Mouse strain differences in locomotor,sensitisation and rewarding effect of heroin; association with alterations in MOP‐r activation and dopamine transporter binding

There is growing agreement that genetic factors play an important role in the risk to develop heroin addiction, and comparisons of heroin addiction vulnerability in inbred strains of mice could provide useful information on the question of individual vulnerability to heroin addiction. This study examined the rewarding and locomotor‐stimulating effects of heroin in male C57BL/6J and DBA/2J mice. Heroin induced locomotion and sensitisation in C57BL/6J but not in DBA/2J mice. C57BL/6J mice developed conditioned place preference (CPP) to the highest doses of heroin, while DBA/2J showed CPP to only the lowest heroin doses, indicating a higher sensitivity of DBA/2J mice to the rewarding properties of heroin vs C57BL/6J mice. In order to investigate the neurobiological substrate underlying some of these differences, the effect of chronic ‘intermittent’ escalating dose heroin administration on the opioid, dopaminergic and stress systems was explored. Twofold higher μ‐opioid receptor (MOP‐r)‐stimulated [³⁵S]GTPγS binding was observed in the nucleus accumbens and caudate of saline‐treated C57BL/6J mice compared with DBA/2J. Heroin decreased MOP‐r density in brain regions of C57BL/6J mice, but not in DBA/2J. A higher density of dopamine transporters (DAT) was observed in nucleus accumbens shell and caudate of heroin‐treated DBA/2J mice compared with heroin‐treated C57BL/6J. There were no effects on D₁ and D₂ binding. Chronic heroin administration decreased corticosterone levels in both strains with no effect of strain. These results suggest that genetic differences in MOP‐r activation and DAT expression may be responsible for individual differences in vulnerability to heroin addiction.     

Cx3cr1‐deficiency exacerbates alpha‐synuclein‐A53T induced neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the degeneration of dopaminergic neurons of the substantia nigra and the accumulation of protein aggregates, called Lewy bodies, where the most abundant is alpha‐synuclein (α‐SYN). Mutations of the gene that codes for α‐SYN (SNCA), such as the A53T mutation, and duplications of the gene generate cases of PD with autosomal dominant inheritance. As a result of the association of inflammation with the neurodegeneration of PD, we analyzed whether overexpression of wild‐type α‐SYN (α‐SYN^WT) or mutated α‐SYN (α‐SYN^A53T) are involved in the neuronal dopaminergic loss and inflammation process, along with the role of the chemokine fractalkine (CX3CL1) and its receptor (CX3CR1). We generated in vivo murine models overexpressing human α‐SYN^WT or α‐SYN^A53T in wild type (Cx3cr1^+/+) or deficient (Cx3cr1^–/–) mice for CX3CR1 using unilateral intracerebral injection of adeno‐associated viral vectors. No changes in CX3CL1 levels were observed by immunofluorescence or analysis by qRT‐PCR in this model. Interestingly, the expression α‐SYN^WT induced dopaminergic neuronal death to a similar degree in both genotypes. However, the expression of α‐SYN^A53T produced an exacerbated neurodegeneration, enhanced in the Cx3cr1^–/– mice. This neurodegeneration was accompanied by an increase in neuroinflammation and microgliosis as well as the production of pro‐inflammatory markers, which were exacerbated in Cx3cr1^–/– mice overexpressing α‐SYN^A53T. Furthermore, we observed that in primary microglia CX3CR1 was a critical factor in the modulation of microglial dynamics in response to α‐SYN^WT or α‐SYN^A53T. Altogether, our study reveals that CX3CR1 plays an essential role in neuroinflammation induced by α‐SYN^A53T.     

