinting of this gene. “Pathways in cancer” and “TGFβ signaling” were the most significant pathways of putative target genes in both the soleus and nucleus accumbens. Our results are the first to consider differential miRNA expression between high- and low-active mice, and suggest that miRNAs may play a role in regulation of physical activity.

Rapid Communications

The mood regulatory mechanisms of deep brain stimulation (DBS)therapy are yet to be fully understood. DBS is shown to have antidepressant actions in severe, treatment-resistant depression (TRD).Interestingly, DBS of mesoaccumbens neurologic targets, includingthe nucleus accumbens (NAc), have also been shown to induce mania in vulnerable individuals. The nucleus accumbens (NAc) is a critical node in the mesocorticolimbic system and plays a major role in mediating antidepressant behavioral responses in the forced swim test (FST), a preclinical screen for antidepressant efficacy. This study investigates the antidepressant effects of NAc DBS in an established animal model of TRD. Wistar rats were divided into 4 groups: TRD-DBS (n = 9), TRD-Sham (n = 8), TRD (n = 10), and Control (n = 10). Bilateral stimulating electrodes were implanted into the NAc of TRD-Sham and TRD-DBS animals. Antidepressant-resistance and depression behaviors were induced through adrenocorticotropic-hormone (ACTH-(1–24); 100 lg/day; 2nd and 3rd weeks) administration and concurrent social isolation (all 3 weeks) respectively. DBS was administered throughout the 2nd week of ACTH treatment via a back mounted rodent DBS system. 24-hour locomotor activity counts were obtained using infrareddetectors and weekly sucrose preference tests were performedthroughout the 3 week protocol. Open field and FST were completedat the end of the 3 weeks. Brains were then removed and stored at 80°C. NAc tissue levels of brain-derived and glialderived neurotrophic factors (BDNF and GDNF, respectively) were quantified using western blot. Results demonstrate significant increases in locomotor activity for TRD-DBS animals (DBS-Vs-Sham: p = 0.0248). Lowered immobility was observed during FST for TRD-DBS animals (DBS-Vs-Sham: p = 0.0188). ACTHinduced BDNF expression increased in the outer region substructure NAc-shell (p = 0.0487) and decreased in the inner region substructure NAc-core (p = 0.0275) compared to controls. These datasupport antidepressant actions of NAc DBS in TRD. Local changes in neurotrophic factors may contribute to these mechanisms. Importantly, observed increases in locomotor activity over the 3 weeks highlight the potential for mesoaccumbens DBS to impact behaviors such as locomotor activity which may contribute to risk for induction of mania. Preliminary analysis of concurrent effects of daily dopamine reuptake inhibitor GBR12909 (16 mg/kg) administration coupled with NAc DBS demonstrates dopamine-mediated augmentation of these mania-like behaviors.

Characterization of [(3) H]LS-3-134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand.

LS-3-134 is a substituted N-phenylpiperazine derivative that has been reported to exhibit: (i) high-affinity binding (Ki value 0.2nM) at human D3 dopamine receptors, (ii) >100-fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low-affinity binding (Ki >5000nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin-dependent activation of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [(3) H]-labeled LS-3-134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10-15min at 37°C) and binds with high affinity to both human (Kd =0.06±0.01nM) and rat (Kd =0.2±0.02nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [(3) H]LS-3-134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [(3) H]LS-3-134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.

감초 메탄올 추출물의 메스암페타민 유도 과다보행활동에 대한 억제작용

Background and objective: Methamphetamine (Meth) is a widely abused psychostimulant that produces hyperlocomotion in rodents. Radix of Glycyrrhizae uralensis comprises a variety of bioactive components that have neuroprotective effects. In a previous study, we have demonstrated methanol extracts from radix of Glycyrrhizae uralensis (MEGR) suppress acute cocaine-induced extracellular dopamine release in the nucleus accumbens. In the present study, we investigated the effect of MEGR on acute Meth-induced hyperlocomotion. Methods: Male Sprague-Dawley rats were orally administered with MEGR (60 mg/kg and 180 mg/kg) 60 min prior to an intraperitoneal injection of Meth (1.0 mg/kg). Results: Behavioral analysis showed acute Meth greatly increased locomotor activities, while pretreatment with MEGR dose dependently inhibited the hyperlocomotion. In parallel, there were markedly increased levels of dopamine and its metabolite 3, 4-dihydroxyphenylacetic acid in the nucleus accumbens tissues in Meth-treated rats, which were also almost completely reversed by 180 mg/kg MEGR. Conclusions: These results showed that radix of Glycyrrhizae uralensis attenuates Meth-induced hyperlocomotion by inhibiting dopamine synthesis and utilization, suggesting that radix of Glycyrrhizae uralensis might be effective in blocking the rewarding effect of Meth.

