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Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death

INTRODUCTION AND OBJECTIVE:Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic urological condition diagnosed in nearly 8 million women in the US. Whether the urinary microbiota plays...

International audience; Numerous lines of evidence support the premise that the misfolding and subsequent accumulation of SNCA/alpha-synuclein is responsible for the underlying neuronal pathology observed in Parkinson disease (PD) and other synucleinopathies. Moreover, the cell-to-cell transfer of these misfolded SNCA species is thought to be responsible for disease progression and the spread of cellular pathology throughout the brain. Previous work has shown that when exogenous, misfolded SNCA fibrils enter cells through endocytosis, they can damage and rupture the membranes of their endocytotic vesicles in which they are trafficked. Rupture of these vesicular membranes exposes intralumenal glycans leading to galectin protein binding, subsequent autophagic protein recruitment, and, ultimately, their introduction into the autophagic-lysosomal pathway. Increasing evidence indicates that both pathological and non-pathological SNCA species undergo autophagy-dependent unconventional secretion. While other proteins have also been shown to be secreted from cells by autophagy, what triggers this release process and how these specific proteins are recruited to a secretory autophagic pathway is largely unknown. Here, we use a human midbrain dopamine (mDA) neuronal culture model to provide evidence in support of a cellular mechanism that explains the cell-to-cell transfer of pathological forms of SNCA that are observed in PD. We demonstrate that LGALS3 (galectin 3) mediates the release of SNCA following vesicular damage. SNCA release is also dependent on TRIM16 (tripartite motif containing 16) and ATG16L1 (autophagy related 16 like 1), providing evidence that secretion of SNCA is mediated by an autophagic secretory pathway

ALIS are large, transient, cytosolic aggregates that serve as storage compartments for ubiquitin-tagged defective ribosomal products. We determined the importance of the protein p62 in the formation of ALIS and demonstrated that two domains of p62—PB1 and UBA—are essential for ALIS assembly. Those two major binding domains of p62, also known as sequestosome 1, were shown to play a critical role in the formation of autophagosomes or cytoplasmic aggregates. Specifically, the PB1 domain is essential for self-oligomerization, and the UBA domain allows p62 to bind to polyubiquitin chains or ubiquitinated proteins. After stimulation of RAW 264.7 macrophages with lipopolysaccharide, we observed a significant decrease in the number of cells with ALIS. Importantly, cells overexpressing either a PB1 mutant or UBA-deleted p62 construct also exhibited a substantially diminished number of cells containing ALIS. Since both p62 and ubiquitin are found in ALIS, we evaluated the dynamics of YFP-tagged p62 in ALIS. In contrast to the findings of a previous study that evaluated GFP-tagged ubiquitin motility in ALIS, we determined that YFP-tagged p62 has very limited mobility. Lastly, we determined that GST–tagged full-length p62 binds to Lys-63-linked polyubiquitin chains but not to Lys-48-linked chains. Overall, our findings provide insight on the essential role that p62, particularly its PB1 and UBA domains, has in the formation of ALIS.

Background and aims Smartphones are ubiquitous. As smartphones increased in popularity, researchers realized that people were becoming dependent on their smartphones. The purpose here was to provide a better understanding of the factors related to problematic smartphone use (PSPU). Methods The participants were 100 undergraduates (25 males, 75 females) whose ages ranged from 18 to 23 (mean age = 20 years). The participants completed questionnaires to assess gender, ethnicity, year in college, father's education level, mother's education level, family income, age, family history of alcoholism, and PSPU. The Family Tree Questionnaire assessed family history of alcoholism. The Mobile Phone Problem Use Scale (MPPUS) and the Adapted Cell Phone Addiction Test (ACPAT) were used to determine the degree of PSPU. Whereas the MPPUS measures tolerance, escape from other problems, withdrawal, craving, and negative life consequences, the ACPAT measures preoccupation (salience), excessive use, neglecting work, anticipation, lack of control, and neglecting social life. Results Family history of alcoholism and father's education level together explained 26% of the variance in the MPPUS scores and 25% of the variance in the ACPAT scores. The inclusion of mother's education level, ethnicity, family income, age, year in college, and gender did not significantly increase the proportion of variance explained for either MPPUS or ACPAT scores. Discussion and conclusions Family history of alcoholism and father's education level are good predictors of PSPU. As 74%-75% of the variance in PSPU scale scores was not explained, future studies should aim to explain this variance.

Extracellular vesicles (EVs) are implicated in a wide variety of biological activities, have been implicated in the pathogenesis of numerous diseases, and have been proposed to serve as potential biomarkers of disease in human patients and animal models. However, characterization of EV populations is often performed using methods that do not account for heterogeneity of EV populations and require comparatively large sample sizes to facilitate analysis. Here, we describe an imaging-based method that allows for the multiplexed characterization of EV populations at the single EV level following centrifugation of EV populations directly onto cover slips, allowing comprehensive analysis of EV populations with relatively small samples. We observe that canonical EV markers are present on subsets of EVs which differ substantially in a producer cell and cargo specific fashion, including differences in EVs containing different HIV-1 proteins previously reported to be incorporated into pathogenic EVs. We also describe a lectin binding assay to interrogate EVs based on their glycan content, which we observe to change in response to pharmacological modulation of secretory autophagy pathways. These studies collectively reveal that multiplexed analysis of EV populations using fluorescent microscopy can reveal differences in specific EV populations that may be used to understand the biogenesis of specific EV populations or to interrogate small subsets of EVs of interest within larger EV populations.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies. IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

Although the role of epigenetics in Parkinson's disease (PD) has not been extensively studied, α-synuclein, the main component of Lewy bodies, decreased histone H3 acetylation. Here, we determined if there were histone acetylation changes in the primary motor cortex which, according to the Braak model, is one of the last brain regions affected in PD. Net histone H3 acetylation, histone H3 lysine 9 (H3K9), histone H3 lysine 14 (H3K14), histone H3 lysine 18 (H3K18), and histone H3 lysine 23 (H3K23) acetylation was assessed in the primary motor cortex of those affected and unaffected by PD. There was net increase in histone H3 acetylation due to increased H3K14 and H3K18 acetylation. There was a decrease in H3K9 acetylation. No between-groups difference was detected in H3K23 acetylation. Relationships between Unified Lewy Body Staging scores and histone H3 acetylation and substantia nigra depigmentation scores and histone H3 acetylation were observed. No relationships were detected between postmortem interval and histone H3 acetylation and expired age and histone H3 acetylation. These correlational data support the notion that the histone H3 acetylation changes observed here are not due to the postmortem interval or aging. Instead, they are due to PD and/or factors that covary with PD. The data suggest enhanced gene transcription in the primary motor cortex of the PD brain due to increase H3K14 and H3K18 acetylation. This effect is partially offset by a decreased H3K9 acetylation, which might repress gene transcription.

