RESUMEN
Drugs of abuse induce sensitization, which is defined as enhanced response to additional drug following a period of withdrawal. Sensitization occurs in both humans and animal models of drug reinforcement and contributes substantially to the addictive nature of drugs of abuse, because it is thought to represent enhanced motivational wanting for drug. The ventral pallidum, a key member of the reward pathway, contributes to behaviors associated with reward, such as sensitization. Dopamine inputs to the ventral pallidum have not been directly characterized. Here we provide anatomical, neurochemical, and behavioral evidence demonstrating that dopamine terminals in the ventral pallidum contribute to reward in mice. We report subregional differences in dopamine release, measured by ex vivo fast-scan cyclic voltammetry: rostral ventral pallidum exhibits increased dopamine release and uptake compared with caudal ventral pallidum, which is correlated with tissue expression of dopaminergic proteins. We then subjected mice to a methamphetamine-sensitization protocol to investigate the contribution of dopaminergic projections to the region in reward related behavior. Methamphetamine-sensitized animals displayed a 508% and 307% increase in baseline dopamine release in the rostral and caudal ventral pallidum, respectively. Augmented dopamine release in the rostral ventral pallidum was significantly correlated with sensitized locomotor activity. Moreover, this presynaptic dopaminergic plasticity occurred only in the ventral pallidum and not in the ventral or dorsal striatum, suggesting that dopamine release in the ventral pallidum may be integrally important to drug-induced sensitization.
Asunto(s)
Trastornos Relacionados con Anfetaminas/metabolismo , Dopaminérgicos/farmacología , Dopamina/metabolismo , Globo Pálido/efectos de los fármacos , Globo Pálido/metabolismo , Metanfetamina/farmacología , Trastornos Relacionados con Anfetaminas/patología , Animales , Estimulantes del Sistema Nervioso Central/farmacología , Modelos Animales de Enfermedad , Globo Pálido/patología , Inmunohistoquímica , Masculino , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Terminales Presinápticos/patologíaRESUMEN
We previously demonstrated that mice with reduced expression of the vesicular monoamine transporter 2 (VMAT2 LO) undergo age-related degeneration of the catecholamine-producing neurons of the substantia nigra pars compacta and locus ceruleus and exhibit motor disturbances and depressive-like behavior. In this work, we investigated the effects of reduced vesicular transport on the function and viability of serotonin neurons in these mice. Adult (4-6 months of age), VMAT2 LO mice exhibit dramatically reduced (90%) serotonin release capacity, as measured by fast scan cyclic voltammetry. We observed changes in serotonin receptor responsivity in in vivo pharmacological assays. Aged (months) VMAT2 LO mice exhibited abolished 5-HT1A autoreceptor sensitivity, as determined by 8-OH-DPAT (0.1 mg/kg) induction of hypothermia. When challenged with the 5HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (1 mg/kg), VMAT2 LO mice exhibited a marked increase (50%) in head twitch responses. We observed sparing of serotonergic terminals in aged mice (18-24 months) throughout the forebrain by SERT immunohistochemistry and [(3)H]-paroxetine binding in striatal homogenates of aged VMAT2 LO mice. In contrast to their loss of catecholamine neurons of the substantia nigra and locus ceruleus, aged VMAT2 LO mice do not exhibit a change in the number of serotonergic (TPH2+) neurons within the dorsal raphe, as measured by unbiased stereology at 26-30 months. Collectively, these data indicate that reduced vesicular monoamine transport significantly disrupts serotonergic signaling, but does not drive degeneration of serotonin neurons.
