RESUMEN
BACKGROUND: The Centers for Disease Control and Prevention recommends initial and follow-up sexually transmitted infection (STI) and HIV testing when taking HIV preexposure prophylaxis (PrEP). We assessed frequencies of STIs and HIV testing and rates of STIs before and after PrEP initiation among men aged ≥18 years. METHODS: We used the OptumLabs database for this cohort study. We measured STI/HIV testing rates and prevalence in 2 time intervals: (1) within 90 days before and on the date of PrEP initiation and (2) within 45 days of the 180th day after the date of PrEP initiation. RESULTS: Of 4210 men who initiated PrEP in 2016 to 2017 and continuously used PrEP for ≥180 days, 45.7%, 45.7%, and 56.0% were tested for chlamydia, gonorrhea, and HIV, respectively, at the second time interval. These percentages were significantly lower than those at the first time interval (58.3%, 57.9%, and 73.5%, respectively; P < 0.01). Chlamydia and gonorrhea prevalence rates at the second time interval were 6.5% and 6.2%, respectively, versus 5.0% and 4.7%, respectively, at the first time interval. Most gonorrhea or chlamydia infections at the second time intervals seem to be new infections new infections. CONCLUSIONS: Sexually transmitted infection/HIV testing for PrEP users in the real-world private settings is much lower than in clinical trials. High STI prevalence before and after PrEP initiation in this study suggests that patients taking PrEP have an increased risk of acquiring STI. Interventions to improve provider adherence for PrEP users are urgently needed.
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Infecciones por VIH , Profilaxis Pre-Exposición , Enfermedades de Transmisión Sexual , Adolescente , Adulto , Estudios de Cohortes , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiología , Infecciones por VIH/prevención & control , Homosexualidad Masculina , Humanos , Masculino , Prevalencia , Enfermedades de Transmisión Sexual/diagnóstico , Enfermedades de Transmisión Sexual/epidemiología , Enfermedades de Transmisión Sexual/prevención & controlRESUMEN
BACKGROUND: The Centers for Disease Control and Prevention (CDC) recommends specific regimens for chlamydia and dual therapy for gonorrhea to mitigate antimicrobial-resistant gonorrhea in the CDC 2015 sexually transmitted disease treatment guidelines. Only limited studies examining adherence to these recommendations have been conducted at private practices in the United States. METHODS: We used the OptumLabs Data Warehouse, a comprehensive, longitudinal data asset with deidentified persons with linked commercial insurance claims and clinical information, to identify persons aged 15 to 60 years who had valid nucleic acid amplification testing results demonstrating urogenital or extragenital gonorrhea or chlamydia in 2016 to 2018. We defined valid laboratory results as positive or negative. We then assessed the time of their first positive test result and the type of treatment within 30 days to determine if there was evidence in the claims record that the CDC-recommended treatment was provided. We defined presumed treatment if the date of treatment was before the date of the positive test result within 30 days. RESULTS: Among 6476 patients with positive gonorrhea test results and 26,847 patients with positive chlamydia test results only, 34.8% and 64.2% had evidence of receiving the CDC-recommended therapy, respectively. Approximately 11.6% of patients with positive gonorrhea test results with recommended dual treatment and 7.1% of patients with positive chlamydia test results only with recommended chlamydia treatment were presumptively treated. CONCLUSION: Analysis of treatment claims and medical records from private settings indicated low rates of recommended gonorrhea and chlamydia treatment. Validation of treatment claims is needed to support further quality of care interventions based on these data.
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Infecciones por Chlamydia , Gonorrea , Enfermedades de Transmisión Sexual , Adolescente , Adulto , Infecciones por Chlamydia/diagnóstico , Infecciones por Chlamydia/tratamiento farmacológico , Infecciones por Chlamydia/epidemiología , Gonorrea/diagnóstico , Gonorrea/tratamiento farmacológico , Gonorrea/epidemiología , Humanos , Laboratorios , Persona de Mediana Edad , Prescripciones , Estados Unidos/epidemiología , Adulto JovenRESUMEN
Short-term synaptic plasticity is induced by calcium (Ca(2+)) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated CaV2.1 Ca(2+) channels by Ca(2+) sensor proteins induces facilitation of Ca(2+) currents and synaptic facilitation in cultured neurons expressing exogenous CaV2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca(2+) sensor binding site. This mutation does not alter voltage dependence or kinetics of CaV2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by â¼ 50%. In the presence of EGTA-AM to prevent global increases in free Ca(2+), the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca(2+) is dependent upon regulation of CaV2.1 channels by Ca(2+) sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10-20 Hz. Evidently, regulation of CaV2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.