Cerebrospinal fluid Tau/α‐synuclein ratio in Parkinson's disease and degenerative dementias

Although alpha‐synuclein is the main constituent of Lewy bodies, cerebrospinal fluid determination on its own does not seem fundamental for the diagnosis of synucleinopathies. We evaluated whether the combination of classical biomarkers, Aβ_1–42, total tau, phosphorylated tau, and α‐synuclein can improve discrimination of Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease, and frontotemporal dementia. Aβ_1–42, total tau, phosphorylated tau, and α‐synuclein were measured in a series of patients with Parkinson's disease (n = 38), dementia with Lewy bodies (n = 32), Alzheimer's disease (n = 48), frontotemporal dementia (n = 31), and age‐matched control patients with other neurological diseases (n = 32). Mean α‐synuclein levels in cerebrospinal fluid were significantly lower in the pathological groups than in cognitively healthy subjects. An inverse correlation of α‐synuclein with total tau (r = ?0.196, P < .01) was observed. In the group of patients with Parkinson's disease, Aβ_1–42, total tau, and phosphorylated tau values were similar to controls, whereas total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios showed the lowest values. Cerebrospinal fluid α‐synuclein alone did not provide relevant information for Parkinson's disease diagnosis, showing low specificity (area under the curve, 0.662; sensitivity, 94%; specificity, 25%). Instead, a better performance was obtained with the total tau/α‐syn ratio (area under the curve, 0.765; sensitivity, 89%; specificity, 61%). Combined determination of α‐synuclein and classical biomarkers in cerebrospinal fluid shows differential patterns in neurodegenerative disorders. In particular, total tau/α‐synuclein and phosphorylated tau/α‐synuclein ratios can contribute to the discrimination of Parkinson's disease. © 2011 Movement Disorder Society     

Sensitivity and specificity of phospho‐Ser129 α‐synuclein monoclonal antibodies

α‐Synuclein (α‐syn) is an abundant presynaptic protein that is the primary constituent of inclusions that define Lewy body diseases (LBDs). In these inclusions, α‐syn is phosphorylated at the serine‐129 residue. Antibodies directed to this phosphorylation site are used to measure inclusion abundance and stage disease progression in preclinical models as well as in postmortem tissues in LBDs. While it is critical to reliably identify inclusions, phospho‐specific antibodies often cross‐react with nonspecific antigens. Four commercially available monoclonal antibodies, two from rabbits (clones EP1536Y and MJF‐R13) and two from mice (81a and pSyn#64), have been the most widely used in detecting pS129‐α‐syn inclusions. Here, we systematically evaluated these antibodies in brain sections and protein lysates from rats and mice. All antibodies detected pS129‐α‐syn inclusions in the brain that were induced by preformed α‐syn fibrils in wild‐type rats and mice. Antibody titrations revealed that clones EP1536Y and 81a comparably labeled inclusions in both the perikarya and neuronal processes in contrast to clones MJF‐R13 and pSyn#64 that incompletely labeled inclusions at various antibody concentrations. Except for EP1536Y, the clones produced nonspecific diffuse neuropil labeling in α‐syn knockout mice as well as mice and rats injected with monomeric α‐syn, with some nonspecific staining titrating with pS129‐α‐syn inclusions. By immunoblot, all the clones cross‐reacted with proteins other than α‐syn, warranting caution in interpretations of specificity. Clone EP1536Y uniquely and robustly detected endogenous pS129‐α‐syn in highly soluble protein fractions from the mouse brain. In summary, EP1536Y had the highest sensitivity and specificity for detecting pS129‐α‐syn.     

α‐Synuclein real‐time quaking‐induced conversion in the cerebrospinal fluid of uncertain cases of parkinsonism

A reliable biomarker is needed for accurate and early differentiation between Parkinson disease and the various forms of atypical parkinsonism. We used a novel real‐time quaking‐induced conversion (RT‐QuIC) assay to detect α‐synuclein (α‐syn) aggregates in cerebrospinal fluid (CSF) of 118 patients with parkinsonism of uncertain clinical etiology and 52 controls. Diagnostic accuracy to distinguish α‐synucleinopathies from non–α‐synucleinopathies and controls was 84% (sensitivity = 75%, specificity = 94%, area under the curve = 0.84, 95% confidence interval = 0.78–0.91, p < 0.0001, positive predictive value = 93%). CSF α‐syn RT‐QuIC could be a useful diagnostic tool to help clinicians differentiate α‐synucleinopathies from other forms of parkinsonism when the clinical picture is uncertain. Ann Neurol 2019;85:777–781     

