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ASCO phenotyping (A: atherosclerosis; S: small-vessel disease; C: cardiac pathology; O: other causes) assigns a degree of likelihood of causal relationship to every potential disease (1 for potentially causal, 2 for causality is uncertain, 3 for unlikely causal but the disease is present, 0 for absence of disease, and 9 for insufficient workup to rule out the disease) commonly encountered in ischemic stroke describing all underlying diseases in every patient. In this new evolution of ASCO called ASCOD, we have added a 'D' for dissection, recognizing that dissection is a very frequent disease in young stroke patients. We have also simplified the system by leaving out the 'levels of diagnostic evidence', which has been integrated into grades 9 and 0. Moreover, we have also changed the cutoff for significant carotid or intracranial stenosis from 70% to more commonly used 50% luminal stenosis, and added a cardiogenic stroke pattern using neuroimaging. ASCOD captures and weights the overlap between all underlying diseases present in ischemic stroke patients.

Background: Early recanalization and increase in collateral blood supply are powerful predictors of favourable outcome in acute ischaemic stroke. The factors contributing to the heterogeneous response to intravenous thrombolysis therapy in individual patients, however, are not fully understood. The on-going single-centre 'MR perfusion imaging during thrombolysis' study uses repetitive arterial spin labelling (ASL) measurements to characterize the haemodynamic processes in acute stroke during therapy. The first milestone was to develop an appropriate infrastructure for thrombolysis in the magnetic resonance imaging (MRI) scanner without time delay and ensuring optimal patient safety and care., Methods: Between February and December 2011, 16 patients with acute neurological symptoms suggestive of hemispheric stroke within 4.5 h after symptom onset were included. In addition to clinical data, we documented the time from onset to arrival at the hospital, start and duration of MRI examination, start of thrombolytic therapy, and complications. The decision to thrombolyse was made after a routine stroke MRI protocol. During the 60-min systemic thrombolysis, repetitive ASL perfusion imaging was acquired, providing non-invasive information on cerebral perfusion. Continuous ECG monitoring, pulse oximetry, blood pressure measurements every 5 min, and short neurological assessments every 15 min were performed in every patient., Results: The median initial NIHSS score of the patients presenting with a mean of 84 min after onset was 4 (range 2-18). MRI examination was initiated within a mean of 45 min after arrival at the hospital. Five patients identified as stroke mimics were not treated with recombinant tissue plasminogen activator (rt-PA), and in 1 case with basilar artery occlusion bridging therapy was performed outside the scanner. In the remaining 10 patients, rt-PA therapy was started in the scanner directly after decision making on the basis of clinical information and baseline MRI. The mean door-to-needle time was 60 min (range 44-115) including approximately 10 min needed for acquiring informed consent. While 4 patients required antihypertensive treatment, no relevant complications were encountered., Conclusions: Fast and safe medical care in patients during systemic thrombolysis in the MRI scanner is feasible. Despite the process of obtaining informed consent, with a dedicated and experienced stroke team the door-to-needle time can be kept in a range recommended by current guidelines. Continuous real-time information about the dynamics of cerebral perfusion from ASL perfusion in acute stroke patients undergoing thrombolysis may provide additional information for the understanding of the events following acute arterial obstruction and its course., (Copyright © 2013 S. Karger AG, Basel.)

Multiple acute ischemic lesions in different hemispheres or vascular territories are mainly considered to be of proximal embolic origin. However, despite careful diagnostic work-up, the etiological classification often stays undetermined. We propose that multiple acute ischemic lesions can sometimes be a phenomenon observed in small vessel disease (SVD). From a prospectively collected database of more than 7,000 stroke patients, 173 patients with acute bihemispheric infarction were identified. We analyzed those subjects with multiple small (< 15 mm Ø) subcortical acute ischemic lesions on diffusion-weighted MRI (DWI) and concomitant severe small vessel disease (Fazekas grades II-III) without a proximal embolic source as evaluated by cardiological investigations. Twenty patients (mean age 66 ± 12 years, 12 men) with a mean number of 2.95 ± 1.24 acute lesions on DWI (range of 2-7 lesions per patient) were identified (n = 5 Fazekas II°, n = 15 Fazekas III°). Most of the lesions were located in typical areas of lacunar infarction. The mean NIHSS score on admission was 2.95 ± 2.0 (range 0-8). Multiple acute ischemic lesions in different vascular territories might not always be of proximal cardiovascular embolic origin. Simultaneous small subcortical ischemic lesions may reflect remote ischemia due to small vessel disease reflecting simultaneous hemorheological dysfunction.

Precise analysis of stroke subtypes is important for clinical treatment decisions, the prognostic evaluation of patients, as well as defining stroke populations in clinical studies. The TOAST classification is the most widely used and approved form for etiologic subtyping. Increasing knowledge about stroke mechanisms and the introduction of new diagnostic techniques have supported the promotion of the new ASCO phenotypic classification, which aims to characterize patients using different grades of evidence for stroke subtypes. We prospectively assigned 103 consecutive patients from our stroke center for subtype classification using ASCO and TOAST. Clinical features and complementary investigations were recorded according to our standardized acute stroke care protocol. Evidence grade 1 with ASCO was assessed in 12.62% for large artery disease (A), 23.30% small-vessel disease (S), 36.89% cardiac source (C) and 1.94% another cause (O). Evidence grades 1-3 were identified in 60.19% A, 75.73% S, 49.51% C, and 3.88% O. A total of 68.93% of the patients were classified in more than one category, and only 3.88% remained completely undetermined. The κ value for inter-rater agreement was 0.92-1. Using TOAST, the distribution was 9.71% A, 23.30% S, 34.95% C, 1.94% O, and 30.10% undetermined. The ASCO classification showed a good concordance with TOAST. The inter-rater agreement was high. The comprehensive character of ASCO allows the recording of important additional information. This may be helpful for a specific treatment adaptation in each individual patient and creation of different etiological profiles in view of adapted clinical trials.

Since decision-making for thrombolysis in acute stroke settings is restricted to a limited time window and based on clinical assessment and CT findings only, thrombolysis is sometimes applied to patients with a final diagnosis other than a stroke. From a prospectively collected stroke/MRI data bank (2004-2010) with 648 suspected ischemic stroke patients treated with rtPA, we identified patients without evidence of acute infarction on follow-up MRI and a final diagnosis other than a stroke or acute cerebrovascular event. We compared demographics, symptoms, complications, and outcome of patients with stroke mimics (SM) to those with acute infarction. In 42 patients, an SM was diagnosed: seizures in 20, conversion disorder in seven, dementia in six, migraine in three, brain tumor in two, and others in four patients. Patients with SM less often had typical stroke symptoms like dysarthria (p < 0.01), facial palsy (p < 0.001), hemiparesis (p < 0.001), horizontal gaze palsy (p < 0.001), and visuospatial neglect (p = 0.03), while aphasia (p = 0.004) and accompanying convulsions (p = 0.01) occurred more often. Independent predictors of SM were known cognitive impairment, aphasia, and accompanying convulsions. Thrombolysis-related complications (orolingual angioedema) occurred in one SM patient and none of the SM patients deteriorated clinically. Stroke mimics comprise neurological/psychiatric disorders and differ from ischemic stroke patients with regard to the clinical presentation at onset. This might be helpful in deciding which patients should undergo acute stroke MRI to rule out SM, facilitate treatment decisions, and reduce the risk of unnecessary therapy.

