RESUMEN

BACKGROUND: Study of synaptic integrity using conventional electrophysiology is a gold standard for quantitative assessment of neurodegeneration. Fluorescence assisted single-synapse long-term potentiation (FASS-LTP) provides a high throughput method to assess the synaptic integrity of neurotransmission within and between different brain regions as a measure of pharmacological efficacy in translational models. NEW METHOD: We adapted the existing method to our purpose by adding a step during the thawing of frozen samples, by an extra step of placing them on a rocker at room temperature for 30 minutes immediately following thawing with constant mixing on a shaker. This allowed for gradual, uniform thawing, effectively separating the synaptosomes. Our study demonstrates FASS-LTP on four brain regions at 6- and 12-month periods in the 3xTg-AD mouse model, treating sibling cohorts with VU0155069 (a small molecule inhibitor) or vehicle (0.9 % saline). RESULTS: Our findings demonstrate the robust ability of the FASS-LTP technique to characterize the functional synaptic integrity maintained by disease-treatment therapies in multiple brain regions longitudinally using frozen brain tissue. COMPARISON WITH EXISTING METHODS: By providing a detailed, user-friendly protocol for this well-known analysis and including a recovery step improved the ability to robustly replicate the FASS-LTP between different brain regions. This may be extrapolated to a translational use on human clinical samples to improve understanding of the therapeutic impact on synaptic performance related to glutamate neurotransmission. CONCLUSIONS: FASS-LTP method offers a robust analysis of synaptosomes isolated from frozen tissue samples, demonstrating greater reproducibility in rodent and human synapses in physiological and pathological states.

Asunto(s)

Encéfalo , Potenciación a Largo Plazo , Sinapsis , Animales , Encéfalo/fisiopatología , Encéfalo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Sinapsis/fisiología , Sinapsis/efectos de los fármacos , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Ratones , Ratones Transgénicos , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Femenino

RESUMEN

CHD8 is a high penetrance, high confidence risk gene for autism spectrum disorder (ASD), a neurodevelopmental disorder that is substantially more prevalent among males than among females. Recent studies have demonstrated variable sex differences in the behaviors and synaptic phenotypes of mice carrying different heterozygous ASD-associated mutations in Chd8. We examined functional and structural cellular phenotypes linked to synaptic transmission in deep layer pyramidal neurons of the prefrontal cortex in male and female mice carrying a heterozygous, loss-of-function Chd8 mutation in the C57BL/6J strain across development from postnatal day 2 to adulthood. Notably, excitatory neurotransmission was decreased only in Chd8+/- males with no differences in Chd8+/- females, and the majority of alterations in inhibitory transmission were found in males. Similarly, analysis of cellular morphology showed male-specific effects of reduced Chd8 expression. Both functional and structural phenotypes were most prominent at postnatal days 14-20, a stage approximately corresponding to childhood. Our findings suggest that the effects of Chd8 mutation are predominantly seen in males and are maximal during childhood.

Asunto(s)

Corteza Prefrontal , Animales , Femenino , Humanos , Masculino , Ratones , Trastorno del Espectro Autista/genética , Haploinsuficiencia , Ratones Endogámicos C57BL , Fenotipo , Corteza Prefrontal/crecimiento & desarrollo

RESUMEN

In recent decades, various subfields within neuroscience, spanning molecular, cellular, and systemic dimensions, have significantly advanced our understanding of the elaborate molecular and cellular mechanisms that underpin learning, memory, and adaptive behaviors. There have been notable advancements in imaging techniques, particularly in reaching superficial brain structures. This progress has led to their widespread adoption in numerous laboratories. However, essential physiological and cognitive processes, including sensory integration, emotional modulation of motivated behavior, motor regulation, learning, and memory consolidation, are intricately encoded within deeper brain structures. Hence, visualization techniques such as calcium imaging using miniscopes have gained popularity for studying brain activity in unrestrained animals. Despite its utility, miniscope technology is associated with substantial brain tissue damage caused by gradient refractive index lens implantation. Furthermore, its imaging capabilities are primarily confined to the neuronal somata level, thus constraining a comprehensive exploration of subcellular processes underlying adaptive behaviors. Consequently, the trajectory of neuroscience's future hinges on the development of minimally invasive optical fiber-based endo-microscopes optimized for cellular, subcellular, and molecular imaging within the intricate depths of the brain. In pursuit of this goal, select research groups have invested significant efforts in advancing this technology. In this review, we present a perspective on the potential impact of this innovation on various aspects of neuroscience, enabling the functional exploration of in vivo cellular and subcellular processes that underlie synaptic plasticity and the neuronal adaptations that govern behavior.