Localization of α‐Synuclein in Teleost Central Nervous System: Immunohistochemical and Western Blot Evidence by 3D5 Monoclonal Antibody in the Common Carp,Cyprinus carpio

Alpha synuclein (α‐syn) is a 140 amino acid vertebrate‐specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α‐syn aggregates suggested a role for α‐syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α‐syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α‐syn. The distribution of α‐syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α‐syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α‐syn‐like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α‐syn‐like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α‐syn expression in cholinergic systems and suggest that α‐syn modulates similar molecular pathways in phylogenetically distant vertebrates. J. Comp. Neurol. 523:1095–1124, 2015. © 2015 Wiley Periodicals, Inc.     

α‐Synuclein activates innate immunity but suppresses interferon‐γ expression in murine astrocytes

Glial activation and neuroinflammation contribute to pathogenesis of neurodegenerative diseases, linked to neuron loss and dysfunction. α‐Synuclein (α‐syn), as a metabolite of neuron, can induce microglia activation to trigger innate immune response. However, whether α‐syn, as well as its mutants (A53T, A30P, and E46K), induces astrocyte activation and inflammatory response is not fully elucidated. In this study, we used A53T mutant and wild‐type α‐syns to stimulate primary astrocytes in dose‐ and time‐dependent manners (0.5, 2, 8, and 20 μg/ml for 24 hr or 3, 12, 24, and 48 hr at 2 μg/ml), and evaluated activation of several canonical inflammatory pathway components. The results showed that A53T mutant or wild‐type α‐syn significantly upregulated mRNA expression of toll‐like receptor (TLR)2, TLR3, nuclear factor‐κB and interleukin (IL)‐1β, displaying a pattern of positive dose–effect correlation or negative time–effect correlation. Such upregulation was confirmed at protein levels of TLR2 (at 20 μg/ml), TLR3 (at most doses), and IL‐1β (at 3 hr) by western blotting. Blockage of TLR2 other than TLR4 inhibited TLR3 and IL‐1β mRNA expressions. By contrast, interferon (IFN)‐γ was significantly downregulated at mRNA, protein, and protein release levels, especially at high concentrations of α‐syns or early time‐points. These findings indicate that α‐syn was a TLRs‐mediated immunogenic agent (A53T mutant stronger than wild‐type α‐syn). The stimulation patterns suggest that persistent release and accumulation of α‐syn is required for the maintenance of innate immunity activation, and IFN‐γ expression inhibition by α‐syn suggests a novel immune molecule interaction mechanism underlying pathogenesis of neurodegenerative diseases.     

Alpha‐synuclein transfers from neurons to oligodendrocytes

The origin of α‐synuclein (α‐syn)‐positive glial cytoplasmic inclusions found in oligodendrocytes in multiple system atrophy (MSA) is enigmatic, given the fact that oligodendrocytes do not express α‐syn mRNA. Recently, neuron‐to‐neuron transfer of α‐syn was suggested to contribute to the pathogenesis of Parkinson's disease. In this study, we explored whether a similar transfer of α‐syn might occur from neurons to oligodendrocytes, which conceivably could explain how glial cytoplasmic inclusions are formed. We studied oligodendrocytes in vitro and in vivo and examined their ability to take up different α‐syn assemblies. First, we treated oligodendrocytes with monomeric, oligomeric, and fibrillar forms of α‐syn proteins and investigated whether α‐syn uptake is dynamin‐dependent. Second, we injected the same α‐syn species into the mouse cortex to assess their uptake in vivo. Finally, we monitored the presence of human α‐syn within rat oligodendroglial cells grafted in the striatum of hosts displaying Adeno‐Associated Virus‐mediated overexpression of human α‐syn in the nigro‐striatal pathway. Here , we show that oligodendrocytes take up recombinant α‐syn monomers, oligomers and, to a lesser extent, fibrils in vitro in a concentration and time‐dependent manner, and that this process is inhibited by dynasore. Further, we demonstrate in our injection model that oligodendrocytes also internalize α‐syn in vivo. Finally, we provide the first direct evidence that α‐syn can transfer to grafted oligodendroglial cells from host rat brain neurons overexpressing human α‐syn. Our findings support the hypothesis of a neuron‐to‐oligodendrocyte transfer of α‐syn, a mechanism that may play a crucial role in the progression and pathogenesis of MSA. GLIA 2014;62:387–398     