In animal models of drug addiction, cocaine exposure has been shown to increase levels of calcium-permeable AMPA receptors (CP-AMPARs) in two brain regions that are critical for motivation and reward-the ventral tegmental area (VTA) and the nucleus accumbens (NAc). This review compares CP-AMPAR plasticity in the two brain regions and addresses its functional significance. In VTA dopamine neurons, cocaine exposure results in synaptic insertion of high conductance CP-AMPARs in exchange for lower conductance calcium-impermeable AMPARs (CI-AMPARs). This plasticity is rapid in onset (hours), GluA2-dependent, and can be observed with a single cocaine injection. Whereas it is short-lived after experimenter-administered cocaine, it persists for months after cocaine self-administration. In addition to strengthening synapses and altering Ca(2+) signaling, CP-AMPAR insertion alters subsequent induction of plasticity at VTA synapses. However, CP-AMPAR insertion is unlikely to mediate the increased DA cell activity that occurs during early withdrawal from cocaine exposure. Metabotropic glutamate receptor 1 (mGluR1) exerts a negative influence on CP-AMPAR accumulation in the VTA. Acutely, mGluR1 stimulation elicits a form of LTD resulting from CP-AMPAR removal and CI-AMPAR insertion. In medium spiny neurons (MSNs) of the NAc, extended access cocaine self-administration is required to increase CP-AMPAR levels. This is first detected after approximately a month of withdrawal and then persists. Once present in NAc synapses, CP-AMPARs mediate the expression of incubation of cue-induced cocaine craving. The mechanism of their accumulation may be GluA1-dependent, which differs from that observed in the VTA. However, similar to VTA, mGluR1 stimulation removes CP-AMPARs from MSN synapses. Loss of mGluR1 tone during cocaine withdrawal may contribute to CP-AMPAR accumulation in the NAc. Thus, results in both brain regions point to the possibility of using positive modulators of mGluR1 as treatments for cocaine addiction.

Our aim was to analyze an important subgroup represented by young adult patients (19-45 years) with acute ischemic stroke according to stroke classification (including the novel ASCO score), infarct types and risk factors. All patients up to 45 years of age with an acute ischemic stroke confirmed by MRI and treated in our stroke unit from 2006 to 2009 were recruited for this study. Patients were neurologically examined and underwent thorough stroke work-up. One hundred four patients (58 women, 46 men) with a mean age of 38 ± 6.9 years were evaluated. The mean NIHSS score (±SD) was 3 ± 5 on admission and 1 ± 4 on discharge. The classification using TOAST/ASCO (grade 1) was as follows: Macroangiopathic 10.6%/8.7%, cardiac origin 21.2%/10.6%, microangiopathic 9.6%/9.6%, other causes 19.2%/13.5% and undetermined 39.4%/19.2% (for A0S0C0O0). The most common risk factors were smoking (55.2%), hypertension (31.4%) and hyperlipidemia (27.6%). Twenty nine of 74 patients with TEE (39.2%) had a patent foramen ovale (PFO). Hypoplastic posterior circulation (21.9%) and migraine (21.0%) were also quite common. Young adult ischemic stroke patients share many of the characteristic risk factors with the general elderly ischemic stroke population. If regular work-up includes TEE, a high percentage of young patients reveal comorbidities with PFO, hypoplasia of the posterior circulation and migraine. The ASCO classification should be favored for a better classification of coexisting stroke subtypes and lower number of undetermined etiologies in this patient group.

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then, cell surface-expressed proteins are covalently cross-linked using the membrane-impermeable, bifunctional cross-linker bis(sulfosuccinimidyl)suberate (BS(3)). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency, and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after cross-linking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants., (© 2012 by John Wiley & Sons, Inc.)

Since many years, clinical decisions about the management of patients with carotid stenosis have been based on the distinction between "asymptomatic" and "symptomatic" presentations. This was also reflected by the design of previous studies on the surgical versus conservative treatment and of current studies on interventional treatment versus surgery. Both terms, however, only address different phases of activity of the one and the same condition and blur the significant message that carotid stenosis is a most important marker of systemic atherosclerosis, which is accompanied by a much higher risk of cardiovascular events rather than stroke. As a consequence, early diagnosis and followup during best medical treatment, life-style management, regular cardiovascular assessment, and good control of all vascular risk factors should be recommended in all patients with carotid stenosis-whether identified in the long-lasting "silent" or short-lasting "vulnerable" period lasting only a few weeks after cerebral ischemia. Patients in this short time window benefit from additional carotid intervention, under the condition of an individually favorable benefit-risk ratio ("individual vulnerability").

We previously showed that the time-dependent intensification ("incubation") of cue-induced cocaine seeking after withdrawal from extended-access cocaine self-administration is accompanied by accumulation of Ca(2+)-permeable AMPA receptors (CP-AMPARs) in the rat nucleus accumbens (NAc). These results suggest an enduring change in Ca(2+) signaling in NAc dendritic spines. The purpose of the present study was to determine if Ca(2+) signaling via NMDA receptors (NMDARs) is also altered after incubation. Rats self-administered cocaine or saline for 10 days (6 h/day). After 45-47 days of withdrawal, NMDAR-mediated Ca(2+) entry elicited by glutamate uncaging was monitored in individual NAc dendritic spines. NMDAR currents were simultaneously recorded using whole cell patch clamp recordings. We also measured NMDAR subunit levels in a postsynaptic density (PSD) fraction prepared from the NAc of identically treated rats. NMDAR currents did not differ between groups, but a smaller percentage of spines in the cocaine group responded to glutamate uncaging with NMDAR-mediated Ca(2+) entry. No significant group differences in NMDAR subunit protein levels were found. The decrease in the proportion of spines showing NMDAR-mediated Ca(2+) entry suggests that NAc neurons in the cocaine group contain more spines which lack NMDARs (non-responding spines). The fact that cocaine and saline groups did not differ in NMDAR currents or NMDAR subunit levels suggests that the number of NMDARs on responding spines is not significantly altered by cocaine exposure. These findings are discussed in light of increases in dendritic spine density in the NAc observed after withdrawal from repeated cocaine exposure.

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 Ca²⁺-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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Following prolonged withdrawal from extended access cocaine self-administration in adult rats, high conductance Ca2+ -ermeable AMPA receptors (CP-AMPARs) accumulate in nucleus accumbens (NAc) synapses and mediate the expression of "incubated" cue-induced cocaine craving. Using patch-clamp recordings from NAc slices prepared after extended access cocaine self-administration and >45 d of withdrawal, we found that group I metabotropic glutamate receptor (mGluR) stimulation using 3,5-dihydroxyphenylglycine (DHPG; 50 μm) rapidly eliminates the postsynaptic CP-AMPAR contribution to NAc synaptic transmission. This is accompanied by facilitation of Ca2+ -impermeable AMPAR (CI-AMPAR)-mediated transmission, suggesting that DHPG may promote an exchange between CP-AMPARs and CI-AMPARs. In saline controls, DHPG also reduced excitatory transmission but this occurred through a CB1 receptor-dependent presynaptic mechanism rather than an effect on postsynaptic AMPARs. Blockade of CB1 receptors had no significant effect on the alterations in AMPAR transmission produced by DHPG in the cocaine group. Interestingly, the effect of DHPG in the cocaine group was mediated by mGluR1 whereas its effect in the saline group was mediated by mGluR5. These results indicate that regulation of synaptic transmission in the NAc is profoundly altered after extended access cocaine self-administration and prolonged withdrawal. Furthermore, they suggest that activation of mGluR1 may represent a potential strategy for reducing cue-induced cocaine craving in abstinent cocaine addicts.