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Canales de Calcio Tipo N/fisiología , Calcio/metabolismo , Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Animales , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Müller glia (MG) that activate the gene encoding the proneural factor Achaete-scute homolog 1 (Ascl1; also known as Mash1 in mammals) and de-differentiate into progenitor cells. By contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether ASCL1 could restore neurogenic potential to mammalian MG, we overexpressed ASCL1 in dissociated mouse MG cultures and intact retinal explants. ASCL1-infected MG upregulated retinal progenitor-specific genes and downregulated glial genes. Furthermore, ASCL1 remodeled the chromatin at its targets from a repressive to an active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers and displayed neuron-like physiological responses. These results indicate that a single transcription factor, ASCL1, can induce a neurogenic state in mature MG.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neuroglía/metabolismo , Regeneración , Retina/citología , Neuronas Retinianas/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores/metabolismo , Proliferación Celular , Células Cultivadas , Reprogramación Celular , Ensamble y Desensamble de Cromatina , Clonación Molecular , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica , Células HEK293 , Histonas/metabolismo , Humanos , Técnicas In Vitro , Lentivirus/genética , Lentivirus/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Neurogénesis , Neuroglía/citología , Técnicas de Placa-Clamp , Retina/metabolismo , Neuronas Retinianas/efectos de los fármacos , Neuronas Retinianas/metabolismo , Proteína Fluorescente RojaRESUMEN
We have identified an asynchronously activated Ca(2+) current through voltage-gated Ca(2+) (Ca(V))-2.1 and Ca(V)2.2 channels, which conduct P/Q- and N-type Ca(2+) currents that initiate neurotransmitter release. In nonneuronal cells expressing Ca(V)2.1 or Ca(V)2.2 channels and in hippocampal neurons, prolonged Ca(2+) entry activates a Ca(2+) current, I(Async), which is observed on repolarization and decays slowly with a half-time of 150-300 ms. I(Async) is not observed after L-type Ca(2+) currents of similar size conducted by Ca(V)1.2 channels. I(Async) is Ca(2+)-selective, and it is unaffected by changes in Na(+), K(+), Cl(-), or H(+) or by inhibitors of a broad range of ion channels. During trains of repetitive depolarizations, I(Async) increases in a pulse-wise manner, providing Ca(2+) entry that persists between depolarizations. In single-cultured hippocampal neurons, trains of depolarizations evoke excitatory postsynaptic currents that show facilitation followed by depression accompanied by asynchronous postsynaptic currents that increase steadily during the train in parallel with I(Async). I(Async) is much larger for slowly inactivating Ca(V)2.1 channels containing ß(2a)-subunits than for rapidly inactivating channels containing ß(1b)-subunits. I(Async) requires global rises in intracellular Ca(2+), because it is blocked when Ca(2+) is chelated by 10 mM EGTA in the patch pipette. Neither mutations that prevent Ca(2+) binding to calmodulin nor mutations that prevent calmodulin regulation of Ca(V)2.1 block I(Async). The rise of I(Async) during trains of stimuli, its decay after repolarization, its dependence on global increases of Ca(2+), and its enhancement by ß(2a)-subunits all resemble asynchronous release, suggesting that I(Async) is a Ca(2+) source for asynchronous neurotransmission.