Plasma α‐synuclein in patients with Parkinson's disease with and without treatment

Alpha‐synuclein (α‐syn) is an intracellular protein with a high tendency to aggregation. It is the major component of Lewy bodies and may play a key role in the pathogenesis of Parkinson's disease (PD). α‐Syn is also released by neurons and can be detected in biological fluids, such as plasma. The purpose of this study was to determine whether plasma α‐syn concentrations are elevated in newly diagnosed PD patients before treatment (nontreated PD group, ntPD; n = 53) and to compare them with concentrations in PD patients with at least 1 year of specific treatment (tPD; n = 42) and in healthy controls (n = 60). Plasma α‐syn concentrations in the ntPD and tPD groups were similar and significantly higher than in healthy controls. In conclusion, α‐syn was elevated early in the development of PD and specific PD treatment did not change plasma α‐syn levels. © 2010 Movement Disorder Society     

Mapping of a FEB3 homologous febrile seizure locus on mouse chromosome 2 containing candidate genes Scn1a and Scn3a

Febrile seizures (FS) are the most common seizure type in children. Recurrent FS are a risk factor for developing temporal lobe epilepsy later in life and are known to have a strong genetic component. Experimental FS (eFS) can be elicited in mice by warm‐air induced hyperthermia. We used this model to screen the chromosome substitution strain (CSS) panel derived from C57BL/6J and A/J for FS susceptibility and identified C57BL/6J‐Chr2^A/NaJ (CSS2), as the strain with the strongest FS susceptibility phenotype. The aim of this study was to map FS susceptibility loci and select candidate genes on mouse chromosome 2. We generated an F₂ population by intercrossing the hybrids (F₁) that were derived from CSS2 and C57BL/6J mice. All CSS2‐F₂ individuals were genotyped and phenotyped for eFS susceptibility, and QTL analysis was performed. Candidate gene selection was based on bioinformatics analyses and differential brain expression between CSS2 and C57BL/6J strains determined by microarray analysis. Genetic mapping of the eFS susceptibility trait identified two significant loci: FS‐QTL2a (LOD‐score 3.6) and FS‐QTL2b (LOD‐score 6.2). FS‐QTL2a contained 44 genes expressed in the brain at post natal day 14. Four of these (Arl6ip6, Cytip, Fmnl2 Ifih1) contained a non‐synonymous SNP comparing CSS2 and C57BL/6J, six genes (March7, Nr4a2, Gpd2, Grb14, Scn1a, Scn3a) were differentially expressed between these strains. A region within FS‐QTL2a is homologous to the human FEB3 locus. The fact that we identify mouse FS‐QTL2a with high FEB3 homology is strong support for the validity of the eFS mouse model to study genetics of human FS.     

Review: Novel treatment strategies targeting alpha‐synuclein in multiple system atrophy as a model of synucleinopathy

Neurodegenerative disorders with alpha‐synuclein (α‐syn) accumulation (synucleinopathies) include Parkinson's disease (PD), PD dementia, dementia with Lewy bodies and multiple system atrophy (MSA). Due to the involvement of toxic α‐syn aggregates in the molecular origin of these disorders, developing effective therapies targeting α‐syn is a priority as a disease‐modifying alternative to current symptomatic treatments. Importantly, the clinical and pathological attributes of MSA make this disorder an excellent candidate as a synucleinopathy model for accelerated drug development. Recent therapeutic strategies targeting α‐syn in in vivo and in vitro models of MSA, as well as in clinical trials, have been focused on the pathological mechanisms of α‐syn synthesis, aggregation, clearance, and/or cell‐to‐cell propagation of its neurotoxic conformers. Here we summarize the most relevant approaches in this direction, with emphasis on their potential as general synucleinopathy modifiers, and enumerate research areas for potential improvement in MSA drug discovery.     