Engineered viral vectors tagged with a fluorescent protein such as enhanced green fluorescent protein (EGFP) have been widely used to study neuronal function after intracranial injection into specific brain regions. A rapid dissection of the virus-expressing region is required for certain biochemical analyses. To improve the accuracy of the rapid dissection, we developed a method that employs a Bluestar flashlight in combination with barrier filter glasses to visualize the expression of EGFP lentivirus that has been microinjected into the nucleus accumbens. Processing of dissected tissue for EGFP immunoblotting further validated the approach., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the rodent nucleus accumbens (NAc), cocaine elevates levels of brain-derived neurotrophic factor (BDNF). Conversely, BDNF can augment cocaine-related behavioral responses. The latter could reflect enhancement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) transmission, because AMPARs in the NAc mediate some cocaine-induced behaviors. Furthermore, in vitro studies in other cell types show that BDNF can promote AMPAR synaptic delivery. In this study, we investigated whether BDNF similarly promotes AMPAR trafficking in the adult rat NAc. After unilateral intracranial injection of BDNF into NAc core or shell, rats were killed at post-injection times ranging from 30 min to 3 days. NAc core or shell tissue from both injected and non-injected hemispheres was analysed by Western blotting. A protein cross-linking assay was used to measure AMPAR surface expression. Assessment of tropomyosin receptor kinase B signaling demonstrated that injected BDNF was biologically active. BDNF injection into NAc core, but not NAc shell, led to a protein synthesis- and extracellular signal-regulated kinase-dependent increase in cell surface GluA1 and a trend towards increased total GluA1. This was detected 30 min post-injection but not at longer time-points. GluA2 and GluA3 were unaffected, suggesting an effect of BDNF on homomeric GluA1 Ca(2+) -permeable AMPARs. These results demonstrate that exogenous BDNF rapidly increases AMPAR surface expression in the rat NAc core, raising the possibility of a relationship between increases in endogenous BDNF levels and alterations in AMPAR transmission observed in the NAc of cocaine-experienced rats., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Repeated noncontingent cocaine injections, which lead to behavioral sensitization, increase AMPA receptor (AMPAR) transmission in the rodent nucleus accumbens (NAc) in a withdrawal-dependent manner. On withdrawal days (WD) 10-21, this is attributable to upregulation of GluA1A2-containing AMPARs. However, synaptic incorporation of GluA2-lacking/Ca(2+)-permeable AMPARs (CP-AMPARs) was observed after longer withdrawal (WD35) from repeated noncontingent cocaine injections in young mice (Mameli et al., 2009). CP-AMPARs had previously been observed in NAc synapses only after prolonged (WD30-WD47) withdrawal from extended-access cocaine self-administration. Our goal was to determine whether rats receiving repeated noncontingent cocaine injections during adulthood similarly exhibit CP-AMPARs in the NAc after prolonged withdrawal. For comparison, we began by evaluating CP-AMPARs on WD35-WD49 after extended-access cocaine self-administration. Confirming our previous results, whole-cell recordings revealed inwardly rectifying AMPAR EPSCs, a hallmark of CP-AMPARs. This was observed in both core and shell. Next, we conducted the same analysis in adult rats treated with eight daily noncontingent cocaine injections and recorded on WD35-WD49. AMPAR EPSCs in core and shell did not show inward rectification and were insensitive to 1-naphthylacetylspermine (a selective antagonist of CP-AMPARs). Locomotor sensitization could still be demonstrated after this long withdrawal period, although the upregulation of GluA1A2-containing AMPARs observed at earlier withdrawal times was no longer detected. In conclusion, in adult rats, accumulation of synaptic CP-AMPARs in the NAc occurs after prolonged withdrawal from extended-access cocaine self-administration but not after prolonged withdrawal from noncontingent cocaine injections.

We characterized the distribution of AMPA receptor (AMPAR) subunits and the transmembrane AMPA receptor regulatory proteins (TARPs) γ-2 and γ-4 in adult rat nucleus accumbens (NAc) using a method that separates plasma membranes into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions. We also measured GluA1 phosphorylated at serine 845 (pS845 GluA1) and serine 831 (pS831 GluA1). GluA1-3 protein levels and pS831 GluA1/total GluA1 were higher in synaptic membranes. However, pS845 GluA1/total GluA1 was higher in extrasynaptic membranes, consistent with a role for S845 phosphorylation in GluA1 insertion at extrasynaptic sites. Homeric GluA1 receptors were detected in extrasynaptic membranes, consistent with evidence for extrasynaptic Ca(2+)-permeable AMPARs in other systems. The TARP γ-2 was enriched in synaptic membranes, whereas γ-4 was mainly found in extrasynaptic membranes, suggesting distinct roles for these proteins in the NAc. These experiments provide fundamental information that will aid in the interpretation of studies on AMPAR-related plasticity in the NAc., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

PCA stroke was under-represented in or excluded from the clinical trials examining thrombolysis based on the PWI-DWI mismatch concept. We present 6 patients with PCA stroke treated with thrombolysis in an extended time window by using MR imaging criteria. Symptoms included aphasia, sensorimotor hemiparesis, hemineglect, and homonymous hemianopia. Initial MR imaging demonstrated circumscribed ischemic lesions in the thalamus or hippocampus; MR angiography showed PCA occlusion with corresponding hypoperfusion. Follow-up MR imaging showed partial/complete recanalization in 4 patients with minor infarction growth, while in 1 patient, PCA occlusion persisted, resulting in a large PCA infarction. Three patients improved within 2 hours; at discharge, homonymous hemianopia had resolved in 3 patients. At 3-month follow-up, 4 patients had an mRS score of 0 or 1. These results support the approach to treat patients with PCA stroke with thrombolysis based on the mismatch concept. Because rehabilitation options for hemianopia are limited, thrombolysis may enhance the chance of a favorable outcome.

The subunit composition of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) is an important determinant of AMPAR biophysical properties and trafficking. To date, AMPAR subunit composition has been quantitatively evaluated only for the hippocampus, where different experimental approaches have yielded different results. Here, we used quantitative co-immunoprecipitation to characterize GluA1-3 associations in the adult rat nucleus accumbens, dorsal striatum, prefrontal cortex, and hippocampus, and blue native electrophoresis (BNE) to study GluA1-3 assembly state. In all brain regions, co-immunoprecipitation experiments showed that ~90% of GluA1 was associated with GluA2 or GluA3 (most was GluA1A2). All regions contained a small number of GluA1A3 receptors. Homomeric GluA1 receptors may also exist. More than half of the GluA2 (53%-65% depending on the region) was not associated with GluA1. However, this represents an over-estimate of the percent of GluA2 present in GluA2A3 receptors, based on BNE results demonstrating that the majority of GluA2 exists as dimers, rather than functional tetrameric receptors. Relatively more GluA1 was present in tetramers. Together with other findings, our results suggest a dominant role for GluA1A2 receptors in all brain regions examined. They also help explain why different results for hippocampal AMPAR subunit composition were obtained using co-immunoprecipitation, which assesses the total cellular pool of AMPARs including partially assembled AMPARs in intracellular compartments, and electrophysiological approaches, which can selectively assess tetrameric (functional) AMPARs on the cell surface., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Changes in α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) surface expression in the rodent nucleus accumbens (NAc) are produced by cocaine exposure and implicated in addiction-related behaviors. The direction of change depends on the animal's prior drug history. However, little is known about the effect of a single exposure to cocaine on AMPAR distribution in the NAc of untreated rats. This is essential information for interpreting the literature on AMPAR trafficking after repeated cocaine exposure. In this study, we used a protein crosslinking assay to determine the effect of a single cocaine injection on surface and intracellular AMPAR subunit levels in the rat NAc. We found increased AMPAR surface expression in the NAc 24 h, but not 30 min or 2 h, after cocaine injection. A major effect of cocaine is to increase extracellular dopamine (DA) levels, leading to DA receptor activation. Therefore, we also evaluated the effects of directly acting DA receptor agonists. In contrast to the effects of cocaine, AMPAR surface expression was significantly decreased 24 h after injection of the D2-class agonist quinpirole, whereas no significant effects were produced by the D1-class agonist SKF 81297 or the mixed DA agonist apomorphine. Our results show that the effects of a single cocaine exposure in drug- and injection-naïve rats are distinct from those previously reported after repeated cocaine administration. They further suggest that cocaine exerts these effects by influencing neuronal circuits rather than simply stimulating NAc DA transmission., (Copyright © 2010 Wiley-Liss, Inc.)