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Canales de Calcio Tipo N/fisiología , Calcio/metabolismo , Activación del Canal Iónico , Neurotransmisores/metabolismo , Animales , Ratones , Transmisión SinápticaRESUMEN
GLT-1, the major glutamate transporter in the adult brain, is abundantly expressed in astrocytic processes enveloping synapses. By limiting glutamate escape into the surrounding neuropil, GLT-1 preserves the spatial specificity of synaptic signaling. Here we show that the amyloid-ß peptide Aß1-42 markedly prolongs the extracellular lifetime of synaptically released glutamate by reducing GLT-1 surface expression in mouse astrocytes and that this effect is prevented by the vitamin E derivative Trolox. These findings indicate that astrocytic glutamate transporter dysfunction may play an important role in the pathogenesis of Alzheimer's disease and suggest possible mechanisms by which several current treatment strategies could protect against the disease.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Fragmentos de Péptidos/metabolismo , Sinapsis/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Antioxidantes/farmacología , Cromanos/farmacología , Interacciones Farmacológicas , Espacio Extracelular/metabolismo , Femenino , Hipocampo/citología , Hipocampo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/farmacología , Sinapsis/efectos de los fármacosRESUMEN
OBJECTIVE: To identify the association between insulin out-of-pocket costs (OOPC) and adherence to insulin in Medicare Advantage (MA) patients. DATA SOURCES AND STUDY SETTING: The study is based on Optum Labs Data Warehouse, a longitudinal, real-world data asset with de-identified administrative claims and electronic health record data. STUDY DESIGN: Using descriptive and multivariable logistic regression analyses, we identified the likelihood of patients with diabetes having ≥60 consecutive days between an expected insulin fill date and the actual fill date (refill lapse) by OOPC, categorized by $0, >$0-$20 (reference), >$20-$35, >$35-$50, and > $50 per 30-day supply. DATA COLLECTION/EXTRACTION METHODS: The study included MA enrollees with type 1 or type 2 diabetes and prescription claims for insulin between 2014 and 2018. PRINCIPAL FINDINGS: Those with average insulin OOPC per 30-day supply >$35 or $0 were more likely to have an insulin refill lapse versus OOPC of >$0 to $20, with odds ratios ranging 1.18 (95% CI 1.13-1.22) to 1.74 (95% CI 1.66-1.83) depending on OOPC group and diabetes type. CONCLUSIONS: Capping average insulin OOPC at $35 per 30-day supply may help avoid cost-related insulin non-adherence in MA patients; efforts to address non-cost barriers to medication adherence remain important.
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Diabetes Mellitus Tipo 2 , Insulinas , Seguro , Medicare Part C , Humanos , Anciano , Estados Unidos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Gastos en Salud , Insulinas/uso terapéuticoRESUMEN
With a multitude of substrates, γ-secretase is poised to control neuronal function through a variety of signaling pathways. Presenilin 1 (PS1) is an integral component of γ-secretase and is also a protein closely linked to the etiology of Alzheimer's disease (AD). To better understand the roles of γ-secretase and PS1 in normal and pathological synaptic transmission, we examined evoked and spontaneous neurotransmitter release in cultured hippocampal neurons derived from PS1 knock-out (KO) mice. We found no changes in the size of evoked synaptic currents, short-term plasticity, or apparent calcium dependence of evoked release. The rate of spontaneous release from PS1 KO neurons was, however, approximately double that observed in wild-type (WT) neurons. This increase in spontaneous neurotransmission depended on calcium influx but did not require activation of voltage-gated calcium channels or presynaptic NMDA receptors or release of calcium from internal stores. The rate of spontaneous release from PS1 KO neurons was significantly reduced by lentivirus-mediated expression of WT PS1 or familial AD-linked M146V PS1, but not the D257A PS1 mutant that does not support γ-secretase activity. Treatment of WT neuronal cultures with γ-secretase inhibitor mimicked the loss of PS1, leading to a selective increase in spontaneous release without any change in the size of evoked synaptic currents. Together, these results identify a novel role for γ-secretase in the control of spontaneous neurotransmission through modulation of low-level tonic calcium influx into presynaptic axon terminals.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Análisis de Varianza , Animales , Western Blotting , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Células Cultivadas , Potenciales Evocados/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Ratones , Ratones Noqueados , Neuronas/citología , Técnicas de Placa-Clamp , Presenilina-1/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