5‐S‐cysteinyldopamine neurotoxicity: Influence on the expression of α‐synuclein and ERp57 in cellular and animal models of Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder whose etiology is still unclear in spite of extensive investigations. It has been hypothesized that 5‐S‐cysteinyldopamine (CysDA), a catechol‐thioether metabolite of dopamine (DA), could be an endogenous parkinsonian neurotoxin. To gain further insight into its role in the neurodegenerative process, both CD1 mice and SH‐SY5Y neuroblastoma cells were treated with CysDA, and the data were compared with those obtained by the use of 6‐hydroxydopamine, a well‐known parkinsonian mimetic. Intrastriatal injection of CysDA in CD1 mice caused a long‐lasting depletion of DA, providing evidence of in vivo neurotoxicity of CysDA. Both in mice and in SH‐SY5Y cells, CysDA treatment induced extensive oxidative stress, as evidenced by protein carbonylation and glutathione depletion, and affected the expression of two proteins, α‐synuclein (α‐Syn) and ERp57, whose levels are modulated by oxidative insult. Real‐time PCR experiments support these findings, indicating an upregulation of both ERp57 and α‐Syn expression. α‐Syn aggregation was also found to be modulated by CysDA treatment. The present work provides a solid background sustaining the hypothesis that CysDA is involved in parkinsonian neurodegeneration by inducing extensive oxidative stress and protein aggregation. © 2013 Wiley Periodicals, Inc.     

Distinct differences in the cannabinoid receptor binding in the brain of C57BL/6 and DBA/2 mice, selected for their differences in voluntary ethanol consumption

The two inbred strains of mice C57BL/6 and DBA/2 mice have been shown to differ significantly in their preference for alcohol (EtOH). These strains of mice have been employed to study various aspects of pharmacological and behavioral effects of EtOH. We have previously demonstrated that chronic EtOH exposure down-regulated cannabinoid receptors (CB1) in mouse synaptic plasma membranes and enhanced the synthesis of endogenous cannabimimetic compound anandamide (AnNH) in human neuroblastoma cells. The purpose of the present study was to investigate whether there were differences in the density and the affinity of CB1 receptors in the brains of the two inbred C57BL/6 (alcohol-preferring) and DBA/2 (alcohol avoiding) mice. The results indicate the presence of specific CB1 receptors in the brain membranes of both the strains. It was also found that the CB1 receptor densities (B(max)) were 25% lower in C57BL/6 (0.66 +/- 0.15 pmol/mg protein) compared with that of DBA/2 (0.88 +/- 0.08 pmol/mg protein) mice. Significant differences in the affinity were also observed between the two lines (K(d), 0.68 +/- 0.15 nM for C57BL/6 and 2.21 +/- 0.56 nM for DBA/2). The competition studies with SR141716A, a CB1 receptor antagonist, and 2-arachidonylglycerol (2-AG) and anandamide (AnNH), known CB1 receptor agonists, all showed a substantial decrease in [(3)H]CP-55,940 binding in both strains of mice with a higher K(i) values in the DBA/2 mice. These results suggest that CB1 receptor signal transduction may play an important role in controlling the voluntary EtOH consumption by these strains of mice.     