RESUMEN

Plasmalogens (Pls) are considered to play a potential role in the treatment of neurodegenerative diseases. In the present study, an Alzheimer's disease (AD) model of zebrafish induced by AlCl3 was established to investigate whether the marine-derived Pls could alleviate cognitive impairments of AD zebrafish. Behavioral tests were carried out to assess the athletic ability. The transcriptional profiles of zebrafish in the control, AD model and AD_PLS group were compared and analyzed to determine the potential mechanisms of dietary Pls on AD. The study found that Pls could reverse athletic impairment in the AD zebrafish model, and the expression levels of genes related to ferroptosis, synaptic dysfunction and apoptosis were significantly altered between experimental groups. Further analysis showed that all of these genes were associated with oxidative stress (OS). These data suggest that healthy protective role of marine-derived Pls on AD zebrafish may result from inhibition of ferroptosis and neuronal apoptosis, restoring synaptic neurotransmission release, and reducing neuroinflammation. Among them, Oxidative stress is acted as the center to connect different regulation pathways. This study provides evidence to support the essential roles of OS in pathogenesis of AD, and the application of Pls in relieving AD.

Asunto(s)

Enfermedad de Alzheimer , Ferroptosis , Fármacos Neuroprotectores , Animales , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Pez Cebra/metabolismo , Plasmalógenos/metabolismo , Plasmalógenos/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo , Apoptosis , Transmisión Sináptica

RESUMEN

Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca2+-triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons.

Asunto(s)

Vesículas Sinápticas , Sinaptotagmina I , Animales , Ratones , Endocitosis/fisiología , Exocitosis/fisiología , Lípidos , Transmisión Sináptica , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo

RESUMEN

An abnormal glutamate signaling pathway has been proposed in the mechanisms of autism spectrum disorder (ASD). However, less is known about the involvement of alterations of glutaminase 1 (GLS1) in the pathophysiology of ASD. We show that the transcript level of GLS1 is significantly decreased in the postmortem frontal cortex and peripheral blood of ASD subjects. Mice lacking Gls1 in CamKIIα-positive neurons display a series of ASD-like behaviors, synaptic excitatory and inhibitory (E/I) imbalance, higher spine density, and glutamate receptor expression in the prefrontal cortex, as well as a compromised expression pattern of genes involved in synapse pruning and less engulfed synaptic puncta in microglia. A low dose of lipopolysaccharide treatment restores microglial synapse pruning, corrects synaptic neurotransmission, and rescues behavioral deficits in these mice. In summary, these findings provide mechanistic insights into Gls1 loss in ASD symptoms and identify Gls1 as a target for the treatment of ASD.

Asunto(s)

Trastorno del Espectro Autista , Ratones , Animales , Trastorno del Espectro Autista/metabolismo , Glutaminasa/genética , Glutaminasa/metabolismo , Neuronas/metabolismo , Transmisión Sináptica/genética , Corteza Prefrontal/metabolismo , Modelos Animales de Enfermedad

RESUMEN

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[14C] glutamate and [3H] GABA, and also [3H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.

Asunto(s)

Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Animales , Membrana Dobles de Lípidos , Ratas , Ratas Wistar , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