BACKGROUND: High out-of-pocket costs (OOPCs) for insulin can lead to cost-related nonadherence and poor outcomes, prompting payers to limit insulin OOPCs. However, data are scarce on whether insulin OOPCs at policy-relevant levels is associated with improved adherence and outcomes. OBJECTIVE: To identify associations between insulin OOPCs and insulin adherence, noninsulin antihyperglycemic (AHG) medication adherence, and diabetes-related emergency department (ED) visits and hospitalizations. METHODS: This retrospective cohort study was conducted using OptumLabs Data Warehouse, a longitudinal, real-world data asset with deidentified administrative claims and electronic health record data. Individuals with type 1 diabetes (T1D) or type 2 diabetes (T2D), insulin use on January 1 of a study year (index date: 2007-2018), continuous commercial health plan eligibility 12 months pre-index and post-index date, and at least 1 insulin claim during the 12-month follow-up period were included. Average insulin OOPCs per 30-day supply in the follow-up period was identified and categorized ($0, > $0-$20 [referent group], > $20-$35, > $35-$50, and > $50). The proportion of patients with a gap in insulin supply of 60 or more continuous days, AHG nonadherence per modified proportion of days covered less than 0.80, and a diabetes-related ED visit or hospitalization were identified and compared by insulin OOPC category vs more than $0 to $20 using pairwise chi-square tests and multivariable logistic regression. RESULTS: The study included 21,085 individuals with T1D and 72,512 with T2D. Patients with average OOPCs more than $50 were more likely to have a gap in insulin supply vs those with OOPCs more than $0 to $20, with an odds ratio (OR) of 1.14 (95% CI =1.05-1.24) and 1.38 (95% CI = 1.32-1.45) for T1D and T2D, respectively. Those with T2D and OOPCs more than $35 were also more likely to have a 60-day gap in insulin supply (OR 1.17; 95% CI = 1.11-1.23). Odds of having a diabetes-related hospitalization or ED visit did not increase with higher OOPCs; rather, associations tended to be inverse. Nonadherence to AHG medications in the T2D cohort was higher with insulin OOPCs more than $20 vs those more than $0-$20 (P < 0.05 for all). CONCLUSIONS: Individuals with T2D were more likely to have a 60-day gap in insulin supply when the OOPC was more than $35 per 30-day supply and with the OOPC more than $50 in those with T1D. These findings suggest that health plans can facilitate adherence to insulin therapy and possibly to noninsulin AHG medications by protecting patients with diabetes from experiencing high insulin OOPC. A study with a longer follow-up period is warranted to fully assess ED and hospitalization outcomes. DISCLOSURES: This study was funded by the Robert Wood Johnson Foundation, Health Data for Action Research Program. The study sponsor played no role in the design or conduct of this study. The views expressed here do not necessarily reflect the views of the Foundation.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Seguro , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Gastos en Salud , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Cumplimiento de la Medicación , Estudios RetrospectivosRESUMEN
PtdIns(4,5)P2 is a signaling lipid central to the regulation of multiple cellular functions. It remains unknown how PtdIns(4,5)P2 fulfills various functions in different cell types, such as regulating neuronal excitability, synaptic release, and astrocytic function. Here, we compared the dynamics of PtdIns(4,5)P2 synthesis in hippocampal neurons and astrocytes with the kidney-derived tsA201 cell line. The experimental approach was to (1) measure the abundance and rate of PtdIns(4,5)P2 synthesis and precursors using specific biosensors, (2) measure the levels of PtdIns(4,5)P2 and its precursors using mass spectrometry, and (3) use a mathematical model to compare the metabolism of PtdIns(4,5)P2 in cell types with different proportions of phosphoinositides. The rate of PtdIns(4,5)P2 resynthesis in hippocampal neurons after depletion by cholinergic or glutamatergic stimulation was three times faster than for tsA201 cells. In tsA201 cells, resynthesis of PtdIns(4,5)P2 was dependent on the enzyme PI4K. In contrast, in hippocampal neurons, the resynthesis rate of PtdIns(4,5)P2 was insensitive to the inhibition of PI4K, indicating that it does not require de novo synthesis of the precursor PtdIns(4)P. Measurement of phosphoinositide abundance indicated a larger pool of PtdIns(4)P, suggesting that hippocampal neurons maintain sufficient precursor to restore PtdIns(4,5)P2 levels. Quantitative modeling indicates that the measured differences in PtdIns(4)P pool size and higher activity of PI4K can account for the experimental findings and indicates that high PI4K activity prevents depletion of PtdIns(4)P. We further show that the resynthesis of PtdIns(4,5)P2 is faster in neurons than astrocytes, providing context to the relevance of cell type-specific mechanisms to sustain PtdIns(4,5)P2 levels.