Genetic variation within the Chrna7 gene modulates nicotine reward‐like phenotypes in mice

Mortality from tobacco smoking remains the leading cause of preventable death in the world, yet current cessation therapies are only modestly successful, suggesting new molecular targets are needed. Genetic analysis of gene expression and behavior identified Chrna7 as potentially modulating nicotine place conditioning in the BXD panel of inbred mice. We used gene targeting and pharmacological tools to confirm the role of Chrna7 in nicotine conditioned place preference (CPP). To identify molecular events downstream of Chrna7 that may modulate nicotine preference, we performed microarray analysis of α7 knock-out (KO) and wild-type (WT) nucleus accumbens (NAc) tissue, followed by confirmation with quantitative polymerase chain reaction (PCR) and immunoblotting. In the BXD panel, we found a putative cis expression quantitative trait loci (eQTL) for Chrna7 in NAc that correlated inversely to nicotine CPP. We observed that gain-of-function α7 mice did not display nicotine preference at any dose tested, whereas conversely, α7 KO mice demonstrated nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the α7 nicotinic acetylcholine receptor (nAChR)-selective agonist, PHA-543613, dose-dependently blocked nicotine CPP, which was restored using the α7 nAChR-selective antagonist, methyllycaconitine citrate (MLA). Our genomic studies implicated a messenger RNA (mRNA) co-expression network regulated by Chrna7 in NAc. Mice lacking Chrna7 demonstrate increased insulin signaling in the NAc, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation.

Enhanced synthesis and release of dopamine in transgenic mice with gain‐of‐function α6* nAChRs

α6β2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9'S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9'S NAc slices compared to control, non-transgenic (non-Tg) slices. Extracellular DA levels in both non-Tg and α6L9'S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9'S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9'S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid compared to non-Tg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9'S compared to non-Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9'S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels.

S-adenosylmethionine modifies cocaine-induced DNA methylation and increases locomotor sensitization in mice

Several studies suggest that individual variability is a critical component underlying drug addiction as not all members of a population who use addictive substance become addicted. There is evidence that the overall epigenetic status of a cell (epigenome) can be modulated by a variety of environmental factors, such as nutrients and chemicals. Based on these data, our aim was to investigate whether environmental factors like S-adenosylmethionine (SAM) via affecting epigenome could alter cocaine-induced gene expression and locomotor sensitization in mice. Our results demonstrate that repeated SAM (10 mm/kg) pretreatment significantly potentiated cocaine-induced locomotor sensitization. Using mouse nucleus accumbens (NAc) tissue, whole-genome gene expression profiling revealed that repeated SAM treatment affected a limited number of genes, but significantly modified cocaine-induced gene expression by blunting non-specifically the cocaine response. At the gene level, we discovered that SAM modulated cocaine-induced DNA methylation by inhibiting both promoter-associated CpG-island hyper- and hypomethylation in the NAc but not in the reference tissue cerebellum. Finally, our in vitro and in vivo data show that the modulating effect of SAM is in part due to decreased methyltransferase activity via down-regulation of Dnmt3a mRNA. Taken together, our results suggest that environmental factors that affect the NAc-cell epigenome may alter the development of psychostimulant-induced addiction and this may explain, at least partly, why some individuals are more vulnerable to drug addiction.

Sex and dose-related differences in methylphenidate adolescent locomotor sensitization and effects on brain-derived neurotrophic factor

This study analyzed repeated methylphenidate (MPH) administration and its effects on brain-derived neurotrophic factor (BDNF) in the dorsal striatum and nucleus accumbens of male and female adolescent rats. In Experiment 1, rats were administered intraperitoneal (ip) saline, 1, 3, or 5 mg/kg dose of MPH every second day from postnatal day (P)33-P49. Locomotor activity was analyzed for 10 min after each administration. Results revealed that the 1 mg/kg dose of MPH produced locomotor suppression, however, the 5 mg/kg dose of MPH produced locomotor sensitization and robust behavioral activation in females as compared to males. In Experiment 2, animals were administered ip saline or the 5 mg/kg dose of MPH using an identical regimen but a 30 min behavioral test was employed. Dorsal striatum and nucleus accumbens tissue was assayed for BDNF at P50. Females demonstrated sensitization to MPH and increased locomotor activation compared to males. Interestingly, females given MPH demonstrated a significant 42% decrease of striatal BDNF whereas males administered MPH demonstrated a significant 50.4% increase of striatal BDNF compared to controls. There were no effects on accumbal BDNF. This report demonstrates robust sex differences in the behavioral response, but sex-dependent changes in striatal BDNF in response to MPH in adolescence.