Asunto(s)
Cuerpo Estriado/metabolismo , Neuronas/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Serotonina/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Anfetaminas/farmacología , Animales , Ratones , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patología , Receptor de Serotonina 5-HT1A/genética , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
The psychostimulant methamphetamine (METH) is highly addictive and neurotoxic to dopamine terminals. METH toxicity has been suggested to be due to the release and accumulation of dopamine in the cytosol of these terminals. The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is a critical mediator of dopamine handling. Mice overexpressing VMAT2 (VMAT2-HI) have an increased vesicular capacity to store dopamine, thus augmenting striatal dopamine levels and dopamine release in the striatum. Based on the altered compartmentalization of intracellular dopamine in the VMAT2-HI mice, we assessed whether enhanced vesicular function was capable of reducing METH-induced damage to the striatal dopamine system. While wildtype mice show significant losses in striatal levels of the dopamine transporter (65% loss) and tyrosine hydroxylase (46% loss) following a 4 × 10 mg/kg METH dosing regimen, VMAT2-HI mice were protected from this damage. VMAT2-HI mice were also spared from the inflammatory response that follows METH treatment, showing an increase in astroglial markers that was approximately one-third of that of wildtype animals (117% vs 36% increase in GFAP, wildtype vs VMAT2-HI). Further analysis also showed that elevated VMAT2 level does not alter the ability of METH to increase core body temperature, a mechanism integral to the toxicity of the drug. Finally, the VMAT2-HI mice showed no difference from wildtype littermates on both METH-induced conditioned place preference and in METH-induced locomotor activity (1 mg/kg METH). These results demonstrate that elevated VMAT2 protects against METH toxicity without enhancing the rewarding effects of the drug. Since the VMAT2-HI mice are protected from METH despite higher basal dopamine levels, this study suggests that METH toxicity depends more on the proper compartmentalization of synaptic dopamine than on the absolute amount of dopamine in the brain.
Asunto(s)
Estimulantes del Sistema Nervioso Central/toxicidad , Metanfetamina/toxicidad , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Animales , Western Blotting , Estimulantes del Sistema Nervioso Central/metabolismo , Inmunohistoquímica , Masculino , Metanfetamina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Attention-deficit hyperactivity disorder (ADHD) is estimated to affect 8-12% of school-age children worldwide. ADHD is a complex disorder with significant genetic contributions. However, no single gene has been linked to a significant percentage of cases, suggesting that environmental factors may contribute to ADHD. Here, we used behavioral, molecular, and neurochemical techniques to characterize the effects of developmental exposure to the pyrethroid pesticide deltamethrin. We also used epidemiologic methods to determine whether there is an association between pyrethroid exposure and diagnosis of ADHD. Mice exposed to the pyrethroid pesticide deltamethrin during development exhibit several features reminiscent of ADHD, including elevated dopamine transporter (DAT) levels, hyperactivity, working memory and attention deficits, and impulsive-like behavior. Increased DAT and D1 dopamine receptor levels appear to be responsible for the behavioral deficits. Epidemiologic data reveal that children aged 6-15 with detectable levels of pyrethroid metabolites in their urine were more than twice as likely to be diagnosed with ADHD. Our epidemiologic finding, combined with the recapitulation of ADHD behavior in pesticide-treated mice, provides a mechanistic basis to suggest that developmental pyrethroid exposure is a risk factor for ADHD.
Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/inducido químicamente , Trastorno por Déficit de Atención con Hiperactividad/patología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Nitrilos/toxicidad , Piretrinas/orina , Receptores de Dopamina D1/metabolismo , Adolescente , Animales , Trastorno por Déficit de Atención con Hiperactividad/metabolismo , Reacción de Prevención/efectos de los fármacos , Western Blotting , Estudios de Casos y Controles , Niño , Cromatografía Líquida de Alta Presión , Estudios Transversales , Femenino , Humanos , Insecticidas/toxicidad , Locomoción/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Ratones , Ratones Endogámicos C57BL , Piretrinas/efectos adversos , Piretrinas/toxicidadRESUMEN
Disruption of neurotransmitter vesicle dynamics (transport, capacity, release) has been implicated in a variety of neurodegenerative and neuropsychiatric conditions. Here, we report a novel mouse model of enhanced vesicular function via bacterial artificial chromosome (BAC)-mediated overexpression of the vesicular monoamine transporter 2 (VMAT2; Slc18a2). A twofold increase in vesicular transport enhances the vesicular capacity for dopamine (56%), dopamine vesicle volume (33%), and basal tissue dopamine levels (21%) in the mouse striatum. The elevated vesicular capacity leads to an increase in stimulated dopamine release (84%) and extracellular dopamine levels (44%). VMAT2-overexpressing mice show improved outcomes on anxiety and depressive-like behaviors and increased basal locomotor activity (41%). Finally, these mice exhibit significant protection from neurotoxic insult by the dopaminergic toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), as measured by reduced dopamine terminal damage and substantia nigra pars compacta cell loss. The increased release of dopamine and neuroprotection from MPTP toxicity in the VMAT2-overexpressing mice suggest that interventions aimed at enhancing vesicular capacity may be of therapeutic benefit in Parkinson disease.