Extracellular vesicles (EVs) have been implicated in a wide variety of biological activities, have been implicated in the pathogenesis of numerous diseases, and have been proposed to serve as potential biomarkers of disease in human patients and animal models. However, characterization of EV populations is often performed using methods that do not account for the heterogeneity of EV populations and require comparatively large sample sizes to facilitate analysis. Here, we describe an imaging-based method that allows for the multiplexed characterization of EV populations at the single EV level following centrifugation of EV populations directly onto cover slips, allowing comprehensive analysis of EV populations with relatively small samples. We observe that canonical EV markers are present on subsets of EVs which differ substantially in a producer cell and cargo specific fashion, including differences in EVs containing different HIV-1 proteins previously reported to be incorporated into pathogenic EVs. We also describe a lectin binding assay to interrogate EVs based on their glycan content, which we observe to change in response to pharmacological modulation of secretory autophagy pathways. These studies collectively reveal that a multiplexed analysis of EV populations using fluorescent microscopy can reveal differences in specific EV populations that may be used to understand the biogenesis of specific EV populations and/or to interrogate small subsets of EVs of interest within larger EV populations in biological samples.

ObjectiveRates of overweight and obesity epidemic have risen significantly in the past few decades, and 34% of adults and 15-20% of children and adolescents in the United States are now obese. Melanocortin receptor 4 (MC4R), contributes to appetite control in hypothalamic neurons and is a target for future anti-obesity treatments (such as setmelanotide) or novel drug development effort. Proper MC4R trafficking regulation in hypothalamic neurons is crucial for normal neural control of homeostasis and is altered in obesity and in presence of lipids. The mechanisms underlying altered MC4R trafficking in the context of obesity is still unclear. Here, we discovered that C2CD5 expressed in the hypothalamus is involved in the regulation of MC4R endocytosis. This study unmasked a novel trafficking protein nutritionally regulated in the hypothalamus providing a novel target for MC4R dependent pathways involved in bodyweight homeostasis and Obesity.MethodsTo evaluate the expression of C2cd5, we first used in situ hybridization and RNAscope technology in combination with electronic microscopy. For in vivo, we characterized the energy balance of wild type (WT) and C2CD5 whole-body knockout (C2CD5KO) mice fed normal chow (NC) and/or western-diet (high-fat/high-sucrose/cholesterol) (WD). To this end, we performed comprehensive longitudinal assessment of bodyweight, energy balance (food intake, energy expenditure, locomotor activity using TSE metabolic cages), and glucose homeostasis. In addition, we evaluated the consequence of loss of C2CD5 on feeding behavior changes normally induced by MC4R agonist (Melanotan, MTII) injection in the paraventricular hypothalamus (PVH). For in vitro approach, we tease out the role of C2CD5 and its calcium sensing domain C2 in MC4R trafficking. We focused on endocytosis of MC4R using an antibody feeding experiment (in a neuronal cell line - Neuro2A (N2A) stably expressing HA-MC4R-GFP; against HA-tag and analyzed by flux cytometry).ResultsWe found that 1) the expression of hypothalamic C2CD5 is decreased in diet-induced obesity models compared to controls, 2) mice lacking C2CD5 exhibit an increase in food intake compared to WT mice, 3) C2CD5 interacts with endocytosis machinery in hypothalamus, 4) loss of functional C2CD5 (lacking C2 domain) blunts MC4R endocytosis in vitro and increases MC4R at the surface that fails to respond to MC4R ligand, and, 5) C2CD5KO mice exhibit decreased acute responses to MTII injection into the PVH.ConclusionsBased on these, we conclude that hypothalamic C2CD5 is involved in MC4R endocytosis and regulate bodyweight homeostasis. These studies suggest that C2CD5 represents a new protein regulated by metabolic cues and involved in metabolic receptor endocytosis. C2CD5 represent a new target and pathway that could be targeted in Obesity.

In 1873 Camillo Golgi discovered a staining technique that allowed for the visualization of whole neurons within the brain, initially termed 'the black reaction' and is now known as Golgi impregnation. Despite the capricious nature of this method, Golgi impregnation remains a widely used method for whole neuron visualization and analysis of dendritic arborization and spine quantification. We describe a series of reliable, modified 'Golgi-Cox' impregnation methods that complement some existing methods and have several advantages over traditional whole brain 'Golgi' impregnation. First, these methods utilize 60-100μm thick brain sections, which allows for fast, reliable impregnation of neurons in rats (7-14 days) and non-human primates (NHP) (30 days) while avoiding the pitfalls of other 'rapid Golgi' techniques traditionally employed with thin sections. Second, these methods employ several common tissue fixatives, resulting in high quality neuron impregnation in brain sections from acrolein, glutaraldehyde, and paraformaldehyde perfused rats, and in glutaraldehyde perfused NHP brain tissue. Third, because thin sections are obtained on a vibratome prior to processing, alternate sections of brain tissue can be used for additional analyses such as immunohistochemistry or electron microscopy. This later advantage allows for comparison of, for example, dendrite morphology in sections adjacent to pertinent histochemical markers or ultrastructural components. Finally, we describe a method for simultaneous light microscopic visualization of both tyrosine hydroxylase immunohistochemistry and Golgi impregnation in the same tissue section. Thus, the methods described here allow for fast, high quality Golgi impregnation and conserve experimental subjects by allowing multiple analyses within an individual animal.