An association between genotypic variations and protein expression of the glial glutamate transporter 2 in the human nucleus accumbens

The glial glutamate transporter EAAT2 is responsible for the majority of synaptic glutamate clearance. Dysfunction of EAAT2 is strongly implicated in neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs) in the EAAT2 gene have been associated with an increased risk of pathological conditions that may result from changes in extracellular glutamate levels. Genetic variation in the metabotropic glutamate receptor 3 (GRM3) gene has been reported to affect EAAT2 mRNA. Therefore, the aim of this study was to investigate the association of EAAT2 (rs4755404 and rs1885343) and GRM3 (rs6465084) SNPs and EAAT2 protein expression in healthy subjects. Postmortem nucleus accumbens tissue from 37 normal subjects had EAAT2 protein determined and was genotyped for three SNPs. Expression of EAAT2 protein was observed in both monomeric (70kDa) and multimeric (150kDa) forms. A significantly lower expression of the monomer (P=0.037) was observed with the GG genotype than in A allele carriers of rs1885343. However, there were no differences in EAAT2 expression associated with genotypes or alleles of rs4755404 and rs6465084. This finding indicates an association between EAAT2 protein expression in the human nucleus accumbens and a genetic polymorphism of EAAT2.

AKAP Signaling in Reinstated Cocaine Seeking Revealed by iTRAQ Proteomic Analysis

To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.

Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca 2+-permeable AMPA receptors during the incubation of cocaine craving

Cue-induced cocaine seeking intensifies or incubates after withdrawal from extended access cocaine self-administration, a phenomenon termed incubation of cocaine craving. The expression of incubated craving is mediated by Ca 2+-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). Thus, CP-AMPARs are a potential target for therapeutic intervention, making it important to understand mechanisms that govern their accumulation. Here we used subcellular fractionation and biotinylation of NAc tissue to examine the abundance and distribution of AMPAR subunits, and GluA1 phosphorylation, in the incubation model. We also studied two transmembrane AMPA receptor regulatory proteins (TARPs), γ-2 and γ-4. Our results, together with earlier findings, suggest that some of the new CP-AMPARs are synaptic. These are probably associated with γ-2, but they are loosely tethered to the PSD. Levels of GluA1 phosphorylated at serine 845 (pS845 GluA1) were significantly increased in biotinylated tissue and in an extrasynaptic membrane-enriched fraction. These results suggest that increased synaptic levels of CP-AMPARs may result in part from an increase in pS845 GluA1 in extrasynaptic membranes, given that S845 phosphorylation primes GluA1-containing AMPARs for synaptic insertion and extrasynaptic AMPARs supply the synapse. Some of the new extrasynaptic CP-AMPARs are likely associated with γ-4, rather than γ-2. The maintenance of CP-AMPARs in NAc synapses during withdrawal is accompanied by activation of CaMKII and ERK2 but not CaMKI. Overall, AMPAR plasticity in the incubation model shares some features with better described forms of synaptic plasticity, although the timing of the phenomenon and the persistence of related neuroadaptations are significantly different. This article is part of a Special Issue entitled ‘Synaptic Plasticity and Addiction’.

Effects of chronic morphine treatment on protein expression in rat dorsal root ganglia

The initial aim of this study was to identify protein changes in the dorsal root ganglia (DRG) associated with long-term morphine treatment. We carried out a differential proteomics analysis on samples from the DRG of control and morphine-dependent rats (5–40 mg/kg, subcutaneously, twice daily for 28 days) after 4 days of morphine withdrawal. Proteins showing a statistically significant variation between the two groups were selected for identification by mass spectrometric analysis. Twelve proteins were unambiguously identified, with the majority being enzymes involved in energy metabolism and protein degradation, signaling and cytoskeletal proteins. Aldolase C and proteasome subunit α type 3 (PRC8 or α7) were further examined by Western blot analysis in the DRG, prefrontal cortex, nucleus accumbens, striatum, hippocampus, ventral tegmental area and locus coeruleus. In addition, expression of PRC8 in the nucleus accumbens tissue after spontaneous or naloxone-precipitated withdrawal was determined using immunohistochemistical staining. Our results indicate that the expression levels of aldolase C and PRC8 proteins were altered in a time- and region-specific manner, and suggest that they could be implicated in the molecular changes underlying adaptations of neurons observed with chronic morphine treatment.