Asunto(s)
Dopamina/metabolismo , Trastornos Parkinsonianos/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/fisiología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Conducta Animal , Cromosomas Artificiales Bacterianos , Cuerpo Estriado/metabolismo , Ratones , Ratones Transgénicos , Trastornos Parkinsonianos/patología , Trastornos Parkinsonianos/fisiopatología , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
Electrochemical detection with carbon-fiber microelectrodes has become an established method to monitor directly the release of dopamine from neurons and its uptake by the dopamine transporter. With constant potential amperometry (CPA) the measured current provides a real time view of the rapid concentration changes, but the method lacks chemical identification of the monitored species and markedly increases the difficulty of signal calibration. Monitoring with fast-scan cyclic voltammetry (FSCV) allows species identification and concentration measurements, but often exhibits a delayed response time due to the time-dependent adsorption/desorption of electroactive species at the electrode. We sought to improve the temporal resolution of FSCV to make it more comparable to CPA by increasing the waveform repetition rate from 10 to 60 Hz with uncoated carbon-fiber electrodes. The faster acquisition led to diminished time delays of the recordings that tracked more closely with CPA measurements. The measurements reveal that FSCV at 10 Hz underestimates the normal rate of dopamine uptake by about 18%. However, FSCV collection at 10 Hz and 60 Hz provide identical results when a dopamine transporter (DAT) blocker such as cocaine is bath applied. To verify further the utility of this method, we used transgenic mice that over-express DAT. After accounting for the slight adsorption delay time, FSCV at 60 Hz adequately monitored the increased uptake rate that arose from overexpression of DAT and, again, was similar to CPA results. Furthermore, the utility of collecting data at 60 Hz was verified in an anesthetized rat by using a higher scan rate (2400 V/s) to increase sensitivity and the overall signal.
RESUMEN
The exposure of the human population to environmental contaminants is recognized as a significant contributing factor for the development of Parkinson's disease (PD) and other forms of parkinsonism. While pesticides have repeatedly been identified as risk factors for PD, these compounds represent only a subset of environmental toxicants that we are exposed to on a regular basis. Thus, non-pesticide contaminants, such as metals, solvents, and other organohalogen compounds have also been implicated in the clinical and pathological manifestations of these movement disorders and it is these non-pesticide compounds that are the subject of this review. As toxic exposures to these classes of compounds can result in a spectrum of PD or PD-related disorders, it is imperative to appreciate shared clinico-pathological characteristics or mechanisms of action of these compounds in order to further delineate the resultant disorders as well as identify improved preventive strategies or therapeutic interventions.
Asunto(s)
Exposición a Riesgos Ambientales , Sustancias Peligrosas/toxicidad , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/etiología , Humanos , Factores de RiesgoRESUMEN
Fast-scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes can be used to measure behaviorally correlated dopamine changes in the extracellular fluid of the brain of freely moving rats. These experiments employ a chronically implanted Ag/AgCl reference electrode. When dopamine measurements are taken 4 days after implantation, there is often a potential shift, typically greater than +0.2 V, in the anodic and cathodic peaks in the cyclic voltammogram for dopamine. In this work, we optimized a method to coat sintered Ag/AgCl reference electrodes with the perfluorinated polymer, Nafion, to prevent this shift. We find that we can stabilize reference electrodes for up to 28 days. Immunohistochemistry of the tissue around the implant site shows extensive glial encapsulation around both bare and Nafion-coated devices. However, the lesion around bare electrodes has a rough texture implying that these cells are strongly adsorbed onto the bare reference electrode, while the lesion around a Nafion-coated electrode shows that cells are more intact implying that they adsorb less strongly. EDS and SEM analysis of the surface of the electrodes confirms this by visualizing a heavy build up of plaques, organic in nature, only on bare electrodes. Impedance spectroscopy indicates no difference between the impedance of bare and Nafion-coated Ag/AgCl electrodes, indicating that glial encapsulation does not lead to an increase in uncompensated resistance between the working and reference electrodes. The electrochemical shift therefore must be due to the unique chemical microenvironment around the reference electrode that alters the chloride equilibrium, a process that the Nafion coating prevents.