Activity-regulated cytoskeleton-associated protein (Arc) integrates information from multiple intracellular signaling cascades and, in turn, regulates cytoskeletal proteins involved in structural synaptic modifications. The purposes of the present study were: (a) to determine if the retrieval of contextual memories would induce Arc in hippocampal and amygdalar neurons; and (b) use unbiased stereology at the ultrastructural level to quantify synapses contacting Arc-labeled (Arc+) and unlabeled (Arc−) postsynaptic structures in brain regions in which the amount of Arc integrated density (ID) correlated strongly with the degree of amphetamine conditioned place preference (AMPH CPP). The retrieval of contextual memories increased the Arc ID in the dentate gyrus, cornu ammonis (CA)1, and CA3 fields of the hippocampus and the basolateral, lateral, and central nuclei of the amygdala but not the primary auditory cortex, a control region. Stereological quantification of Arc+ and Arc− synapses in the basolateral nucleus of the amygdala (BLA) was undertaken because the strongest relationship between the amount of Arc ID and AMPH CPP was observed in the BLA. The retrieval of contextual memories increased the number and density of asymmetric (presumed excitatory) synapses contacting Arc+ spines and dendrites of BLA neurons, symmetric (presumed inhibitory or modulatory) synapses contacting Arc+ dendrites of BLA neurons, and multisynaptic boutons contacting Arc+ postsynaptic structures. Thus, the retrieval of contextual memories increases Arc in the amygdala and hippocampus, an effect that could be important for approach behavior to a drug-associated context.

The basolateral amygdala (BLA) plays an important role in the formation of associations between context and drug. BLA activity and BLA-dependent drug-seeking behavior are driven by excitatory inputs. Drug-seeking behavior driven by context involves participation of the BLA, and plasticity of excitatory inputs to the BLA may contribute to this behavior. In this study, amphetamine conditioned place preference (AMPH CPP) was used to model the formation of context-drug associations. Learning-induced changes of excitatory synapses within the BLA were examined. Male Sprague-Dawley rats were assigned to one of three groups, the experimental group (AMPH CPP) or one of two control groups (saline or AMPH delayed pairing). Approximately 24 h after testing their preference, spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs, respectively) in BLA pyramidal neurons were investigated using whole-cell patch-clamp recordings. There were no between-groups differences in the amplitude or frequency of sEPSCs or mEPSCs. In a higher osmolarity solution to increase release, there was a significantly greater frequency of the mEPSCs in neurons from AMPH CPP animals compared with controls. This was observed with no change detected in the probability of glutamate release. Together, these data demonstrate no evidence for increased synaptic strength, but are consistent with an increase in the number of synapses in the BLA after AMPH CPP. These findings may underlie increased excitatory drive of the BLA after AMPH CPP, and contribute to the animals' preference for the AMPH-paired compartment.

The goal of researchers and clinicians interested in re-instituting cell based therapies for PD is to develop an effective and safe surgical approach to replace dopamine (DA) in individuals suffering from Parkinson’s disease (PD). Worldwide clinical trials involving transplantation of embryonic DA neurons into individuals with PD have been discontinued because of the often devastating post-surgical side-effect known as graft-induced dyskinesia (GID). There have been many review articles published in recent years on this subject. There has been a tendency to promote single factors in the cause of GID. In this review, we contrast the pros and cons of multiple factors that have been suggested from clinical and/or preclinical observations, as well as novel factors not yet studied that may be involved with GID. It is our intention to provide a platform that might be instrumental in examining how individual factors that correlate with GID and/or striatal pathology might interact to give rise to dysfunctional circuit remodeling and aberrant motor output.

Drug seeking and the vulnerability to relapse occur when individuals are exposed to an environment with sensory cues in which drug taking has occurred. Memory formation is thought to require plasticity in synaptic circuits, and so we examined whether the memory for a drug-paired environment correlates with changes in the synaptic circuits of the basolateral amygdala (BLA), in which emotional learning is a recognized phenomenon. We used amphetamine (AMPH) as the unconditioned stimulus in the conditioned place preference (CPP) paradigm. Rats were conditioned with 1.0 mg/kg AMPH and tested, drug free, 72 h after the last conditioning session. Controls included a saline-conditioned group and a home cage AMPH injection group, whose exposure to the CPP apparatus was delayed by 4 h, long enough to clear the AMPH from the brain. We counted excitatory synapses in the BLA using the electron microscope and the physical disector design (stereology). Rats that expressed AMPH CPP had an increase in excitatory synapses compared with controls. Excitatory synaptic activity was measured usingin vivointracellular recordings from the BLA in anesthetized rats. We found that AMPH CPP, but not drug alone, increased measures of synaptic drive, including the frequency of synaptic events, and the paired-pulse ratio of synaptic inputs to BLA pyramidal neurons. Thein vivofindings suggest that the increase in BLA neuronal excitatory drive reflects the change in excitatory synapse number. Thus, context–drug associations are accompanied by structural and functional plasticity in the BLA, findings that have important implications for drug-seeking behavior.

Psychostimulant drug experience leads not only to long-lasting changes in behavior but also modifications in the activity and morphology of pyramidal neurons in the medial prefrontal cortex (mPFC). The objective of this study was to establish whether repeated treatment of rats with amphetamine (AMPH) is accompanied by changes in the pattern or types of synapses in the mPFC and, specifically, onto neurons that project to the lateral hypothalamus, where our earlier work has shown increased markers of neuronal activity after repeated AMPH treatment (Morshedi and Meredith [2008] Psychopharmacology (Berl) 197:179–189). Rats were treated with a behaviorally sensitizing regimen of AMPH, following which synapses in the infralimbic and prelimbic cortices of the mPFC, were analyzed with unbiased stereology (physical disector and electron microscopy). All synapses were counted and their targets were identified by standard methodological criteria. Repeated AMPH administration was associated with a significant increase in the number of asymmetric axospinous synapses, no change in axodendritic or axosomatic contacts, and no change in the total number of synapses on corticolateral hypothalamic pyramidal neurons compared to vehicle-treated rats. Therefore, behavioral sensitization as a result of repeated exposure to AMPH is accompanied by the increased formation of spine, but not dendritic, synapses onto pyramidal neurons in the mPFC. Synapse 63:126–135, 2009. ©2008 Wiley-Liss, Inc.