Progression of intestinal permeability changes and alpha‐synuclein expression in a mouse model of Parkinson's disease

Parkinson's disease (PD) is a multifocal degenerative disorder for which there is no cure. The majority of cases are sporadic with unknown etiology. Recent data indicate that untreated patients with de novo PD have increased colonic permeability and that both de novo and premotor patients have pathological expression of α‐synuclein (α‐syn) in their colon. Both endpoints potentially can serve as disease biomarkers and even may initiate PD events through gut‐derived, lipopolysaccharide (LPS)‐induced neuronal injury. Animal models could be ideal for interrogating the potential role of the intestines in the pathogenesis of PD; however, few current animal models of PD encompass these nonmotor features. We sought to establish a progressive model of PD that includes the gastrointestinal (GI) dysfunction present in human patients. C57/BL6 mice were systemically administered one dose of either LPS (2.5 mg/kg) or saline and were sacrificed in monthly intervals (n = 5 mice for 5 months) to create a time‐course. Small and large intestinal permeability was assessed by analyzing the urinary output of orally ingested sugar probes through capillary column gas chromatography. α‐Syn expression was assessed by counting the number of mildly, moderately, and severely affected myenteric ganglia neurons throughout the GI tract, and the counts were validated by quantitative optical density measurements. Nigrostriatal integrity was assessed by tyrosine hydroxylase immunohistochemistry stereology and densitometry. LPS caused an immediate and progressive increase in α‐syn expression in the large intestine but not in the small intestine. Intestinal permeability of the whole gut (large and small intestines) progressively increased between months 2 and 4 after LPS administration but returned to baseline levels at month 5. Selective measurements demonstrated that intestinal permeability in the small intestine remained largely intact, suggesting that gut leakiness was predominately in the large intestine. Phosphorylated serine 129‐α‐syn was identified in a subset of colonic myenteric neurons at months 4 and 5. Although these changes were observed in the absence of nigrostriatal degeneration, an abrupt but insignificant increase in brainstem α‐syn was observed that paralleled the restoration of permeability. No changes were observed over time in controls. LPS, an endotoxin used to model PD, causes sequential increases in α‐syn immunoreactivity, intestinal permeability, and pathological α‐syn accumulation in the colon in a manner similar to that observed in patients with PD. These features are observed without nigrostriatal degeneration and incorporate PD features before the motor syndrome. This allows for the potential use of this model in testing neuroprotective and disease‐modifying therapies, including intestinal‐directed therapies to fortify intestinal barrier integrity. © 2013 International Parkinson and Movement Disorder Society     

Dopaminergic lesioning impairs adult hippocampal neurogenesis by distinct modification of α‐synuclein

Nonmotor symptoms of cognitive and affective nature are present in premotor and motor stages of Parkinson's disease (PD). Neurogenesis, the generation of new neurons, persists throughout the mammalian life span in the hippocampal dentate gyrus. Adult hippocampal neurogenesis may be severely affected in the course of PD, accounting for some of the neuropsychiatric symptoms such as depression and cognitive impairment. Two important PD‐related pathogenic factors have separately been attributed to contribute to both PD and adult hippocampal neurogenesis: dopamine depletion and accumulation of α‐synuclein (α‐syn). In the acute 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine model, altered neurogenesis has been linked merely to a reduced dopamine level. Here, we seek to determine whether a distinct endogenous α‐syn expression pattern is associated, possibly contributing to the hippocampal neurogenic deficit. We observed a persistent reduction of striatal dopamine and a loss of tyrosine hydroxylase‐expressing neurons in the substantia nigra pars compacta in contrast to a complete recovery of tyrosine hydroxylase‐immunoreactive dopaminergic fibers within the striatum. However, dopamine levels in the hippocampus were significantly decreased. Survival of newly generated neurons was significantly reduced and paralleled by an accumulation of truncated, membrane‐associated, insoluble α‐syn within the hippocampus. Specifically, the presence of truncated α‐syn species was accompanied by increased activity of calpain‐1, a calcium‐dependent protease. Our results further substantiate the broad effects of dopamine loss in PD‐susceptible brain nuclei, gradually involved in the PD course. Our findings also indicate a detrimental synergistic interplay between dopamine depletion and posttranslational modification of α‐syn, contributing to impaired hippocampal plasticity in PD. © 2015 Wiley Periodicals, Inc.     