RESUMEN
BACKGROUND: Prostatic acid phosphatase (PAP) and ecto-5'-nucleotidase (NT5E, CD73) produce extracellular adenosine from the nucleotide AMP in spinal nociceptive (pain-sensing) circuits; however, it is currently unknown if these are the main ectonucleotidases that generate adenosine or how rapidly they generate adenosine. RESULTS: We found that AMP hydrolysis, when measured histochemically, was nearly abolished in dorsal root ganglia (DRG) neurons and lamina II of spinal cord from Pap/Nt5e double knockout (dKO) mice. Likewise, the antinociceptive effects of AMP, when combined with nucleoside transport inhibitors (dipyridamole or 5-iodotubericidin), were reduced by 80-100% in dKO mice. In addition, we used fast scan cyclic voltammetry (FSCV) to measure adenosine production at subsecond resolution within lamina II. Adenosine was maximally produced within seconds from AMP in wild-type (WT) mice but production was reduced >50% in dKO mice, indicating PAP and NT5E rapidly generate adenosine in lamina II. Unexpectedly, we also detected spontaneous low frequency adenosine transients in lamina II with FSCV. Adenosine transients were of short duration (<2 s) and were reduced (>60%) in frequency in Pap-/-, Nt5e-/- and dKO mice, suggesting these ectonucleotidases rapidly hydrolyze endogenously released nucleotides to adenosine. Field potential recordings in lamina II and behavioral studies indicate that adenosine made by these enzymes acts through the adenosine A1 receptor to inhibit excitatory neurotransmission and nociception. CONCLUSIONS: Collectively, our experiments indicate that PAP and NT5E are the main ectonucleotidases that generate adenosine in nociceptive circuits and indicate these enzymes transform pulsatile or sustained nucleotide release into an inhibitory adenosinergic signal.
Asunto(s)
5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Nucleótidos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , 5'-Nucleotidasa/genética , Fosfatasa Ácida , Adenosina Monofosfato/metabolismo , Animales , Dipiridamol/farmacología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Masculino , Ratones , Ratones Noqueados , Nocicepción/efectos de los fármacos , Dolor/metabolismo , Proteínas Tirosina Fosfatasas/genética , Receptor de Adenosina A1/genética , Receptor de Adenosina A1/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Tubercidina/análogos & derivados , Tubercidina/farmacologíaRESUMEN
BACKGROUND: Low-level laser therapy (LLLT) is commonly used in medical applications, but scientific studies of its efficacy and the mechanism by which it causes loss of fat from fat cells for body contouring are lacking. This study examined the effectiveness and mechanism by which 635680 nm LLLT acts as a non-invasive body contouring intervention method. METHODS: Forty healthy men and women ages 1865 years with a BMI <30 kg/m2 were randomized 1:1 to laser or control treatment. Subject's waistlines were treated 30 min twice a week for 4 weeks. Standardized waist circumference measurements and photographs were taken before and after treatments 1, 3, and 8. Subjects were asked not to change their diet or exercise habits. In vitro assays were conducted to determine cell lysis, glycerol, and triglyceride release. RESULTS: Data were analyzed for those with body weight fluctuations within 1.5 kg during 4 weeks of the study. Each treatment gave a 0.40.5 cm loss in waist girth.Cumulative girth loss after 4 weeks was −2.15 cm (−0.78 ± 2.82 vs. 1.35 ± 2.64 cm for the control group,p < 0.05). A blinded evaluation of standardized pictures showed statistically significant cosmetic improvement after 4 weeks of laser treatment. In vitro studies suggested that laser treatment increases fat loss from adipocytes by release of triglycerides, without inducing lipolysis or cell lysis. CONCLUSIONS: LLLT achieved safe and significant girth loss sustained over repeated treatments and cumulative over 4 weeks of eight treatments. The girth loss from the waist gave clinically and statistically significant cosmetic improvement.