Endocannabinoid signaling has been implicated in habituation to repeated stress. The hypothesis that repeated exposures to stress alters endocannabinoid signaling in the limbic circuit was tested by restraining male mice for 30 min/day for 1, 7, or 10 days and measuring brain endocannabinoid content. Amygdalar N-arachidonylethanolamine was decreased after 1, 7, and 10 restraint episodes; 2-arachidonylglycerol was increased after the 10th restraint. A similar pattern occurred in the medial prefrontal cortex (mPFC): N-arachidonylethanolamine was decreased after the 7th and 10th restraints and 2-arachidonylglycerol was increased after the 10th restraint. In the ventral striatum, the pattern reversed: N-arachidonylethanolamine was increased after the 10th restraint and 2-arachidonylglycerol was decreased after the 7th restraint. Palmitoylethanolamide contents changed in parallel with N-arachidonylethanolamine in the amygdala and ventral striatum. A single restraint episode did not affect the activity of fatty acid amide hydrolase (FAAH) in any of the brain regions examined. After the 10th restraint, both V(max) and K(m) for N-arachidonylethanolamine were increased in the mPFC; while only the V(max) was increased in the amygdala. On the other hand, the V(max) of FAAH was decreased in ventral striatum after the 10th restraint. After the 10th restraint, the maximum velocity for 2-oleoylglycerol hydrolysis was increased in mPFC; no other changes in 2-oleoylglycerol hydrolysis occurred. Repeated exposure to restraint produced no changes in CB(1) receptor density in any of the areas examined. These studies are consistent with the hypothesis that stress exposure alters endocannabinoid signaling in the brain and that alterations in endocannabinoid signaling occur during habituation to stress.

We tested the hypothesis that amphetamine (AMPH)-induced conditioned motor sensitization is accompanied by cellular activation (measured by Fos immunoreactivity) and synaptophysin immunoreactivity in reward-related brain areas. Forty-eight rats were tested for conditioned motor sensitization using a conditioning paradigm that was performed in a three-chambered apparatus. Rats underwent two drug pairings with 1.0 mg/kg AMPH in one outer chamber and, on alternate days, were paired with saline in the other. On the fifth day, relative to the first AMPH treatment, AMPH administration increased motor activity in the AMPH-paired context but not in the saline-paired context. Relative to the first saline treatment, saline on the fifth day produced a conditioned increase in motor activity when given in the chamber previously paired with AMPH, and saline given in the saline-paired context produced a conditioned decrease in motor activity. AMPH administered in the AMPH-paired context increased the density of both Fos and synaptophysin immunoreactivity in the dentate gyrus, cornu ammonis (CA)1, CA3, basolateral amygdala and dorsolateral striatum. This pairing between context and drug increased Fos but not synaptophysin immunoreactivity in the nucleus accumbens core and shell. Saline administered in the AMPH-paired context increased the density of Fos immunoreactivity in the basolateral amygdala and nucleus accumbens core. These data indicate that the basolateral amygdala-nucleus accumbens core pathway is necessary for the context-elicited conditioned motor responses, while the hippocampus encodes the spatial context.

Since endocannabinoids modulate reward processing and the stress response, we tested the hypothesis that endocannabinoids regulate stress-induced decreased sensitivity to natural reward. Restraint was used to produce stress-induced reductions in sucrose consumption and preference in male mice. Central cannabinoid receptor (CB(1)) signaling was modulated pharmacologically prior to the application of stress. The preference for sucrose over water was significantly decreased in mice exposed to restraint. Treatment of mice with a cannabinoid receptor agonist (CP55940) or fatty acid amide hydrolase inhibitor (URB597) attenuated, while the CB(1) receptor antagonist/inverse agonist, rimonabant (SR141716), enhanced, stress-induced decreases in sucrose preference. These data are consistent with a tonically active, stress-inhibitory role for the CB(1) receptor. Mice treated with 10 daily episodes of restraint showed reduced sucrose preference that was unaffected by CP55940 and URB597. However, rimonabant produced a greater reduction in sucrose preference on day 10 compared to day 1. These data suggest that on day 10, endocannabinoid signaling is maximally activated and essential for reward sensitivity. The findings of the present study indicate that the CB(1)/endocannabinoid signaling system is an important allostatic mediator that both modulates the responses of mice to stress and is itself modulated by stress.

Advanced age is the primary risk factor for Parkinson's disease (PD). In PD patients and rodent models of PD, advanced age is associated with inferior symptomatic benefit following intrastriatal grafting of embryonic dopamine (DA) neurons, a pattern believed to result from decreased survival and reinnervation provided by grafted neurons in the aged host. To help understand the capacity of the aged, parkinsonian striatum to be remodeled with new DA terminals, we used a grafting model and examined whether increasing the number of grafted DA neurons in aged rats would translate to enhanced behavioral recovery. Young (3 months), middle-aged (15 months), and aged (22 months) parkinsonian rats were grafted with proportionately increasing numbers of embryonic ventral mesencephalic (VM) cells to evaluate whether the limitations of the graft environment in subjects of advancing age can be offset by increased numbers of transplanted neurons. Despite robust survival of grafted neurons in aged rats, reinnervation of striatal neurons remained inferior and amelioration of levodopa-induced dyskinesias (LID) was delayed or absent. This study demonstrates that: 1) counter to previous evidence, under certain conditions the aged striatum can support robust survival of grafted DA neurons; and 2) unknown factors associated with the aged striatum result in inferior integration of graft and host, and continue to present obstacles to full therapeutic efficacy of DA cell-based therapy in this model of aging.