Changes in the levels or biochemistry of cerebrospinal fluid (CSF) neuropeptides with opioid-like properties have been suggested to reflect alterations in specific biological processes. We have determined various kinetic parameters for methionine-enkephalin (MET) degradation by CSF samples from nonneurological patients. Study subjects included 9 males (51-67 years of age) and 5 females (47-61 years of age). Aliquots, removed from an incubation vessel containing buffer, CSF, and peptide [tyr-3',5'-H(N)MET], were analyzed for tyrosine and other degradation products. Essentially all of the labeled tyrosine from the added MET was recovered as free amino acid after 60 minutes of incubation (1:2 ratio, vol:vol; optimum pH 7.4; and temperature 37°C); other possible peptide metabolites (>3%) were not detected. Irrespective of age or gender, the peptide's degradation half-life and initial velocity values were in a limited range; t1/2 26.2 ± 5.5 and 20.8-33.8 minutes, and Iv 0.03 ±0.01 and 0.02-0.03 pg of peptide per milligram protein per minute. Km and Vmax values were 0.19 ± 0.02 and 0.17-0.21 mM, and 9.8 ± 2.2 and 7.6-12.0 μmol·L·min, respectively. Neither CSF sample storage time (up to a year) nor repeated freezing and thawing (up to 3 times over a year) altered the kinetics or products of this reaction. These preliminary findings might serve as reference values when conducting similar studies using CSF from patients diagnosed with specific neurological conditions; significant alterations in MET degradation profile in such a population could provide valuable biological markers for diagnostic and treatment purposes.

Current diagnostic criteria for headaches are based on the International Classification for Headache Disorders, second edition, which is largely built on data obtained from clinical examinations and patients' medical histories. Despite decades of vigorous basic and clinical research, we still lack reliable clinical laboratory diagnostic markers for headaches, which clearly obstructs the physician's ability to optimize and follow the patient's response to treatment protocols as well as holds back the discovery and implementation of new therapeutic modalities. In this paper, we review and discuss current efforts to identify and characterize biochemical and immunological changes in biological fluids and tissue that may be specifically associated with the etiology and/or pathophysiology of migraine and cluster headaches; we also discuss some of the recent genetic findings and ion channel modulation studies that may help to distinguish among various headache populations.

This review focuses on cocaine-induced postsynaptic plasticity in the nucleus accumbens (NAc) involving changes in AMPA receptor (AMPAR) transmission. First, fundamental properties of AMPAR in the NAc are reviewed. Then, we provide a detailed and critical analysis of literature demonstrating alterations in AMPAR transmission in association with behavioral sensitization to cocaine and cocaine self-administration. We conclude that cocaine exposure leads to changes in AMPAR transmission that depend on many factors including whether exposure is contingent or non-contingent, the duration of withdrawal, and whether extinction training has occurred. The relationship between changes in AMPAR transmission and responding to cocaine or cocaine-paired cues can also be affected by these variables. However, after prolonged withdrawal in the absence of extinction training, our findings and others lead us to propose that AMPAR transmission is enhanced, resulting in stronger responding to drug-paired cues. Finally, many results indicate that the state of synaptic transmission in the NAc after cocaine exposure is associated with impairment of AMPAR-dependent plasticity. This may contribute to a broad range of addiction-related behavioral changes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Nucleus accumbens (NAc) neurons are excited primarily by AMPA-type glutamate receptors (AMPAR). This is required for cocaine seeking in animal models of cocaine addiction, suggesting AMPAR transmission in the NAc as a key control point for cocaine-related behaviors. This review will briefly describe AMPAR properties and trafficking, with a focus on studies in NAc neurons, and then consider mechanisms by which cocaine may alter AMPAR transmission. Two examples will be discussed that may be important in two different stages of addiction: learning about drugs and drug-related cues during the period of drug exposure, and persistent vulnerability to craving and relapse after abstinence is achieved. The first example is drawn from studies of cultured NAc neurons. Elevation of dopamine levels (as would occur following cocaine exposure) facilitates activity-dependent strengthening of excitatory synapses onto medium spiny neurons, the main cell type and projection neuron of the NAc. This occurs because activation of D1-class dopamine receptors primes AMPAR for synaptic insertion. This may create a temporal window in which stimuli related to cocaine-taking are more efficacious at eliciting synaptic plasticity and thus being encoded into memory. The second example involves rat models of cocaine addiction. Cell surface and synaptic expression of AMPAR on NAc neurons is persistently increased after withdrawal from repeated cocaine exposure. We hypothesize that this increases the reactivity of NAc neurons to glutamate inputs from cortex and limbic structures, facilitating the ability of these inputs to trigger cocaine seeking and thus contributing to the persistent vulnerability to relapse that characterizes addiction.

Activation of medium spiny neurons (MSNs) of the nucleus accumbens is critical for goal-directed behaviors including cocaine seeking. Studies in cocaine-experienced rodents have revealed three major categories of neuroadaptations that influence the ability of glutamate inputs to activate MSNs: changes in synaptic AMPA receptor levels, changes in extracellular non-synaptic glutamate levels and changes in MSN intrinsic membrane excitability. Most studies have focused on one of these adaptations. This review will consider the possibility that they are causally related and speculate about how time-dependent changes in their interactions may regulate MSN output during early and late withdrawal from repeated cocaine exposure., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Dopamine receptors (DARs) in the nucleus accumbens (NAc) are critical for cocaine's actions but the nature of adaptations in DAR function after repeated cocaine exposure remains controversial. This may be due in part to the fact that different methods used in previous studies measured different DAR pools. In the present study, we used a protein crosslinking assay to make the first measurements of DAR surface expression in the NAc of cocaine-experienced rats. Intracellular and total receptor levels were also quantified. Rats self-administered saline or cocaine for 10 days. The entire NAc, or core and shell subregions, were collected one or 45 days later, when rats are known to exhibit low and high levels of cue-induced drug seeking, respectively. We found increased cell surface D1 DARs in the NAc shell on the first day after discontinuing cocaine self-administration (designated withdrawal day 1, or WD1) but this normalized by WD45. Decreased intracellular and surface D2 DAR levels were observed in the cocaine group. In shell, both measures decreased on WD1 and WD45. In core, decreased D2 DAR surface expression was only observed on WD45. Similarly, WD45 but not WD1 was associated with increased D3 DAR surface expression in the core. Taking into account many other studies, we suggest that decreased D2 DAR and increased D3 DAR surface expression on WD45 may contribute to enhanced cocaine-seeking after prolonged withdrawal, although this is likely to be a modulatory effect, in light of the mediating effect previously demonstrated for AMPA-type glutamate receptors., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) surface expression in the nucleus accumbens (NAc) is enhanced after withdrawal from repeated cocaine exposure. However, it is unclear whether this contributes to the expression of locomotor sensitization and whether similar changes can be observed in other striatal regions. In this study we examined the relationship between AMPAR surface expression in the NAc and locomotor sensitization. We also examined AMPAR distribution in the dorsolateral striatum (DS) and NMDA receptor (NMDAR) distribution in the NAc and DS. Trends but no significant changes in NMDAR distribution were found in the NAc after withdrawal. No NMDAR changes were observed in the DS. AMPAR surface expression was increased in the NAc 15 days after the last exposure to cocaine, but decreased in the DS. Re-exposure to cocaine on withdrawal day 14 decreased AMPAR surface expression in the NAc 24 h, but not 30 min, after challenge, but increased it in the DS 24 h and 30 min after challenge. Locomotor sensitization was evaluated at times associated with increased or decreased AMPAR surface expression in the NAc. The magnitude of sensitization did not vary with changes in the level of AMPAR surface expression, nor was it significantly reduced by decreasing AMPAR transmission through intra-NAc infusion of CNQX before cocaine challenge. On the basis of our results, and other findings, we suggest that the expression of sensitization has no clear relationship to altered AMPAR surface expression in the NAc, although the latter may have a role in the enhanced pursuit and self-administration of drugs observed in sensitized rats.