Asunto(s)
Adipocitos/efectos de la radiación , Distribución de la Grasa Corporal , Técnicas Cosméticas , Láseres de Semiconductores/uso terapéutico , Terapia por Luz de Baja Intensidad , Sobrepeso/radioterapia , Grasa Subcutánea Abdominal/efectos de la radiación , Adipocitos/metabolismo , Adolescente , Adulto , Anciano , Método Doble Ciego , Femenino , Humanos , Lipólisis/efectos de la radiación , Masculino , Persona de Mediana Edad , Triglicéridos/metabolismo , Circunferencia de la Cintura , Adulto JovenRESUMEN
Synapsins are a family of synaptic vesicle proteins that are important for neurotransmitter release. Here we have used triple knock-out (TKO) mice lacking all three synapsin genes to determine the roles of synapsins in the release of two monoamine neurotransmitters, dopamine and serotonin. Serotonin release evoked by electrical stimulation was identical in substantia nigra pars reticulata slices prepared from TKO and wild-type mice. In contrast, release of dopamine in response to electrical stimulation was approximately doubled in striatum of TKO mice, both in vivo and in striatal slices, in comparison to wild-type controls. This was due to loss of synapsin III, because deletion of synapsin III alone was sufficient to increase dopamine release. Deletion of synapsins also increased the sensitivity of dopamine release to extracellular calcium ions. Although cocaine did not affect the release of serotonin from nigral tissue, this drug did enhance dopamine release. Cocaine-induced facilitation of dopamine release was a function of external calcium, an effect that was reduced in TKO mice. We conclude that synapsins play different roles in the control of release of dopamine and serotonin, with release of dopamine being negatively regulated by synapsins, specifically synapsin III, while serotonin release appears to be relatively independent of synapsins. These results provide further support for the concept that synapsin function in presynaptic terminals varies according to the neurotransmitter being released.
Asunto(s)
Dopamina/metabolismo , Neostriado/metabolismo , Serotonina/metabolismo , Sustancia Negra/metabolismo , Sinapsinas/metabolismo , Animales , Calcio/metabolismo , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Estimulación Eléctrica , Femenino , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Dinámicas no Lineales , Terminales Presinápticos/metabolismo , Análisis de Regresión , Vesículas Sinápticas/metabolismoRESUMEN
Vesicular monoamine transporters (VMATs) are responsible for the packaging of neurotransmitters such as dopamine, serotonin, norepinephrine, and epinephrine into synaptic vesicles. These proteins evolved from precursors in the major facilitator superfamily of transporters and are among the members of the toxin extruding antiporter family. While the primary function of VMATs is to sequester neurotransmitters within vesicles, they can also translocate toxicants away from cytosolic sites of action. In the case of dopamine, this dual role of VMAT2 is combined-dopamine is more readily oxidized in the cytosol where it can cause oxidative stress so packaging into vesicles serves two purposes: neurotransmission and neuroprotection. Furthermore, the deleterious effects of exogenous toxicants on dopamine neurons, such as MPTP, can be attenuated by VMAT2 activity. The active metabolite of MPTP can be kept within vesicles and prevented from disrupting mitochondrial function thereby sparing the dopamine neuron. The highly addictive drug methamphetamine is also neurotoxic to dopamine neurons by using dopamine itself to destroy the axon terminals. Methamphetamine interferes with vesicular sequestration and increases the production of dopamine, escalating the amount in the cytosol and leading to oxidative damage of terminal components. Vesicular transport seems to resist this process by sequestering much of the excess dopamine, which is illustrated by the enhanced methamphetamine neurotoxicity in VMAT2-deficient mice. It is increasingly evident that VMAT2 provides neuroprotection from both endogenous and exogenous toxicants and that while VMAT2 has been adapted by eukaryotes for synaptic transmission, it is derived from phylogenetically ancient proteins that originally evolved for the purpose of cellular protection.
Asunto(s)
Monoaminas Biogénicas/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/metabolismo , Animales , Monoaminas Biogénicas/química , Humanos , Intoxicación por MPTP/prevención & control , Neuronas/citología , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
Computerized treadmill gait analysis in models of toxicant exposure and neurodegenerative disorders holds much potential for detection and therapeutic intervention in these models, and researchers must validate the technology that assists in that data collection and analysis. The present authors used a commercially available computerized gait analysis system that used (a) a motorized treadmill on retired breeder male C57BL/6J mice, (b) the toxicant-induced (1-methyl-1-, 2-, 3-, 6-tetrahydropyridine) MPTP mouse model of Parkinson's disease (PD), and (c) the superoxide dismutase 1 (SOD1) G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS). The authors compared the detection of deficits by computerized treadmill gait analysis in MPTP-treated mice with inked-paw stride length and correlated these measures to dopamine (DA) loss. The authors found that the computerized treadmill gait analysis system did not distinguish MPTP-treated mice from vehicle controls, despite a nearly 90% deficit of striatal DA. In contrast, decreases in inked-paw stride length correlated strongly with DA losses in these same animals. Computerized treadmill gait analysis could neither reliably distinguish SOD1 G93A mutant mice from controls from 6 to 12 weeks of age nor detect any consistent early motor deficits in these mice. On the basis of the authors' findings, they inferred that computerized gait analysis on a motorized treadmill is not suited to measuring motor deficits in either the MPTP mouse model of PD or the SOD1 G93A mouse model of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/diagnóstico , Prueba de Esfuerzo/métodos , Marcha/fisiología , Actividad Motora/fisiología , Trastornos Parkinsonianos/diagnóstico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Análisis por Apareamiento , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/fisiopatología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5-10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.