Here, carbon nanodots synthesized from ß-alanine (Ala-CDs) and detonation nanodiamonds (NDs) were assessed using (1) radiolabeled excitatory neurotransmitters L-[14C]glutamate, D-[2,33H]aspartate, and inhibitory ones [3H]GABA, [3H]glycine for registration of their extracellular concentrations in rat cortex nerve terminals; (2) the fluorescent ratiometric probe NR12S and pH-sensitive probe acridine orange for registration of the membrane lipid order and synaptic vesicle acidification, respectively; (3) suspended bilayer lipid membrane (BLM) to monitor changes in transmembrane current. In nerve terminals, Ala-CDs and NDs increased the extracellular concentrations of neurotransmitters and decreased acidification of synaptic vesicles, whereas have not changed sufficiently the lipid order of membrane. Both nanoparticles, Ala-CDs and NDs, were capable of increasing the conductance of the BLM by inducing stable potential-dependent cation-selective pores. Introduction of divalent cations, Zn2+ or Cd2+ on the particles` application side (cis-side) increased the rate of Ala-CDs pore-formation in the BLM. The application of positive potential (+100 mV) to the cis-chamber with Ala-CDs or NDs also activated the insertion as compared with the negative potential (-100 mV). The Ala-CD pores exhibited a wide-range distribution of conductances between 10 and 60 pS and consecutive increase in conductance of each major peak by ~10 pS, which suggest the clustering of the same basic ion-conductive structure. NDs also formed ion-conductive pores ranging from 6 pS to 60 pS with the major peak of conductance at ~12 pS in cholesterol-containing membrane. Observed Ala-CDs and NDs-induced increase in transmembrane current coincides with disturbance of excitatory and inhibitory neurotransmitter transport in nerve terminals.

Asunto(s)

Corteza Cerebral/metabolismo , Nanopartículas/química , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Vesículas Sinápticas/química , Alanina/síntesis química , Alanina/química , Animales , Ácido Aspártico/síntesis química , Ácido Aspártico/química , Ácido Aspártico/farmacología , Carbono/química , Carbono/farmacología , Radioisótopos de Carbono/química , Radioisótopos de Carbono/farmacología , Cationes/farmacología , Corteza Cerebral/efectos de la radiación , Colesterol/química , Ácido Glutámico/síntesis química , Ácido Glutámico/química , Ácido Glutámico/farmacología , Membrana Dobles de Lípidos/química , Nanodiamantes/química , Neurotransmisores/química , Neurotransmisores/farmacología , Ratas , Sinapsis/química , Transmisión Sináptica/fisiología , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Ácido gamma-Aminobutírico/síntesis química , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/farmacología

RESUMEN

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca2+ and a number of Ca2+ sensors including synaptotagmins (Syts) and Double C2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C2 domain architecture, but are functionally diverse in their subcellular location, Ca2+-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca2+-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca2+ sensors that may be implicated in the spontaneous release of glutamate.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de Unión al Calcio/fisiología , Calcio/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Sinaptotagmina I/fisiología , Proteínas de Transporte Vesicular/fisiología , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Células Cultivadas , Secuencia Conservada , Ácido Glutámico/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Potenciales Postsinápticos Miniatura/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Dominios Proteicos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sinaptotagmina I/química , Sinaptotagmina I/deficiencia , Sinaptotagmina I/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genética , Rabfilina-3A

RESUMEN

Synaptic neurotransmission has recently been proposed to function via either a membrane-independent or a membrane-dependent mechanism, depending on the neurotransmitter type. In the membrane-dependent mechanism, amphipathic neurotransmitters first partition to the lipid headgroup region and then diffuse along the membrane plane to their membrane-buried receptors. However, to date, this mechanism has not been demonstrated for any neurotransmitter-receptor complex. Here, we combined isothermal calorimetry measurements with a diverse set of molecular dynamics simulation methods to investigate the partitioning of an amphipathic neurotransmitter (dopamine) and the mechanism of its entry into the ligand-binding site. Our results show that the binding of dopamine to its receptor is consistent with the membrane-dependent binding and entry mechanism. Both experimental and simulation results showed that dopamine favors binding to lipid membranes especially in the headgroup region. Moreover, our simulations revealed a ligand-entry pathway from the membrane to the binding site. This pathway passes through a lateral gate between transmembrane alpha-helices 5 and 6 on the membrane-facing side of the protein. All in all, our results demonstrate that dopamine binds to its receptor by a membrane-dependent mechanism, and this is complemented by the more traditional binding mechanism directly through the aqueous phase. The results suggest that the membrane-dependent mechanism is common in other synaptic receptors, too.