Background: In large territorial stroke of the anterior and the posterior circulation, the extent of affected tissue can be characterized by the demonstration of vessel occlusion on MR angiography (MRA), while the extent of hypoperfusion can be shown on dynamic susceptibility contrast perfusion-weighted MRI (PWI). The ability of MRA and conventional MRI sequences to demonstrate branches of the basilar artery (BA) is very limited. This study analyzes the value of the combined use of diffusion-weighted MRI (DWI), MRA and PWI in acute pontine stroke., Methods: A series of 24 consecutive patients with acute pontine stroke received an extensive MRI stroke workup including DWI, PWI and MRA., Results: In 11/24 patients visual analysis of PWI demonstrated persisting hypoperfusion, and in 1/24 patients indication of hyperperfusion was found. Vessel abnormalities were seen in 19/24 patients (15/24 hypoplastic vertebral artery, 9/24 stenosis or occlusion of the BA, 1/20 ectatic BA). Persistent pontine hypoperfusion was more frequently associated with BA pathology (9/11 vs. 1/13, p = 0.001), large-vessel disease (8/11 vs. 1/13; p = 0.001) and a more pronounced clinical deficit (NIHSS score on day 1: 7 vs. 3, p = 0.01)., Conclusions: In pontine ischemia areas of hypoperfusion can be identified due to the strong contrast induced by ischemia on PWI and can be easily related to DWI lesion size. This is of use particularly as small vessels are frequently missed by MRA and occlusion of the BA can be better characterized with the help of PWI., ((c) 2010 S. Karger AG, Basel.)

Select phenothiazine drugs significantly decrease, in a dose-dependent manner, the rate of methione-enkephalin (MET) degradation by discrete human brain areas, for example, putamen and hippocampus. This pentapeptide is rapidly, and essentially completely, hydrolyzed at the tyrosine-glycine bond by bacitracin-sensitive aminopeptidase(s) (AP); neither dipeptidyl peptidase(s) (N-carboxyphenylmethyl leucine and captopril) nor peptidyl dipeptidase(s) (thiorphan) inhibitors altered the kinetics of MET degradation. Half-life (t1/2) and initial velocity (Iv) of this reaction were significantly increased and decreased, respectively, by fluphenazine > prochlorpherazine > chlorpromazine > thioridazine > promethazine > ethopropazine; brain A hippocampus (t1/2, control 2.8 and 60.2, 22.9, 15.4, 10.0, 9.9, and 4.8 minutes; and Iv, control 59.0 and 3.1, 10.7, 21.3, 22.8, 23.8, and 36.9 pg MET/mg brain tissue/minute) and putamen (t1/2, control 2.5 and 52.4, 19.9, 12.4, 8.2, 8.4, and 4.1 minutes; and Iv, control 53.1 and 2.7, 9.3, 17.1, 18.6, 20.1, and 31.7 pg MET/mg brain tissue/minute). Bacitracin was used for comparison purposes; hippocampus (t1/2 and Iv of 64.2 minutes and 4.3 pg MET/mg brain tissue/minute, respectively). Results using brain B tissue followed a comparable pattern, providing similar results. Hippocampus and putamen samples from both brains showed similar Km (mean 25.2, muM; range, 23.3-27.2 mum), Vmax (mean 108 nmol/mg protein/minute; range, 103-115 nmol/mg), and IC50 values (bacitracin, mean 14.4 muM; range, 13.8-14.7 mum) for MET AP degradation. Neither the phenothiazines methotrimeprazine or trifluoperazine nor other commonly used nonphenothiazine antipsychotic tested, for example, clozapine, haloperidol, loxapine, molindone, sulpiride and thiothixine, significantly altered the kinetics of this reaction. The presence of the phenothiazine molecule appears to be necessary for AP inhibition; however, our results failed to show s correlation among chemical structure, pharmacologic profile, and tested compound ability to inhibit MET degradation. This research provides initial information that could lead to the rational design of agents capable of modulate the bioavailability of enkephalins and other AP-metabolized biologically active compounds. Whether their development could find useful pharmacologic applications remains to be explored.

Increased reactivity of certain frontal cortical brain regions to cocaine re-exposure or drug-associated cues in cocaine-abstinent human addicts is linked to drug craving. Similarly, in rats tested after withdrawal from repeated cocaine exposure, cocaine or other strong excitatory stimuli produce greater activation of pyramidal neurons in the medial prefrontal cortex (mPFC). Our recent findings indicate that the increased mPFC neuronal activation depends primarily upon enhanced voltage-sensitive Ca(2+) influx, most likely through high-voltage activated (HVA) L-type Ca(2+) channels, but the mechanism underlying the enhanced Ca(2+) currents is unknown. In this study, we used a protein crosslinking assay to show that repeated cocaine injections, resulting in behavioral sensitization, increased total protein levels and cell surface expression of HVA-Ca(v)1.2 L-type channels in pyramidal neurons in deep layers of the mPFC. These changes in Ca(v)1.2 L-channels were time dependent and subtype specific (i.e., differed from those observed for Ca(v)1.3 L-channels). Furthermore, we found enhanced PKA activity in the mPFC of cocaine-sensitized rats that persisted for 21 days after withdrawal. PKA phosphorylation of L-channels increases their activity, so Ca(2+) currents after cocaine withdrawal could be enhanced as a result of both increased activity and number of HVA-Ca(v)1.2 L-channels on the cell surface. By increasing the suprafiring threshold excitability of mPFC pyramidal neurons, excessive upregulation of HVA L-channel activity and number may contribute to the cortical hyper-responsiveness that enhances vulnerability to cocaine craving and relapse. More generally, our results are the first to demonstrate that repeated cocaine exposure alters the membrane trafficking of a voltage-sensitive ion channel.

Synaptic scaling has been proposed as a form of plasticity that may contribute to drug addiction but it has not been previously demonstrated in the nucleus accumbens (NAc), a critical region for addiction. Here we demonstrate bidirectional synaptic scaling in postnatal rat NAc neurons that were co-cultured with prefrontal cortical neurons to restore excitatory input. Prolonged activity blockade (1-3 days) with an AMPA receptor antagonist increased cell surface (synaptic and extrasynaptic) glutamate receptor 1 (GluR1) and GluR2 but not GluR3, as well as GluR1/2 co-localization on the cell surface and total GluR1 and GluR2 protein levels. A prolonged increase in activity (bicuculline, 48 h) produced opposite effects. These results suggest that GluR1/2-containing AMPA receptors undergo synaptic scaling in NAc neurons. GluR1 and GluR2 surface expression was also increased by tetrodotoxin alone or in combination with an N-methyl-d-aspartate receptor or AMPA receptor antagonist but not by the l-type Ca(2+) channel antagonist nifedipine. A cobalt-quenching assay confirmed the immunocytochemical results indicating that synaptic scaling after activity blockade did not involve a change in abundance of GluR2-lacking AMPA receptors. Increased AMPA receptor surface expression after activity blockade required protein synthesis and was occluded by inhibition of the ubiquitin-proteasome system. Repeated dopamine (DA) treatment, which leads to upregulation of surface GluR1 and GluR2, occluded activity blockade-induced synaptic scaling. These latter results indicate an interaction between cellular mechanisms involved in synaptic scaling and adaptive mechanisms triggered by repeated DA receptor stimulation, suggesting that synaptic scaling may not function normally after exposure to DA-releasing drugs such as cocaine.

Behavioral sensitization is an animal model for aspects of cocaine addiction. Cocaine-sensitized rats exhibit increased AMPA receptor (AMPAR) surface expression in the nucleus accumbens (NAc) which may in turn enhance drug seeking. To identify signaling pathways contributing to AMPAR up-regulation, we measured AMPAR surface expression and signaling pathway activation in the NAc of cocaine-sensitized rats, cocaine-exposed rats that failed to sensitize and saline controls on withdrawal days (WD) 1, 7, and 21. We focused on calcium/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated protein kinase (ERK), and protein kinase A (PKA). In sensitized rats, AMPAR surface expression was elevated on WD7 and WD21 but not WD1. ERK2 activation followed a parallel time-course, suggesting a role in AMPAR up-regulation. Both sensitized and non-sensitized rats exhibited CaMKII activation on WD7, suggesting that CaMKII activation is not sufficient for AMPAR up-regulation. PKA phosphorylation, measured using an antibody recognizing phosphorylated PKA substrates, increased gradually over withdrawal in sensitized rats, from below control levels on WD1 to significantly greater than controls on WD21. Using proteomics, novel sensitization-related PKA substrates were identified, including two structural proteins (CRMP-2 and alpha-tubulin) that we speculate may link PKA signaling to previously reported dendritic remodeling in NAc neurons of cocaine-sensitized rats.