The role of endocannabinoid (eCB) signalling in restraint stress-induced neuronal activation was studied. Male mice exposed to 30 min of restraint exhibit increased Fos protein within prefrontal cortex (PFC), lateral septum (LS), nucleus accumbens (Acb) and medial amygdala. SR141716 (2 mg/kg) itself had no effect on Fos but pretreatment with SR141716 significantly potentiated restraint-induced Fos expression in cingulate, LS and Acb. SR141716 also significantly increased the time spent in active escape behaviours during the restraint. In restraint-habituated mice (mice exposed to four previous restraint episodes), the fifth restraint exposure resulted in decreased expression of active escape behaviours compared to the first exposure and only induced Fos protein in the central and medial amygdala. Administration of SR141716 prior to the fifth restraint episode resulted in greater potentiation of restraint-induced Fos induction than the first; significant increases occurred within all regions of PFC examined, LS and Acb. Brain regional eCB content was measured immediately after restraint. N-arachidonylethanolamine content within the amygdala was significantly decreased after both restraint episodes. 2-Arachidonylglycerol content was significantly increased in both the limbic forebrain and amygdala after the fifth restraint but not the first. Restraint had no effect on cerebellar eCB content. These data suggest that eCB activation of CB 1 receptors opposes the behavioural and neuronal responses to aversive stimuli. Because repeated homotypic stress increased both limbic 2-AG and resulted in a greater effect of SR141716 on limbic Fos expression, we hypothesize that increased CB 1 receptor activity contributes to the expression of habituation to homotypic stress.

N-Arachidonylethanolamine (AEA) accumulates during brain injury and postmortem. Because fatty acid amide hydrolase (FAAH) regulates brain AEA content, the purpose of this study was to determine its role in the postmortal accumulation of AEA using FAAH null mice. As expected, AEA content in immediately frozen brain tissue was significantly greater in FAAH-deficient (FAAH−/−) than in wild-type mice. However, AEA content was significantly lower in brains from FAAH−/− mice at 5 and 24 h postmortem. Similarly, wild-type mice treated in vivo with a FAAH inhibitor (URB532) had significantly lower brain AEA content 24 h postmortem compared with controls. These data indicate that FAAH contributes significantly to the postmortal accumulation of AEA. In contrast, the accumulations of two other N-acylethanolamines, N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), were not reduced at 24 h postmortem in either the FAAH−/− mice or mice treated with URB532. FAAH−/− mice accumulated significantly less ethanolamine at 24 h postmortem compared with wild-type mice, suggesting that FAAH activity plays a role in the accumulation of ethanolamine postmortem. These data demonstrate that FAAH activity differentially affects AEA and OEA/PEA contents postmortem and suggest that AEA formation specifically occurs via an ethanolamine-dependent route postmortem.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for the adaptation and survival of animals upon exposure to stressful stimuli, and data suggest that endocannabinoid (eCB) signaling modulates neuroendocrine function. We have explored the role of eCB signaling in the modulation of stress-induced HPA axis activation. Administration of the CB1 receptor antagonist/inverse agonist SR141716 (0.01, 0.1, 1, and 5 mg/kg, ip) to male mice produced a small, dose-dependent increase in the serum corticosterone (CORT) concentration. Despite this effect, the highest dose of SR141716 did not significantly increase neuronal activity within the paraventricular nucleus of the hypothalamus, as measured by the induction of Fos protein. Similarly, exposure of mice to 30 min of restraint increased serum CORT concentrations, but did not produce a consistent, statistically significant increase in Fos expression within the PVN. However, pretreatment of mice with SR141716 before restraint stress robustly potentiated restraint-induced CORT release and Fos expression within the PVN. Pretreatment of mice with either the CB1 receptor agonist CP55940, the eCB transport inhibitor AM404, or the fatty acid amide hydrolase inhibitor URB597 significantly decreased or eliminated restraint-induced CORT release. Upon exposure to acute restraint, hypothalamic 2-arachidonylglycerol content was reduced compared with the control value; however, after 5 d of restraint exposure (which resulted in an attenuated CORT response), the hypothalamic 2-arachidonylglycerol content was increased compared with the control value. These data indicate that eCB signaling negatively modulates HPA axis function in a context-dependent manner and suggest that pharmacological augmentation of eCB signaling could serve as a novel approach to the treatment of anxiety-related disorders.

We have established a clonal cell line derived from rat microglia that proliferates in response to macrophage-colony stimulating factor (CSF-1). Like primary neonatal microglia, these cells (named RTMGL1) exhibit a ramified morphology, bind isolectin B4, express CD68 and are weakly positive for CD11b and MHC class II. CSF-1-dependent proliferation requires intact signal transduction through several pathways. RTMGL1 synthesize multiple cyclooxygenase (COX) products including 11- and 15-hydroxyeicosatetraenoic acid (HETE) and express COX-2. RTMGL1 synthesize 5-HETE from arachidonic acid (AA) likely via a 5-lipoxygenase (LO). Thus, RTMGL1 have morphological and histological characteristics of primary microglia and metabolize AA via both COX and LO pathways.

Cannabinoids have been shown to modulate central autonomic regulation and baroreflex control of blood pressure (BP). The presence of cannabinoid CB1 receptors on fibers in the nucleus tractus solitarius (NTS) suggests that some presynaptic modulation of transmitter release could occur in this region, which receives direct afferent projections from arterial baroreceptors and cardiac mechanoreceptors. This study, therefore, was performed to determine the mechanism(s) of effects of microinjection of an endocannabinoid, arachidonylethanolamide (anandamide, AEA), into the NTS on baroreflex sympathetic nerve responses produced by phenylephrine-induced pressure changes in anesthetized rats. AEA prolonged reflex inhibition of renal sympathetic nerve activity (RSNA), suggesting an increase in baroreflex sensitivity. This effect of AEA was blocked by prior microinjection of SR-141716 to block cannabinoid CB1 receptors. To determine whether this baroreflex enhancement by AEA involved a GABAA mechanism, the baroreflex response to AEA was tested after prior blockade of postsynaptic GABAA receptors by bicuculline, which would eliminate any effects due to modulation of GABA activity. After bicuculline, which alone prolonged the baroreflex inhibition of RSNA, AEA shortened the duration of RSNA inhibition, suggesting a possible presynaptic inhibition of glutamate release previously obscured by a more dominant GABAA effect. To support a possible physiological role for AEA, AEA concentration in the NTS was measured after a phenylephrine-induced increase in BP. AEA content in the NTS was increased significantly over that in normotensive animals. These results support the hypothesis that AEA content is increased by brief periods of hypertension and suggest that AEA can modulate the baroreflex through activation of CB1 receptors within the NTS, possibly modulating effectiveness of GABA and/or glutamate neurotransmission.