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Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositoles , Hipocampo/metabolismo , Neuronas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismoRESUMEN
In this issue of Neuron, Südhof and colleagues determine which of the eight Ca(2+)-binding synaptotagmin isoforms expressed in brain can support synchronous neurotransmitter release at mammalian CNS synapses. Unexpectedly, only three-synaptotagmin-1, -2, and -9-can serve as Ca(2+) sensors for fast transmission. Further characterization reveals the unique ability of each isoform to shape neurotransmission.
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Calcio/metabolismo , Neuronas/citología , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Ratones , Sinaptotagminas/metabolismo , Sinaptotagminas/fisiologíaRESUMEN
In this issue of Neuron, Harata et al. use a novel quenching technique to provide compelling evidence that kiss-and-run is the dominant mode of vesicle fusion at hippocampal synapses and that the prevalence of kiss-and-run can be modulated by stimulus frequency. The increased incidence of kiss-and-run at lower frequencies may ensure that vesicles are available for use during periods of high demand.
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Vesículas Sinápticas/fisiología , Electrofisiología , Hipocampo/fisiología , Humanos , Neurotransmisores/fisiologíaRESUMEN
BACKGROUND: To evaluate real-world pharmacologic treatment of mixed dyslipidemia in patients with diabetes mellitus (DM). METHODS: All commercial health plan members in a large US managed care database with complete lipid panel results (HDL-C, LDL-C, TG) between 1/1/2006 and 12/31/2006 were identified (N = 529,236). DM patients (N = 53,679) with mixed dyslipidemia were defined as having any 2 suboptimal lipid parameters (N = 28,728). Lipid treatment status 6 months pre- and post-index date was determined using pharmacy claims for any lipid therapy. RESULTS: Post-index, 41.1% of DM patients with 2 abnormal lipid parameters and 45.1% with 3 abnormal lipid parameters did not receive lipid-modifying treatment. Post-index treatment rates were 57.4%, 63.6%, and 66.4% for patients with LDL-C, HDL-C, and TG in the most severe quartiles, respectively. Statin monotherapy was the primary lipid-modifying regimen prescribed (54.8% and 47.8% of patients with any 2 and all 3 lipids not at goal, respectively). Less than 30% of treated patients received combination therapy. CONCLUSION: Over 40% of DM patients with mixed dyslipidemia received no lipid-modifying therapy during the follow-up period. Those who were treated were primarily prescribed statin monotherapy. This study suggests that DM patients are not being treated to ADA-suggested targets.
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Complicaciones de la Diabetes/sangre , Complicaciones de la Diabetes/tratamiento farmacológico , Dislipidemias/complicaciones , Dislipidemias/tratamiento farmacológico , Hipolipemiantes/uso terapéutico , Anciano , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Enfermedad Coronaria/sangre , Enfermedad Coronaria/tratamiento farmacológico , Enfermedad Coronaria/etiología , Enfermedad Coronaria/prevención & control , Bases de Datos Factuales , Dislipidemias/sangre , Femenino , Humanos , Masculino , Programas Controlados de Atención en Salud , Persona de Mediana Edad , Guías de Práctica Clínica como Asunto , Estudios Retrospectivos , Factores de Riesgo , Triglicéridos/sangre , Estados UnidosRESUMEN
Synaptotagmin is a synaptic vesicle protein that has been proposed to be the calcium sensor responsible for fast neurotransmitter release at synapses. Synaptotagmin's two C2 domains, C2A and C2B, each provide a calcium binding pocket lined with negative charges contributed by five conserved aspartates. We find that even when all of C2A's conserved aspartates are neutralized by replacement with asparagines, neurotransmitter release still occurs at hippocampal synapses in culture. Because exocytosis continues to be dependent on extracellular calcium concentration, the C2A domain cannot represent the entire calcium sensor. C2A does appear to be part of the calcium sensor, however, because substitution of D232 alters the calcium dependence of release, perhaps by reducing the number of calcium ions that must bind to trigger exocytosis. We conclude that neutralization of the negative charge at D232 by coordination of a calcium ion is necessary--but not sufficient--for fast neurotransmission at mammalian CNS synapses.