We examined whether behavioral sensitization to amphetamine is associated with redistribution of glutamate receptors (GluR) in the rat nucleus accumbens (NAc) or dorsolateral striatum (DLSTR). Following repeated amphetamine treatment and 21 days of withdrawal, surface and intracellular levels of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or NMDA receptor subunits were determined using a protein cross-linking assay. In contrast to our previous results in cocaine-sensitized rats, we did not observe redistribution of GluR1 or GluR2 to the cell surface in the NAc after amphetamine withdrawal, although a small increase in total GluR1 was found in the shell subregion. Nor did we observe activation of signaling pathways associated with cocaine-induced AMPA receptor trafficking or changes in NMDA receptor subunits. No significant changes were observed in the DLSTR. We also investigated the effect of administering a challenge injection of amphetamine to amphetamine-sensitized rats 24 h prior to biochemical analysis based on prior studies showing that cocaine challenge decreases AMPA receptor surface expression in the NAc of cocaine-sensitized rats. GluR1 and GluR2 were not significantly altered in either NAc or DLSTR, although a modest effect on GluR3 cannot be ruled out. Our results suggest that glutamate transmission in the NAc is dramatically different in rats sensitized to amphetamine versus cocaine.

Interactions between dopamine (DA) and glutamate systems in the prefrontal cortex (PFC) are important in addiction and other psychiatric disorders. Here, we examined DA receptor regulation of NMDA receptor surface expression in postnatal rat PFC neuronal cultures. Immunocytochemical analysis demonstrated that surface expression (synaptic and non-synaptic) of NR1 and NR2B on PFC pyramidal neurons was increased by the D1 receptor agonist SKF 81297 (1 microM, 5 min). Activation of protein kinase A (PKA) did not alter NR1 distribution, indicating that PKA does not mediate the effect of D1 receptor stimulation. However, the tyrosine kinase inhibitor genistein (50 microM, 30 min) completely blocked the effect of SKF 81297 on NR1 and NR2B surface expression. Protein cross-linking studies confirmed that SKF 81297 (1 microM, 5 min) increased NR1 and NR2B surface expression, and further showed that NR2A surface expression was not affected. Genistein blocked the effect of SKF 81297 on NR1 and NR2B. Surface-expressed immunoreactivity detected with a phospho-specific antibody to tyrosine 1472 of NR2B also increased after D1 agonist treatment. Our results show that tyrosine phosphorylation plays an important role in the trafficking of NR2B-containing NMDA receptors in PFC neurons and the regulation of their trafficking by DA receptors.

Relapse to cocaine use after prolonged abstinence is an important clinical problem. This relapse is often induced by exposure to cues associated with cocaine use. To account for the persistent propensity for relapse, it has been suggested that cue-induced cocaine craving increases over the first several weeks of abstinence and remains high for extended periods. We and others identified an analogous phenomenon in rats that was termed 'incubation of cocaine craving': time-dependent increases in cue-induced cocaine-seeking over the first months after withdrawal from self-administered cocaine. Cocaine-seeking requires the activation of glutamate projections that excite receptors for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the nucleus accumbens. Here we show that the number of synaptic AMPA receptors in the accumbens is increased after prolonged withdrawal from cocaine self-administration by the addition of new AMPA receptors lacking glutamate receptor 2 (GluR2). Furthermore, we show that these new receptors mediate the incubation of cocaine craving. Our results indicate that GluR2-lacking AMPA receptors could be a new target for drug development for the treatment of cocaine addiction. We propose that after prolonged withdrawal from cocaine, increased numbers of synaptic AMPA receptors combined with the higher conductance of GluR2-lacking AMPA receptors causes increased reactivity of accumbens neurons to cocaine-related cues, leading to an intensification of drug craving and relapse.

Postsynaptic interactions between dopamine (DA) and glutamate receptors in the nucleus accumbens (NAc) are critical for addiction. To determine the effect of acute and repeated DA receptor stimulation on AMPA receptor (AMPAR) synaptic targeting in medium spiny NAc neurons, we developed a model system consisting of rat NAc neurons cocultured with prefrontal cortex neurons from enhanced green fluorescent protein-expressing mice. Cortical neurons restore excitatory input onto NAc neurons but are distinguishable based on fluorescence. First, we showed that brief D1-like agonist exposure increased AMPAR insertion onto extrasynaptic regions of NAc neuronal processes through a mechanism requiring protein kinase A. This facilitated the Ca2+/calmodulin dependent protein kinase II (CaMKII)-dependent synaptic incorporation of AMPARs in response to subsequent NMDA receptor (NMDAR) stimulation. Through this mechanism, DA may promote reward- and drug-related plasticity in the NAc. Then, to model effects of repeated in vivo cocaine exposure, we treated cocultures with DA (1 microm, 30 min) on days 7, 9, and 11 in culture. On day 15, NAc neurons exhibited increased synaptic AMPAR levels. This was associated with CaMKII activation and was blocked by the CaMKII inhibitor KN-93 (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide phosphate salt). Furthermore, D1-like agonist exposure on day 15 no longer increased AMPAR surface expression. This refractoriness was associated with decreased D1 receptor surface expression. NMDAR surface expression was not altered by acute or repeated DA receptor stimulation. These results suggest that (1) after repeated DA treatment, NAc neurons are more responsive to glutamate inputs but D(1)-like receptor regulation of plasticity is impaired, and (2) NAc/prefrontal cortex cocultures are useful for studying dopamine-induced neuroadaptations.

Aims: To assess visual acuity (VA) objectively using visual evoked potentials (VEPs), avoiding subjective trace evaluation and providing an acuity estimate with associated confidence limits., Methods: 40 normal subjects and 24 patients (with corneal and retinal diseases, decimal VA range 0.15-1.1 (= 0.8(logMAR) to -0.04(logMAR))) participated in the study. Checkerboard stimuli with six check sizes covering 0.05-0.4 degrees (or 0.09-0.8 degrees for visual acuities below 0.35 (= 0.46(logMAR)) were presented in brief-onset mode (40 ms on, 93 ms off) at 7.5 Hz. In normal subjects, the stimuli were also optically degraded by frosted occluders resulting in a decimal VA range of 0.13-2.8 (= 0.9(logMAR) to -0.45(logMAR)). Altogether, 108 steady-state VEPs were recorded with a Laplacian montage (2xOz-(RO+LO)). Fourier analysis yielded the magnitude (A) at the stimulus frequency, and the average of the two neighboring frequencies as noise estimate (N). A and N determine the significance level p of the response, and from their ratio the non-noise-contaminated response (A*) can be calculated. Tuning curves were obtained by plotting A* vs the dominant spatial frequency of the corresponding checkerboard. A fully automatic algorithm used the significance level (p<5%) and A* to automatically select an appropriate region in the high spatial-frequency range on which a linear regression was performed, yielding a zero-amplitude extrapolated spatial frequency SF0. Subjective VA was obtained with the automated "Freiburg Acuity Test"., Results: The brief-onset presentation evoked high VEP amplitudes; however, many tuning curves displayed the well-known "notch" at intermediate check sizes. The fully automated analysis algorithm succeeded in 107 of 108 cases and effectively ignored the notch, if present. The relation between logVA and log(SF0) was a constant factor throughout the range tested: logVA = log(SF0)/17.6 cpd. In more than 95% of all cases, the acuity predicted from SF0 coincided within a factor of two (up and down, or +/-0.3 logMAR) with subjective VA with a coefficient of correlation of 0.90., Conclusion: The fully automated analysis avoided subjective problems in peak-trough assessment. The results provide quantitative limits to assess patients with possible malingering.