1. Propofol (2,6-diisopropylphenol) is widely used as a general anesthetic and for the maintenance of long-term sedation. We have tested the hypothesis that propofol alters endocannabinoid brain content and that this effect contributes to its sedative properties. 2. A sedating dose of propofol in mice produced a significant increase in the whole-brain content of the endocannabinoid, N-arachidonylethanolamine (anandamide), when administered intraperitoneally in either Intralipid or emulphor-ethanol vehicles. 3. In vitro, propofol is a competitive inhibitor (IC(50) 52 micro M; 95% confidence interval 31, 87) of fatty acid amide hydrolase (FAAH), which catalyzes the degradation of anandamide. Within a series of propofol analogs, the critical structural determinants of FAAH inhibition and sedation were found to overlap. Other intravenous general anesthetics, including midazolam, ketamine, etomidate, and thiopental, do not affect FAAH activity at sedative-relevant concentrations. Thiopental, however, is a noncompetitive inhibitor of FAAH at a concentration of 2 mM. 4. Pretreatment of mice with the CB(1) receptor antagonist SR141716 (1 mg kg(-1), i.p.) significantly reduced the number of mice that lost their righting reflex in response to propofol. Pretreatment of mice with the CB(1) receptor agonist, Win 55212-2 (1 mg kg(-1), i.p.), significantly potentiated the loss of righting reflex produced by propofol. These data indicate that CB(1) receptor activity contributes to the sedative properties of propofol. 5. These data suggest that propofol activation of the endocannabinoid system, possibly via inhibition of anandamide catabolism, contributes to the sedative properties of propofol and that FAAH could be a novel target for anesthetic development.

Glutamatergic synaptic transmission within the striatum and prefrontal cortex regulates the neuronal synthesis of endocannabinoids. Because a primary role of dopamine is to modulate this excitatory transmission, we tested the hypothesis that dopaminergic transmission modulates endocannabinoid content in the limbic forebrain. Liquid chromatography/mass spectrometry was used to determine endogenous anandamide and 2-arachidonylglycerol (2-AG) contents within the limbic forebrain of mice after pharmacological manipulation of dopaminergic transmission. Increasing synaptic dopamine concentrations with methylphenidate significantly and dose dependently decreased both anandamide and 2-AG content. The selective dopamine reuptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909) also significantly decreased anandamide and tended to decrease 2-AG content. The D1 receptor antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) increased and the D1 receptor agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 33939) decreased anandamide content. 2-AG content was unaffected by SCH 23390 but was significantly increased by the D2 receptor antagonist eticlopride, which had no effect on anandamide content. The D2 agonist quinpirole had a biphasic effect on anandamide content with low, autoreceptor-preferring doses increasing anandamide and higher doses decreasing it back toward control. Quinpirole did not significantly affect 2-AG content. Together, these data indicate that endogenous dopamine exerts a differential, net suppressive effect upon anandamide and 2-AG content via activation of D1 and D2 receptors, respectively. These data are consistent with the hypothesis that modulation of endocannabinoid content by dopamine is secondary to changes in glutamatergic transmission, and they provide a pharmacological framework for the rational development of endocannabinoid-based therapeutic interventions for dopamine-related neuropsychiatric disorders.

Baroreceptor afferent fibers synapse in the nucleus tractus solitarius (NTS) of the medulla. Neuronal cannabinoid (CB)1 receptors are expressed in the NTS and central administration of CB1 receptor agonists affect blood pressure (BP) and heart rate. In addition, there is evidence that endocannabinoids are produced in the brain stem. This study examined whether changes in CB1 receptor activity in the NTS modulated the baroreceptor reflex, contributing to changes seen in BP and heart rate. Baroreflexes were evoked in anesthetized dogs by pressure ramp stimulations of the isolated carotid sinus before and after microinjection of CB1 receptor agonist WIN-55212-2 (1.25–1.50 pmol) or antagonist SR-141716 (2.5–3.0 pmol) into cardiovascular regions of the NTS. Microinjection of the SR-141716 did not affect baseline BP or baroreflex sensitivity. However, SR-141716 significantly prolonged the time needed to return to the baseline level of BP after the pressure ramp. Microinjection of WIN-55212-2 had no effect on the baroreflex. These data suggest that endocannabinoids can modulate the excitability of NTS neurons involved in the baroreceptor reflex, leading to modulation of baroreflex regulation.

The lead (Pb) chelator, meso-2,3-dimercaptosuccinic acid (DMSA) may be effective in reversing some of the adverse effects of Pb exposure. Pb-induced behavioral deficits observed in fish are due to disruptions in the integrative functioning of the medulla, cerebellum, and optic tectum. Pb exposure increased serotonin (5-HT) content in all three brain regions without an effect on 5-hydroxy-3-indoleacetic acid (5-HIAA). Pb exposure followed by no Pb in the diet increased 5-HT and 5-HIAA content in all three brain regions. The replacement of dietary Pb with DMSA had no effect on 5-HT and increased 5-HIAA content. DMSA increased 5-HIAA content in all three brain regions and increased 5-HT content only the optic tectum. Treatment with DMSA may be more effective than removal of Pb from the diet in reversing Pb-induced alterations in 5-HT.