Asunto(s)
Dopamina/metabolismo , Gliosis/inducido químicamente , Metanfetamina/toxicidad , Degeneración Nerviosa/inducido químicamente , Vesículas Sinápticas/efectos de los fármacos , Proteínas de Transporte Vesicular de Monoaminas/efectos de los fármacos , Trastornos Relacionados con Anfetaminas/metabolismo , Trastornos Relacionados con Anfetaminas/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Compartimento Celular/efectos de los fármacos , Compartimento Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Fiebre/inducido químicamente , Fiebre/metabolismo , Fiebre/fisiopatología , Gliosis/metabolismo , Gliosis/fisiopatología , Ratones , Ratones Noqueados , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
The aim of this study was to evaluate the lipolytic potential of solutions used in the practice of cosmetic mesotherapy to stimulate lipolysis, cause local fat reduction and reduce the appearance of cellulite. The mesotherapy solutions were tested in a human fat cell assay using the fold induction of glycerol generation as a measure of lipolysis. The following mesotherapy solutions were tested: aminophylline; yohimbine; isoproterenol; melilotus; aminophylline with melilotus; aminophylline with isoproterenol; aminophylline with isoproterenol and yohimbine; aminophylline with isoproterenol and lidocaine; and aminophylline with isoproterenol, yohimbine and lidocaine. Isoproterenol (P<0.002), aminophylline (P<0.00004) and yohimbine (P<0.001) stimulated lipolysis compared to the buffer control. The lipolysis stimulated by melilotus (P<0.01) and isoproterenol (P<0.002) was enhanced by aminophylline (P<0.001 and P<0.001, respectively). The lipolytic stimulation by aminophylline and isoproterenol (P<0.0009), and by aminophylline and isoproterenol with yohimbine (P<0.0007) was inhibited by lidocaine, not significant compared to buffer control for aminophylline and isoproterenol, but aminophylline, isoproterenol and yohimbine still stimulated lipolysis more than control, P<0.05). Isoproterenol, aminophylline, yohimbine and melilotus stimulate lipolysis alone, and lipolysis is further enhanced by combining lipolytic stimulators in mesotherapy solutions. Lidocaine is antilipolytic and should be removed from mesotherapy solutions designed for local fat reduction.
Asunto(s)
Tejido Adiposo/patología , Lipólisis/efectos de los fármacos , Obesidad/patología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Aminofilina/farmacología , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Glicerol/metabolismo , Humanos , Inyecciones Subcutáneas , Isoproterenol/farmacología , Melilotus , Obesidad/metabolismo , Extractos Vegetales/farmacología , Yohimbina/farmacologíaRESUMEN
BACKGROUND: Inhibition of angiogenesis reverses rodent obesity. A validated assay in human fat tissue is needed to study the role of angiogenesis in human obesity. METHODS: Human fat tissue fragments from surgery were placed in 96-well plates, embedded in fibrin thrombin clot and overlaid with cell culture media containing 20% fetal bovine serum. After 15 days, the clots were examined by histology and electron microscopy. The effect of taxol, cobalt chloride and a heparin-steroid combination was tested in the fat tissue assay and compared to the validated human placental vein angiogenesis model (HPVAM). RESULTS: Blood vessels initiated growth and elongated from the fat tissue fragments over 15 days. Presence of blood vessels was confirmed with histology and electron microscopy. Taxol at 10(-6) and 10(-7) M completely inhibited angiogenesis, while Taxol 10(-8) and 10(-9) M and the heparin-steroid partially inhibited angiogenesis. The response to taxol and heparin-steroid was similar to that of the HPVAM, a validated angiogenesis assay. Cobalt chloride, a stimulator of vascular endothelial growth factor (VEGF) stimulated angiogenesis initiation at 10(-9) M in fat tissue and the HPVAM, but at 10(-10) M blood vessel growth was stimulated only in the fat assay. CONCLUSION: This angiogenesis assay based on human fat tissue uses three-dimensionally intact human tissue. The vessels are derived from quiescient vessels within the fat. These properties allow the angiogenic switch to be evaluated in an in vitro setting. The angiogenic response of fat tissue is not identical to placental tissue. This assay allows exploration of angiogenesis in fat tissue.