Prefrontal glutamate correlates of methamphetamine sensitization and preference

Methamphetamine (MA) is a widely misused, highly addictive psychostimulant that elicits pronounced deficits in neurocognitive function related to hypo‐functioning of the prefrontal cortex (PFC). Our understanding of how repeated MA impacts excitatory glutamatergic transmission within the PFC is limited, as is information about the relationship between PFC glutamate and addiction vulnerability/resiliency. In vivo microdialysis and immunoblotting studies characterized the effects of MA (ten injections of 2 mg/kg, i.p.) upon extracellular glutamate in C57BL/6J mice and upon glutamate receptor and transporter expression, within the medial PFC. Glutamatergic correlates of both genetic and idiopathic variance in MA preference/intake were determined through studies of high vs. low MA‐drinking selectively bred mouse lines (MAHDR vs. MALDR, respectively) and inbred C57BL/6J mice exhibiting spontaneously divergent place‐conditioning phenotypes. Repeated MA sensitized drug‐induced glutamate release and lowered indices of N‐methyl‐d ‐aspartate receptor expression in C57BL/6J mice, but did not alter basal extracellular glutamate content or total protein expression of Homer proteins, or metabotropic or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid glutamate receptors. Elevated basal glutamate, blunted MA‐induced glutamate release and ERK activation, as well as reduced protein expression of mGlu2/3 and Homer2a/b were all correlated biochemical traits of selection for high vs. low MA drinking, and Homer2a/b levels were inversely correlated with the motivational valence of MA in C57BL/6J mice. These data provide novel evidence that repeated, low‐dose MA is sufficient to perturb pre‐ and post‐synaptic aspects of glutamate transmission within the medial PFC and that glutamate anomalies within this region may contribute to both genetic and idiopathic variance in MA addiction vulnerability/resiliency.     

Decay‐accelerating factor 1 deficiency exacerbates Trypanosoma cruzi‐induced murine chronic myositis

Introduction: Murine infection with Trypanosoma cruzi (Tc) has been used to study the role of T‐cells in the pathogenesis of human inflammatory idiopathic myositis. Absence of decay‐accelerating factor 1 (Daf1) has been shown to enhance murine T‐cell responses and autoimmunity. Methods: To determine whether Daf1 deficiency can exacerbate Tc‐induced myositis, C57BL/6 DAF^+/+ and DAF^?/? mice were inoculated with 5 × 10⁴ trypomastigotes, and their morbidity, parasitemia, parasite burden, histopathology, and T‐cell expansion were studied in the acute and chronic stages. Results: DAF^?/? mice had lower parasitemia and parasite burden but higher morbidity, muscle histopathology, and increased number of CD44⁺ (activated/memory phenotype) splenic CD4⁺ and CD8⁺ T‐cells. Conclusions: An enhanced CD8⁺ T‐cell immune‐specific response may explain the lower parasitemia and parasite burden levels and the increase in histopathological lesions. We propose that Tc‐inoculated DAF^?/? mice are a useful model to study T‐cell mediated immunity in skeletal muscle tissues. Muscle Nerve 46: 582–587, 2012     

Lentivirus‐Mediated α‐Melanocyte‐Stimulating Hormone Overexpression in the Hypothalamus Decreases Diet Induced Obesity in Mice

Melanocyte stimulating hormone (MSH) derived from the pro‐hormone pro‐opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long‐term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long‐term anti‐obesity effects of hypothalamic α‐MSH overexpression in mice. An α‐MSH lentivirus (LVi‐α‐MSH‐EGFP) vector carrying the N‘‐terminal part of POMC and the α‐MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi‐α‐MSH‐EGFP or control LVi‐EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high‐fat diet. The effects of 6‐week‐treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14‐day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi‐α‐MSH‐EGFP treatment is dependent on decreased food intake. The 6‐week LVi‐α‐MSH‐EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi‐EGFP treated mice. Food intake was decreased during the first week in the LVi‐α‐MSH‐EGFP treated mice but subsequently increased to the level of LVi‐EGFP treated mice. The LVi‐EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi‐α‐MSH‐EGFP treated mice. We demonstrate that gene transfer of α‐MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α‐MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site‐specific effects of POMC peptides.