The PKC inhibitor Ro31-8220 blocks acute amphetamine-induced dopamine overflow in the nucleus accumbens

Acute administration of the psychostimulant amphetamine increases extracellular levels of dopamine (DA) by reversing the DA transporter on ascending midbrain DA neurons. In vitro studies using striatal synaptosomal, slice and nucleus accumbens (NAcc) tissue preparations have implicated protein kinase C (PKC) in this effect. The present study further examined this effect in vivo by assessing the ability of the PKC inhibitor, Ro31-8220 (10 μM), to inhibit acute amphetamine-induced DA overflow when applied with this drug to the NAcc via reverse dialysis. Amphetamine was applied at a concentration of 30 μM, and the core and shell subregions of the NAcc were assayed separately in freely moving rats. These brain regions play a role in the acute locomotor-activating and motivational effects of amphetamine. Consistent with the findings of previous in vitro experiments, reverse dialysis of Ro31-8220 with amphetamine robustly attenuated the ability of this drug to increase extracellular levels of dopamine in both the core and shell subregions of the NAcc. These results confirm that amphetamine stimulates dopamine overflow via a PKC-dependent mechanism.

Desipramine Reduces Stress-Activated Dynorphin Expression and CREB Phosphorylation in NAc Tissue

The nucleus accumbens (NAc) is a critical brain area for reward and motivated behavior. Accumulating evidence suggests that altered function of the transcription factor cAMP response element binding protein (CREB) within the NAc is involved in depressive behavior. In rats, stress activates CREB within the NAc, and elevation of CREB expression in this region produces depressive-like behaviors that are accompanied by activation of CREB-regulated target genes. The depressive-like behaviors seem to be due, at least in part, to CREB-mediated increases in dynorphin function, because they are mimicked by kappa-opioid receptor (KOR) agonists and attenuated by KOR antagonists. We hypothesized that if CREB-mediated dynorphin expression in the NAc contributes to depressive behavior, then antidepressants might reduce dynorphin function in this region. Here, we demonstrate that desipramine (DMI), a norepinephrine reuptake inhibitor that has been used for decades to treat clinical depression, blocks swim stress-induced activation of prodynorphin (encodes dynorphin) in the NAc. In primary cultures of NAc and striatum, DMI decreases basal and stimulated CREB phosphorylation by causing reductions in intracellular calcium (Ca(2+)) availability that are independent of norepinephrine or other monoaminergic inputs, identifying a potential mechanism for alterations in CREB-mediated gene expression. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, has similar effects in culture, suggesting a common intracellular effect of these antidepressants. These findings raise the possibility that a therapeutically relevant mechanism of action of DMI occurs through attenuation of CREB-mediated gene transcription, which is mediated via previously uncharacterized mechanisms that occur directly within the NAc.

ΔFosB induction in orbitofrontal cortex potentiates locomotor sensitization despite attenuating the cognitive dysfunction caused by cocaine

The effects of addictive drugs change with repeated use: many individuals become tolerant of their pleasurable effects but also more sensitive to negative sequelae (e.g., anxiety, paranoia, and drug craving). Understanding the mechanisms underlying such tolerance and sensitization may provide valuable insight into the basis of drug dependency and addiction. We have recently shown that chronic cocaine administration reduces the ability of an acute injection of cocaine to affect impulsivity in rats. However, animals become more impulsive during withdrawal from cocaine self-administration. We have also shown that chronic administration of cocaine increases expression of the transcription factor ΔFosB in the orbitofrontal cortex (OFC). Mimicking this drug-induced elevation in OFC ΔFosB through viral-mediated gene transfer mimics these behavioural changes: ΔFosB over-expression in OFC induces tolerance to the effects of an acute cocaine challenge but sensitizes rats to the cognitive sequelae of withdrawal. Here we report novel data demonstrating that increasing ΔFosB in the OFC also sensitizes animals to the locomotor-stimulant properties of cocaine. Analysis of nucleus accumbens tissue taken from rats over-expressing ΔFosB in the OFC and treated chronically with saline or cocaine does not provide support for the hypothesis that increasing OFC ΔFosB potentiates sensitization via the nucleus accumbens. These data suggest that both tolerance and sensitization to cocaine's many effects, although seemingly opposing processes, can be induced in parallel via the same biological mechanism within the same brain region, and that drug-induced changes in gene expression within the OFC play an important role in multiple aspects of addiction.

Integrin expression is altered after acute and chronic cocaine

Cocaine addiction is associated with an increase in actin cycling and alterations in dendritic spines in the nucleus accumbens. Both actin polymerization and spine morphology are regulated in part by beta-(β) integrins. Mice were administered acute or daily injections of cocaine or saline for 7 days. After 3 weeks of withdrawal, the level of β-integrins in the postsynaptic density enriched subfraction from nucleus accumbens tissue was quantified by immunoblotting at 0, 30 or 120 min following an a cocaine challenge injection. After chronic treatment and withdrawal the basal level of β1-integrin was increased while β3-integrin was unaltered. However, following a cocaine challenge in chronic cocaine, but not saline-treated animals, β3-integrin was transiently up-regulated while β1-integrin was transiently downregulated. These data demonstrate a bidirectional regulation of β-integrins by chronic cocaine treatment that may contribute to cocaine-induced changes in actin cycling and dendrite morphology.