Asunto(s)
Bioensayo/métodos , Neovascularización Fisiológica/fisiología , Grasa Subcutánea Abdominal/irrigación sanguínea , Ácido Aminocaproico , Moduladores de la Angiogénesis/farmacología , Fibrinógeno , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Obesidad Mórbida/patología , Placenta/irrigación sanguínea , Reproducibilidad de los Resultados , Grasa Subcutánea Abdominal/efectos de los fármacos , Grasa Subcutánea Abdominal/patología , Trombina , Técnicas de Cultivo de TejidosRESUMEN
The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is responsible for packaging dopamine into vesicles for subsequent release and has been suggested to serve a neuroprotective role in the dopamine system. Here, we show that mice that express approximately 5% of normal VMAT2 (VMAT2 LO) display age-associated nigrostriatal dopamine dysfunction that ultimately results in neurodegeneration. Elevated cysteinyl adducts to L-DOPA and DOPAC are seen early and are followed by increased striatal protein carbonyl and 3-nitrotyrosine formation. These changes were associated with decreased striatal dopamine and decreased expression of the dopamine transporter and tyrosine hydroxylase. Furthermore, we observed an increase in alpha-synuclein immunoreactivity and accumulation and neurodegeneration in the substantia nigra pars compacta in aged VMAT2 LO mice. Thus, VMAT2 LO animals display nigrostriatal degeneration that begins in the terminal fields and progresses to eventual loss of the cell bodies, alpha-synuclein accumulation, and an L-DOPA responsive behavioral deficit, replicating many of the key aspects of Parkinson's disease. These data suggest that mishandling of dopamine via reduced VMAT2 expression is, in and of itself, sufficient to cause dopamine-mediated toxicity and neurodegeneration in the nigrostriatal dopamine system. In addition, the altered dopamine homeostasis resulting from reduced VMAT2 function may be conducive to pathogenic mechanisms induced by genetic or environmental factors thought to be involved in Parkinson's disease.
Asunto(s)
Dopamina/metabolismo , Neostriado/metabolismo , Degeneración Nerviosa/metabolismo , Sustancia Negra/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismo , Animales , Dopamina/genética , Dopamina/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neostriado/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Sustancia Negra/patología , Vesículas Sinápticas/genética , Proteínas de Transporte Vesicular de Monoaminas/antagonistas & inhibidores , Proteínas de Transporte Vesicular de Monoaminas/genéticaRESUMEN
Numerous epidemiological studies have shown an association between pesticide exposure and an increased risk of developing Parkinson's disease (PD). Here, we provide evidence that the insecticide dieldrin causes specific oxidative damage in the nigrostriatal dopamine (DA) system. We report that exposure of mice to low levels of dieldrin for 30 days resulted in alterations in dopamine-handling as evidenced by a decrease in dopamine metabolites, DOPAC (31.7% decrease) and HVA (29.2% decrease) and significantly increased cysteinyl-catechol levels in the striatum. Furthermore, dieldrin resulted in a 53% decrease in total glutathione, an increase in the redox potential of glutathione, and a 90% increase in protein carbonyls. Alpha-synuclein protein expression was also significantly increased in the striatum (25% increase). Finally, dieldrin caused a significant decrease in striatal expression of the dopamine transporter as measured by (3)H-WIN 35,428 binding and (3)H-dopamine uptake. These alterations occurred in the absence of dopamine neuron loss in the substantia nigra pars compacta. These effects represent the ability of low doses of dieldrin to increase the vulnerability of nigrostriatal dopamine neurons by inducing oxidative stress and suggest that pesticide exposure may act as a promoter of PD.