The effects of cocaine, amperozide, clozapine, olanzapine and cocaine/atypical antipsychotic combinations on aggression, affiliation and defensive behaviors was examined. Acute cocaine (30.0 mg/kg) decreased basal aggression and affiliation yet increased basal defense. Amperozide (1.0, 3.0 and 5.0 mg/kg) decreased basal aggression, affiliation and defense had no effect on the cocaine-induced decrease in affiliation, and accentuated the cocaine-induced decrease in aggression. Near basal levels of defense were observed for animals treated with either amperozide, clozapine (3.0 and 10.0 mg/kg but not 30.0 mg/kg) or olanzapine followed by cocaine. Clozapine (3.0, 10.0 and 30.0 mg/kg) decreased basal aggression and affiliation. Clozapine (30.0 mg/kg but not 3.0 or 10.0 mg/kg) decreased basal defense. Clozapine attenuated the cocaine-induced decrease in aggression. Although 3.0 and 10.0 mg/kg clozapine attenuated the cocaine-induced decrease in affiliation, 30.0 mg/kg clozapine accentuated this cocaine-induced effect. Olanzapine (1.0, 3.0 and 10.0 mg/kg) decreased basal aggression, affiliation and defense. Olanzapine had no effect on the cocaine-induced decrease in aggression. Olanzapine (3.0 mg/kg but not 1.0 or 10.0 mg/kg) attenuated the cocaine-induced decrease in affiliation. Thus, acute cocaine administration had an antiaggressive effect, suppressed affiliative behavior and enhanced defensive behavior. Amperozide, clozapine and olanzapine have anticonflict and anxiolytic effects, as well as potent and specific antiaggressive effects.

Administration of the non-selective opioid receptor antagonists, naloxone and naltrexone, attenuate the rewarding effects of cocaine. The relative contributions of specific opioid receptor subtypes that underlie this effect have not been well characterized. Administration of 20.0 mg/kg cocaine resulted in a conditioned place preference. Pretreatment with 20.0 mg/kg but neither 10.0 nor 1.0 mg/kg of the selective mu(1)-opioid receptor antagonist, naloxonazine, blocked cocaine-induced conditioned place preference. On the days in which rats received cocaine only, locomotor behavior was elevated. Pretreatment with the selective mu(1)-opioid receptor antagonist, naloxonazine, regardless of dose, had no effect on cocaine-induced hyperlocomotion. These findings indicate that the rewarding effects of cocaine can be blocked solely by mu(1)-opioid receptor antagonism and are consistent with the view that the locomotor and rewarding effects of drugs can be dissociated.

Context-drug learning produces structural and functional synaptic changes in the circuitry of the basolateral nucleus of the amygdala (BLA). However, how the synaptic changes translated to the neuronal targets was not established. Thus, in the present study, immunohistochemistry with a cell specific marker and the stereological quantification of synapses was used to determine if context-drug learning increases the number of excitatory and inhibitory/modulatory synapses contacting the gamma-aminobutyric acid (GABA) interneurons and/or the pyramidal neurons in the BLA circuitry. Amphetamine conditioned place preference (AMPH CPP) increased the number of asymmetric (excitatory) synapses contacting the spines and dendrites of pyramidal neurons and the number of multisynaptic boutons contacting pyramidal neurons and GABA interneurons. Context-drug learning increased asymmetric (excitatory) synapses onto dendrites of GABA interneurons and increased symmetric (inhibitory or modulatory) synapses onto dendrites but not perikarya of these same interneurons. The formation of context-drug associations alters the synaptic connectivity in the BLA circuitry, findings that have important implications for drug-seeking behavior.

N-Methyl-D-aspartate (NMDA) ([R]-2-[Methylamino]succinic acid) is a specific excitatory amino acid. Two experiments were conducted to determine the rewarding properties of this compound using the conditioned place preference paradigm. In the first experiment, 40 male Sprague-Dawley rats received place preference conditioning for a 4 day period. The conditioned place preference apparatus consisted of two chambers with distinct visual and tactile cues, separated by a removable door. On days 2 and 4, rats were systemically administered NMDA (1.0, 15.0, and 30.0 mg/kg) paired with one chamber. On days 3 and 5, rats were systemically administered saline paired with the other chamber. Day 6 was the test day, and the rat was allowed free run of the entire apparatus in a drug-free state. Time spent in each side of the apparatus was computer recorded. NMDA produced a significant increase in the amount of time spent on the side previously paired with drug for 15.0 and 30.0, but not 1.0 mg/kg NMDA. In the second experiment, systemic administration of NMDA (30.0 mg/kg) paired with the noncompetitive NMDA receptor antagonist, MK-801 (0.5 mg/kg), resulted in neither place preference nor place aversion.

The effects of methylmercury (MeHg) exposure on neurotransmitter (NT) levels in larval mummichogs ( Fundulus heteroclitus ) obtained from a mercury-polluted site (Piles Creek (PC), NJ) and a reference site (Tuckerton (TK), NJ) were examined. Population differences between PC and TK larvae in neurochemical composition and in neurochemical changes in response to MeHg intoxication were found. Heads of untreated PC larvae (7 days posthatch (dph)) contained considerably higher levels of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) than TK. However, they had comparable levels of serotonin (5-hydroxytryptamine (5-HT)) and 5-hyroxy-3-indoleacetic acid (5-HIAA)/5-HT ratios. Changes in NTs with age were noticed, especially in PC larvae. Exposure of larvae to 10 μg/l MeHg induced neurochemical alterations. A significant increase in DA and 5-HT, as well as depressed dopaminergic and serotonergic activity (i.e. decreased DOPAC/DA, HVA/DA and 5-HIAA/5-HT ratios) were seen in TK larvae. Exposure of PC larvae to 10 μg/l MeHg reduced 5-HT at 14 dph, increased serotonergic activity at 7 dph, and altered dopaminergic activity (i.e. increased DOPAC/DA ratios, but decreased HVA/DA ratios). Changes in DA levels were inconsistent over time. The DA level, which was considerably higher than the control at 7 dph, was significantly lower than the control at 14 dph. For the two populations, the level of 5-HT and serotonergic activity, as well as DOPAC and HVA levels, were correlated with previously noted spontaneous activity. The changes in NT levels after exposure to MeHg are an indication of neurological dysfunction in larvae.