After 10-minute incubation of [3H]-tyrosine methionine-enkephalin (MET) with 100,000 x g supernatant from select brain regions of patients with chronic schizophrenia (n = 3), essentially all of the labeled tyrosine was recovered as the free amino acid. Initial velocity and half-life of MET degradation obtained from different brain areas (limbic system, thalamus, basal ganglia, cerebellum, and cortex) of individual brains or from equivalent sections from different brains were scattered and considerable spread out (brains A, B, and C: 21.7-60.2 and 2.1-14.3, 25.6-88.7 and 1.6-14.1, 24.5-56.1 and 2.6-14.3 pg MET/mg brain tissue/min and min, respectively; brains A-C range, 21.7-88.7 pg MET/mg brain tissue/min and 1.6-14.3 min, respectively). These results failed to identify consistent differences in peptide degradation kinetics between the various brains areas studied from the same individual or from equivalent section from different subjects. MET metabolic rate was pH and temperature-dependent (optimum 7.4 degrees C and 37 degrees C), reduced by the aminopeptidase inhibitors puromycin, bacitracin, and bestatin, and to a lesser extent by thioridazine. However, peptide metabolism was not significantly affected by differences in tissue storage time or repeated freezing and thawing; by preincubation with N-carboxymethyl phenyl leucine, captopril, or thiorphan (dipeptidyl peptidase[s] or peptidyl dipeptidase[s] inhibitors, respectively); or by the many different drugs used by the patients with chronic schizophrenia. Our findings, although of a preliminary nature and generally similar to those recently reported for comparable studies on nonneuropsychiatric patients, provide a much needed understanding of the mechanisms regulating brain MET metabolism. Whether these results may contribute to the rational design of pharmacologic strategies for the treatment of pathologies associated with alterations in the enkephalinergic system needs further research.

Incubation of [3H]-tyrosine methionine5-enkephalin (MET) with human brain preparations (100,000g supernatant; sections of the limbic system, thalamus, basal ganglia, cerebellum, and cortex) results in its rapid and complete degradation; over 95% of the initial labeled tyrosine is recovered as the free aminoacid within 10 min. Results show a considerable range in the peptide initial velocity (Iv) and half-life (t1/2) degradation values obtained from different brain sections of individual brains, either from the same or from different main brain areas. This relatively wide range of values was scattered, failing to identify consistent differences between the various brains areas studied. Differences in brain tissue storage time or repeated sample freezing and thawing failed to alter significantly either of these kinetic parameters of MET metabolism. Peptide degradation rate (optimum pH and temperature of 7.4 and 37 degrees C, respectively) was concentration-dependent inhibited by known aminopeptidase inhibitors (puromycin, bacitracin, and bestatin, and to a lesser extent by thioridazine). However, it was not significantly affected by either N-carboxymethyl phenyl leucine, captopril or thiorphan [dipeptidyl peptidase(s) or peptidyl dipeptidase(s) inhibitors, respectively]. A better understanding of the mechanisms regulating brain MET metabolism may contribute to the rational design of pharmacological strategies based in the modulation of its bioavailability.

Excitatory synapses onto dopamine (DA) neurons of the ventral tegmental area (VTA) represent a critical site of psychostimulant-induced synaptic plasticity. This plasticity involves alterations in synaptic strength through AMPA receptor (AMPAR) redistribution. Here, we report an in vitro model for studying regulation of AMPAR trafficking in DA neurons under control conditions and after elevation of DA levels, mimicking cocaine exposure. We used cocultures containing rat VTA neurons and prefrontal cortex (PFC) neurons from enhanced cyan fluorescent protein-expressing mice. In VTA-PFC cocultures, D1 receptor activation (10 min) increased synaptic and nonsynaptic glutamate receptor subunit 1 (GluR1) and GluR2 surface expression on DA neurons. NMDA or AMPA receptor antagonists blocked this effect, and it was not observed in pure VTA cultures, suggesting that DA agonists acted on D1 receptors on PFC neurons, altering their excitatory transmission onto VTA DA neurons and, thus, influencing AMPARs. To mimic the longer elevation in extracellular DA levels produced by systemic cocaine, cocultures were incubated with DA for 1 h. Synaptic GluR1 was increased 24 h later, reminiscent of the increased AMPA/NMDA ratio at excitatory synapses onto VTA DA neurons 24 h after cocaine injection (Ungless et al., 2001). In contrast, GluR2 was unchanged. Analysis of colocalization of surface GluR1-3 labeling suggested that control DA neurons express a substantial number of GluR1/2, GluR2/3, and homomeric GluR1 receptors and that the increase in surface AMPARs 24 h after DA exposure may in part reflect increased GluR1/3-containing receptors. These results help define the cellular basis for plasticity underlying the development of behavioral sensitization.

Although some studies report increased responsiveness of nucleus accumbens (NAc) AMPA receptors (AMPARs) after withdrawal from repeated cocaine treatment, others report decreased responsiveness after withdrawal plus cocaine challenge. Here we examine this apparent contradiction by quantifying cell surface and intracellular AMPAR subunits in the NAc before and after a challenge injection in behaviorally sensitized rats. Because MAPKs (mitogen-activated protein kinases) regulate AMPAR trafficking and are implicated in addiction, we also evaluated phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. Glutamate receptor 1 (GluR1) and GluR2 surface/intracellular (S/I) ratios were increased after 14 d of withdrawal in sensitized rats but were decreased 24 h after challenge with cocaine (which elicited a sensitized locomotor response) or saline (which elicited conditioned locomotion). These findings suggested redistribution of GluR1/2-containing receptors, a possibility supported by immunoprecipitation experiments indicating that most AMPARs in the NAc are GluR1/2 or GluR2/3, with few homomeric GluR1 or GluR1/3 receptors. In sensitized rats, ERK phosphorylation in the NAc increased during withdrawal and normalized after cocaine challenge. JNK phosphorylation also increased after withdrawal, but after cocaine challenge, it was inversely related to GluR1 and GluR2 S/I ratios. After saline challenge, p38 phosphorylation was increased. In summary, surface expression of GluR1/2-containing AMPARs increased in the NAc of sensitized rats, but AMPARs internalized after a single reexposure to cocaine or cocaine-related cues. ERK phosphorylation paralleled AMPAR surface expression. Although JNK results were complex, JNK and p38 may be involved in AMPAR internalization after cocaine or saline challenge, respectively.

A critical event in the development of behavioral sensitization is a transient increase in excitatory drive to dopamine neurons of the ventral tegmental area (VTA). This is likely to be due, in part, to the ability of drugs of abuse to produce long-term potentiation, expressed as increased AMPA receptor transmission, at excitatory synapses onto VTA dopamine neurons. We investigated the role of the laterodorsal tegmentum (LDT) in behavioral sensitization because LDT neurons provide an important source of excitatory drive to VTA dopamine neurons, through mixed glutamate and cholinergic inputs. To test the role of the LDT in amphetamine sensitization, ibotenic acid or sham lesions of the LDT were performed 1 week before the first of six daily amphetamine injections. When challenged with amphetamine 13 days after the last injection, sham rats expressed sensitization of stereotypy and post-stereotypy locomotor hyperactivity, whereas the latter was attenuated by ibotenic acid lesions of the LDT. To determine whether plasticity occurs in the LDT during amphetamine sensitization, we used a previously developed microdialysis assay in which increased ability of AMPA to activate a pathway serves as a marker for long-term potentiation. Two days after discontinuing repeated saline or amphetamine injections, the responsiveness of LDT-VTA neurons to AMPA was determined by microinjecting AMPA (0.4 nmol) into the LDT and measuring glutamate efflux in the ipsilateral VTA. Glutamate efflux was transiently increased in both groups but a delayed group difference was apparent with relatively higher glutamate efflux in amphetamine rats 30-60 min after AMPA injection. In parallel experiments, dopamine efflux in the nucleus accumbens (NAc) following intra-LDT AMPA declined in saline rats but remained relatively stable in amphetamine rats. Both results suggest relatively greater excitability of the LDT-VTA-NAc pathway after repeated amphetamine treatment. Our results provide the first evidence that neuronal plasticity in the LDT contributes to behavioral sensitization.