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Proteínas de Unión al Calcio , Calcio/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Estructura Terciaria de Proteína/fisiología , Transmisión Sináptica/fisiología , Oxidorreductasas de Alcohol , Animales , Arginina/genética , Ácido Aspártico/genética , Sitios de Unión , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electrofisiología , Embrión de Mamíferos , Potenciales Postsinápticos Excitadores/genética , Hipocampo/fisiología , Magnesio/metabolismo , Glicoproteínas de Membrana/clasificación , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/clasificación , Neuronas/metabolismo , Recuperación de la Función/genética , Sinaptotagmina II , Sinaptotagminas , Transfección/veterinariaRESUMEN
Synaptotagmin IV (Syt IV) is a brain-specific isoform of the synaptotagmin family, the levels of which are strongly elevated after seizure activity. The dominant hypothesis of Syt IV function states that Syt IV upregulation is a neuroprotective mechanism for reducing neurotransmitter release. To test this hypothesis in mammalian CNS synapses, Syt IV was overexpressed in cultured mouse hippocampal neurons, and acute effects on fast excitatory neurotransmission were assessed. We found neurotransmission unaltered with respect to basal release probability, Ca2+ dependence of release, short-term plasticity, and fusion pore kinetics. In contrast, expression of a mutant Syt I with diminished Ca2+ affinity (R233Q) reduced release probability and altered the Ca2+ dependence of release, thus demonstrating the sensitivity of the system to changes in neurotransmission resulting from changes to the Ca2+ sensor. Together, these data refute the dominant model that Syt IV functions as an inhibitor of neurotransmitter release in mammalian neurons.
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Neuronas/metabolismo , Neurotransmisores/metabolismo , Sinaptotagminas/fisiología , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Células Cultivadas/metabolismo , Exocitosis/genética , Exocitosis/fisiología , Hipocampo/citología , Ratones , Plasticidad Neuronal , Técnicas de Placa-Clamp , Mutación Puntual , Unión Proteica/genética , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/fisiología , Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética , Sinaptotagminas/genéticaRESUMEN
We report a thorough analysis of neurotransmission in cultured hippocampal neurons lacking synaptic vesicle protein 2 (SV2), a membrane glycoprotein present in all vesicles that undergo regulated secretion. We found that SV2 selectively enhances low-frequency neurotransmission by priming morphologically docked vesicles. Loss of SV2 reduced initial release probability during a train of action potentials but had no effect on steady-state responses. The amount and decay rate of asynchronous release, two measures sensitive to presynaptic calcium concentrations, are not altered in SV2 knock-outs, suggesting that SV2 does not act by modulating presynaptic calcium. Normal neurotransmission could be temporarily recovered by delivering an exhaustive stimulus train. Our results indicate that SV2 primes vesicles in quiescent neurons and that SV2 function can be bypassed by an activity-dependent priming mechanism. We propose that SV2 action modulates synaptic networks by ensuring that low-frequency neurotransmission is faithfully conveyed.