Cocaine decreases social interactions in both humans and animals, but it is not known whether the drugged animal withdraws from an undrugged conspecific, the undrugged conspecific avoids the drugged animal, or a combination of these two factors occurs. In the first experiment, the source of cocaine-induced social withdrawal was determined using a tether paradigm, in which the movement of one of the rats was restricted to one half of the observation chamber, such that the freely moving rat had the option of escaping social interactions. There were decreases in social interactions in the condition in which both rats were freely moving, and in the condition in which the undrugged rat was tethered, but not when the drugged rat was tethered and could not escape social contact. A second experiment was conducted to test the efficacy of the potent serotonin receptor antagonist, amperozide, in attenuating cocaine-induced social withdrawal using the condition in which the drugged rat was freely moving. Either amperozide (1.0, 3.0, and 5.0 mg/kg) or saline vehicle was injected into rats 1 h before receiving a 30.0 mg/kg cocaine dose. Cocaine decreased social interactions. Amperozide restored social interactions to near control levels and elevated social interactions in the animals treated with saline vehicle.

We examined the structural plasticity of excitatory synapses from corticostriatal and thalamostriatal pathways and their postsynaptic targets in adult Sprague-Dawley rats to understand how these striatal circuits change in l-DOPA-induced dyskinesias (LIDs). We present here detailed electron and light microscopic analyses that provide new insight into the nature of the structural and synaptic remodeling of medium spiny neurons in response to LIDs. Numerous studies have implicated enhanced glutamate signaling and persistent long-term potentiation as central to the behavioral sensitization phenomenon of LIDs. Moreover, experience-dependent alterations in behavior are thought to involve structural modifications, specifically alterations in patterns of synaptic connectivity. Thus, we hypothesized that in the striatum of rats with LIDs, one of two major glutamatergic pathways would form new or altered contacts, especially onto the spines of medium spiny neuron (MSNs). Our data provide compelling evidence for a dramatic rewiring of the striatum of dyskinetic rats and that this rewiring involves corticostriatal but not thalamostriatal contacts onto MSNs. There is a dramatic increase in corticostriatal contacts onto spines and dendrites that appear to be directly linked to dyskinetic behaviors, since they were not seen in the striatum of animals that did not develop dyskinesia. There is also an aberrant increase in spines receiving more than one excitatory contact(i.e., multisynaptic spines) in the dyskinetic animals compared with the 6-hydroxydopamine-treated and control rats. Such alterations could substantially impair the ability of striatal neurons to gate cortically driven signals and contribute to the loss of bidirectional synaptic plasticity.

Among the most widely used models of Parkinson’s disease (PD) are those that employ toxins, especially 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Depending on the protocol used, MPTP yields large variations in nigral cell loss, striatal dopamine loss and behavioral deficits. Motor deficits do not fully replicate those seen in PD. Nonetheless, MPTP mouse models mimic many aspects of the disease and are therefore important tools for understanding PD. In this review, we will discuss the ability of MPTP mouse models to replicate the pathophysiology of PD, the mechanisms of MPTP-induced neurotoxicity, strain differences in susceptibility to MPTP, and the models’ roles in testing therapeutic approaches.

The affective aspects of D: -amphetamine (AMPH) may be mediated, in part, by cocaine- and amphetamine-regulated transcript (CART) peptides in the basolateral amygdala (BLA). The formation of context-drug associations produces either conditioned place preference (CPP) or conditioned place aversion (CPA).The aim of the present study was to determine whether intra-BLA infusions of CART 55-102 are either rewarding or aversive and modulate AMPH reward.Rats were implanted with bilateral cannulae in the BLA, were subjected to place conditioning, and were tested for CPP or CPA. Rats were conditioned with either intra-BLA infusions of artificial cerebral spinal fluid or one of three dose of CART 55-102 (1, 2, or 4 microg/side), intra-BLA infusions of a subrewarding dose of CART 55-102 (1 microg/side) plus injections of a subrewarding dose of AMPH (0.1 mg/kg, i.p.), or intra-BLA infusions of an aversive dose of CART 55-102 (4 microg/side) plus injections of a rewarding dose of AMPH (1.0 mg/kg, i.p.).Intra-BLA infusions of 2 microg/side CART 55-102 produced CPP, 4 microg/side produced CPA, and 1 microg/side produced neither CPP nor CPA. Intra-BLA infusions of a subrewarding dose of CART 55-102 (1 microg/side) plus injections of a subrewarding dose of AMPH (0.1 mg/kg, i.p.) produced CPP. Intra-BLA infusions of an aversive dose of CART 55-102 (4 microg/side) plus injections of a rewarding dose of AMPH (1.0 mg/kg, i.p.) produced neither CPP nor CPA.Both the affective properties of intra-BLA CART 55-102 and its ability to either facilitate or block AMPH reward are dose dependent.

We tested the hypothesis that amphetamine- (AMPH) induced conditioned motor sensitization is accompanied by cellular activation (measured by Fos immunoreactivity) and synaptophysin immunoreactivity in reward-related brain areas. Forty-eight rats were tested for conditioned motor sensitization using a conditioning paradigm that was performed in a three-chambered apparatus. Rats underwent two drug pairings with 1.0 mg/kg AMPH in one outer chamber and, on alternate days, were paired with saline in the other. On the fifth day, relative to the first AMPH treatment, AMPH administration increased motor activity in the AMPH-paired context but not in the saline-paired context. Relative to the first saline treatment, saline on the fifth day produced a conditioned increase in motor activity when given in the chamber previously paired with AMPH, and saline given in the saline-paired context produced a conditioned decrease in motor activity. AMPH administered in the AMPH-paired context increased the density of both Fos and synaptophysin immunoreactivity in the dentate gyrus, cornu ammonis (CA)1, CA3, basolateral amygdala, and dorsolateral striatum. This pairing between context and drug increased Fos but not synaptophysin immunoreactivity in the nucleus accumbens core and shell. Saline administered in the AMPH-paired context increased the density of Fos immunoreactivity in the basolateral amygdala and nucleus accumbens core. These data indicate that the basolateral amygdala-nucleus accumbens core pathway is necessary for the context-elicited conditioned motor responses, while the hippocampus encodes the spatial context.