Background: Little is known about how childhood asthma affects immigrant Hispanic families in the United States. Qualitative research is effective for understanding the social, cultural, functional, and structural aspects of asthma in the family context. Furthermore, such knowledge is necessary to develop culturally appropriate interventions for these families., Objectives: To describe participants' perceptions of their roles in caring for an asthmatic child, to compare family patterns of caring for an asthmatic child by parents' country of origin, to identify barriers to caring for an asthmatic child, and to evaluate specific coping needs of low-income immigrant Hispanic families caring for an asthmatic child., Methods: Five focus groups were conducted with low-income, immigrant, Spanish-speaking Hispanic adults caring for an asthmatic child, including community health workers, mothers, fathers, and grandparents, along with women with asthma. Audiotaped focus groups were transcribed verbatim in Spanish, forward translated into English, and back translated into Spanish. Data analysis was performed using qualitative analytic methods., Results: Forty-one participants represented a range of countries of origin. Different themes emerged for community health workers vs parents and grandparents and for women vs men caring for a child with asthma. All the participants reported strong beliefs in using folk medicines. Barriers identified included language, culture, poverty, lack of health insurance, and poor living conditions., Conclusions: Results highlight the lack of asthma self-management skills, diagnostic uncertainty, and the use of folk medicine as factors that should be taken into consideration when tailoring interventions to improve asthma outcomes in this vulnerable population.

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.

Adaptations in dopamine (DA) transmission in the prefrontal cortex (PFC) are thought to be critical to the development and persistence of drug addiction. Our previous findings showed that medial PFC (mPFC) neurons in rats treated repeatedly with amphetamine exhibit a decreased inhibitory response to iontophoretically applied DA, demonstrating altered DA receptor transmission. To determine the role postsynaptic DA D1 receptors play in this effect, we used whole-cell patch-clamp recordings of acutely dissociated pyramidal mPFC neurons and inhibition of transient voltage-sensitive sodium current (INaT) as a measure of D1 receptor function. After 3 d of withdrawal, neurons recorded from amphetamine-treated rats (5 mg/kg for 5 d) demonstrated a significant decrease in whole-cell INaT density and in the ability of D1 receptor stimulation to inhibit INaT. Application of a protein kinase A (PKA) inhibitor blocked the ability of D1 receptor activation to inhibit INaT and increased the current density of both groups to similar values. These results suggest that repeated amphetamine exposure results in subsensitivity of the INaT to D1 receptor-mediated inhibition because of a possible increase in basal PKA activity. This adaptation may contribute to perseverative behaviors in animals that self-administer psychostimulants as well as compromised PFC-dependent behaviors in human addicts.

Regulation of AMPA receptor trafficking is important for many forms of neuronal plasticity. In this study, a protein cross-linking assay was used to evaluate the contribution of AMPA receptor trafficking to plasticity associated with behavioral sensitization, an animal model of drug addiction. Cross-linking was used to distinguish between cell surface and intracellular AMPA receptors in nucleus accumbens (NAc) tissue obtained from rats treated repeatedly with saline or cocaine. Surface/intracellular (S/I) ratios for glutamate receptor 1 (GluR1) and GluR2/3 subunits were increased 21 d after the last injection in cocaine-sensitized rats but not rats that failed to sensitize, and the magnitude of the S/I ratio for cocaine-sensitized rats was positively correlated with the magnitude of behavioral sensitization. At the 1 d withdrawal time, cocaine did not alter S/I ratios, and there was no correlation between S/I ratios and behavioral sensitization. The majority of surface-expressed GluR1 detected with this assay was associated with synapses, based on coimmunoprecipitation with postsynaptic density protein of 95 kDa. These findings suggest that behavioral sensitization to cocaine is associated with a slowly developing redistribution of AMPA receptors to the surface of NAc neurons. Motor execution of drug-seeking responses depends on activation of AMPA receptors on NAc neurons by glutamate afferents originating in cortical and limbic regions. We propose that drug-seeking responses are more effectively triggered in cocaine-sensitized rats because of increased cell surface expression of AMPA receptors.

Addiction is believed to involve glutamate-dependent forms of synaptic plasticity that promote the formation of new habits focused on drug seeking. We used primary cultures of rat prefrontal cortex (PFC) neurons to explore mechanisms by which dopamine-releasing psychomotor stimulants such as cocaine and amphetamine influence synaptic plasticity, focusing on AMPA receptor trafficking because of its key role in long-term potentiation (LTP). Brief stimulation of D1 dopamine receptors increased surface expression of glutamate receptor 1 (GluR1)-containing AMPA receptors through a protein kinase A-dependent mechanism, by increasing their rate of externalization at extrasynaptic sites. Newly externalized GluR1 remained extrasynaptic under basal conditions but could be translocated into synapses by subsequent NMDA receptor activation. These results suggest that D1 receptors may facilitate LTP by increasing the AMPA receptor pool available for synaptic insertion. However, stimulation of D2 receptors decreased surface and synaptic GluR1 expression. These findings are discussed in the context of evidence that D1 and D2 receptors act independently rather than antagonistically in the intact PFC. D1 receptor facilitation of AMPA receptor synaptic insertion helps explain D1 receptor-dependent facilitation of LTP and learning in the normal brain. Abnormal engagement of this mechanism during unregulated dopamine release may account for maladaptive plasticity after repeated exposure to cocaine or amphetamine.

We identified a number of clinically used drugs and biologically active endogenous peptides able to significantly decrease the rate of human plasmatic aminopeptidase (AP) leucine-enkephalin (LEU) degradation. Bacitracin, bestatin, fluvoxamine, and each of 4 peptides tested significantly increased, in a dose-dependent manner (10-10 M), LEU degradation half-life (t1/2) in each of 5 plasma samples studied. Each sample was obtained by pooling equal volume of 6 randomly selected, individual plasmas (4 male and 2 female healthy, drug-free volunteers). Thirty subjects (20 females and 10 males) participated in this study. With the exception of fluvoxamine, this inhibitory effect was lacking in various other commonly used drugs with widely different chemical structures and pharmacological profiles, eg, antidepressants (SSRIs, imipramine-like tricyclics, MAOIs), acute antimigraine agents (triptan class drugs), the nonselective beta-adrenergic antagonist propranolol, and serotonin receptor agonists and antagonists. Agents (concentration 10 M used as illustration), listed in decreasing order of LEU-AP inhibitory activity: substance P > angiotensin III > methionine-enkephalin > angiotensin II > fluvoxamine > bestatin gave t1/2 values (+/- SD) of 39.3 +/- 1.1, 29.4 +/- 0.8, 28.3 +/- 0.8, 27.4 +/- 0.7, 24.5 +/- 1.5, and 23.6 +/- 0.9 minutes, respectively. Control, bacitracin, and fluphenazine (known LEU-AP inhibitors were used for comparison) values of 11.8 +/- 1.0, 31.3 +/- 0.7, and 19.6 +/- 1.0 minutes, respectively. As expected, these drugs significantly decreased the initial velocity of peptide degradation; Iv values (+/- SD) of: 0.17 +/- 0.1 (0.02 +/- 0.01), 0.23 +/- 0.2 (0.02 +/- 0.01), 0.25 +/- 0.2 (0.02 +/- 0.01), 0.26 +/- 0.2 (0.03 +/- 0.01), 0.31 +/- 0.1 (0.03 +/- 0.01), and 0.33 +/- 0.1 (0.03 +/- 0.01), respectively; control, bacitracin, and fluphenazine: 1.10 +/- 0.3 (0.12 +/- 0.03), 0.20 +/- 0.1 (0.02 +/- 0.01), and 0.82 +/- 0.2 (0.08 +/- 0.02) pg LEU/min (pg LEU/mg protein/min), respectively. Results emphasize the selective nature of chemical structures required to significantly inhibit AP activity and provided information that could help the rational design of agents with high specificity in a biologic milieu containing multiple peptidases. In this case, targeted modulation of the bioavailability of LEU and other endogenous AP-degraded hormonal and nonhormonal peptides could be useful in the treatment of the pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.