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Hipocampo/citología , Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Neurotransmisores/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Potenciales de Acción , Animales , Astrocitos/fisiología , Calcio/farmacología , Células Cultivadas , Potenciales Evocados/fisiología , Genotipo , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Vesículas Sinápticas/ultraestructuraRESUMEN
The 5-HT6 receptor has been implicated in a variety of cognitive processes including habitual behaviors, learning, and memory. It is found almost exclusively in the brain, is expressed abundantly in striatum, and localizes to neuronal primary cilia. Primary cilia are antenna-like, sensory organelles found on most neurons that receive both chemical and mechanical signals from other cells and the surrounding environment; however, the effect of 5-HT6 receptor function on cellular morphology has not been examined. We confirmed that 5-HT6 receptors were localized to primary cilia in wild-type (WT) but not 5-HT6 knockout (5-HT6KO) in both native mouse brain tissue and primary cultured striatal neurons then used primary neurons cultured from WT or 5-HT6KO mice to study the function of these receptors. Selective 5-HT6 antagonists reduced cilia length in neurons cultured from wild-type mice in a concentration and time-dependent manner without altering dendrites, but had no effect on cilia length in 5-HT6KO cultured neurons. Varying the expression levels of heterologously expressed 5-HT6 receptors affected the fidelity of ciliary localization in both WT and 5-HT6KO neurons; overexpression lead to increasing amounts of 5-HT6 localization outside of the cilia but did not alter cilia morphology. Introducing discrete mutations into the third cytoplasmic loop of the 5-HT6 receptor greatly reduced, but did not entirely eliminate, trafficking of the 5-HT6 receptor to primary cilia. These data suggest that blocking 5-HT6 receptor activity reduces the length of primary cilia and that mechanisms that regulate trafficking of 5-HT6 receptors to cilia are more complex than previously thought.
Asunto(s)
Cilios/metabolismo , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Neuronas/citología , Neuronas/metabolismo , Receptores de Serotonina/metabolismo , Animales , Células Cultivadas , Cilios/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Expresión Génica , Metilaminas/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Piperazinas/farmacología , Piridinas/farmacología , Receptores de Serotonina/genética , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Sulfonamidas/farmacología , Factores de TiempoRESUMEN
Overexpression of proteins is a powerful way to determine their function. Until recently, the low efficiency of neuronal transfection has made it difficult to use overexpression and structure-function studies to investigate the role of neuronal proteins in their native environment. The development of neurotrophic viral systems has overcome the obstacle of low efficiency and allows for unprecedented opportunities to use biochemical and electrophysiological techniques to assess the effects of overexpressing wild-type or mutant proteins in neurons. Here, a general protocol for the production of replication-deficient Semliki Forest virus constructs directing the overexpression of proteins of interest in cultured mammalian neurons is described.
Asunto(s)
Virus Defectuosos/genética , Virus Defectuosos/fisiología , Expresión Génica , Neuronas/metabolismo , Proteínas/genética , Animales , Células Cultivadas , Clonación Molecular , Cricetinae , ADN/genética , ADN Complementario/genética , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Virus de los Bosques Semliki/fisiología , Transcripción Genética , Virión , Activación Viral , Replicación ViralRESUMEN
Presynaptic receptors that are coupled to heterotrimeric G-proteins are found throughout the brain and are responsible for modulating synaptic transmission. At least 10 G-protein-coupled receptors (GPCRs) reduce transmission in hippocampal neurons. Additionally, hippocampal neurons express up to 17 different Galpha, Gbeta, and Ggamma subunits, making for a striking array of possible heterotrimer compositions and GPCR-heterotrimer interactions. The identity of the Galpha subunit is likely a critical determinant in coupling specificity between GPCRs and their molecular effectors mediating presynaptic inhibition. We studied the role of four Galpha(i/o) subunits (Galpha(o1), Galpha(i1,) Galpha(i2), and Galpha(i3)) in mediating presynaptic inhibition in hippocampal neurons by expressing pertussis toxin-insensitive (PTx-ins) Galpha(i/o) mutants. PTx treatment of these cells disrupts coupling of endogenous subunits, leaving only the mutant Galpha subunits to couple with native GPCRs and betagamma subunits. Successful rescue of presynaptic inhibition indicates that the expressed mutant Galpha subunit can couple to the GPCR of interest. All four PTx-ins Galpha subunits rescued presynaptic inhibition by adenosine A1 receptors. A PTx-ins Galpha subunit also rescued adenosine A1-mediated inhibition of spontaneous vesicle fusion frequency. Of the remaining GPCRs tested, cannabinoid CB1, somatostatin, and GABA(B) receptors displayed an alpha subunit-dependent selectivity in binding to G-protein heterotrimers, whereas group III metabotropic glutamate receptor-mediated inhibition was not rescued by expression of any of the four PTx-ins Galpha subunits. Differential coupling of G-protein alpha subunits may be a means of achieving specificity between different GPCRs and their molecular targets for mediating presynaptic inhibition.