Asunto(s)

Dopamina , Simulación de Dinámica Molecular , Sitios de Unión , Membrana Celular/metabolismo , Dopamina/metabolismo , Membrana Dobles de Lípidos/metabolismo , Unión Proteica , Transmisión Sináptica

RESUMEN

Synaptic neurotransmission is generally considered as a function of membrane-embedded receptors and ion channels in response to the neurotransmitter (NT) release and binding. This perspective aims to widen the protein-centric view by including another vital component-the synaptic membrane-in the discussion. A vast set of atomistic molecular dynamics simulations and biophysical experiments indicate that NTs are divided into membrane-binding and membrane-nonbinding categories. The binary choice takes place at the water-membrane interface and follows closely the positioning of the receptors' binding sites in relation to the membrane. Accordingly, when a lipophilic NT is on route to a membrane-buried binding site, it adheres on the membrane and, then, travels along its plane towards the receptor. In contrast, lipophobic NTs, which are destined to bind into receptors with extracellular binding sites, prefer the water phase. This membrane-based sorting splits the neurotransmission into membrane-independent and membrane-dependent mechanisms and should make the NT binding into the receptors more efficient than random diffusion would allow. The potential implications and notable exceptions to the mechanisms are discussed here. Importantly, maintaining specific membrane lipid compositions (MLCs) at the synapses, especially regarding anionic lipids, affect the level of NT-membrane association. These effects provide a plausible link between the MLC imbalances and neurological diseases such as depression or Parkinson's disease. Moreover, the membrane plays a vital role in other phases of the NT life cycle, including storage and release from the synaptic vesicles, transport from the synaptic cleft, as well as their synthesis and degradation.

Asunto(s)

Metabolismo de los Lípidos/fisiología , Lípidos , Neurotransmisores/metabolismo , Transmisión Sináptica/fisiología , Animales , Humanos , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo

RESUMEN

Migraine appears to be the most common neurological syndrome in primary care. Pain in migraine is mediated by the release of inflammatory mediators at the level of nerves and blood vessels. The antioxidant and neuroprotective effects of vitamin D in the central nervous system suggest that deficiency of this vitamin can be involved in migraine. Moreover, low serum levels of vitamin D correlates with a higher incidence of chronic pain, including migraine and in co-administered with anti-migraine treatment reduces the frequency of migraine attacks. We report a 46-year old woman affected by migraine, anxiety and mild depressive mood (MSQ score: 24; BDI score: 34; VAS score: 8) that partially improved with pregabalin treatment (VAS: 5). Laboratory findings documented low serum levels of vitamin D (25-hydroxy-vitamin D: 12 ng/mL; normal range: 20-100 ng/mL; 1-25 di-hydroxy-vitamin D: 19 ng/mL, normal range: 25-66 ng/mL). The treatment with 10,000 UI vitamin D during pregabalin therapy induced an improvement of clinical symptoms (pain, anxiety and depression) and of the quality of life. This case report suggest that in chronic migraine patient with anxiety and mild mood depression in treatment with pregabalin a supplementation of vitamin D improvement the clinical symptoms of migraine and a modulation of inhibitoy synaptic neurotransmission may explain this effect in our migraine patient.

Asunto(s)

Analgésicos/administración & dosificación , Trastornos Migrañosos/tratamiento farmacológico , Pregabalina/administración & dosificación , Vitamina D/administración & dosificación , Ansiedad/tratamiento farmacológico , Depresión/tratamiento farmacológico , Femenino , Humanos , Persona de Mediana Edad , Trastornos Migrañosos/fisiopatología , Trastornos Migrañosos/psicología , Calidad de Vida , Resultado del Tratamiento , Vitamina D/sangre , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/tratamiento farmacológico

RESUMEN

Conventional serotonin-enhancing antidepressants including selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs) have shown effectiveness in the treatment of major depression, but their significant limitations such as slowness of action have led to intensive research efforts to develop new antidepressants. Increased synaptic neurotransmission of serotonin (5-hdroxytryptamine; 5-HT) through orchestration of stimulation and blockade of various subtypes of 5-HT receptors is involved in the mechanisms of action of SSRIs. Agonists at the 5-HT1A, 5-HT1B, 5-HT2C, 5-HT4, and 5-HT6 receptors and antagonists at the 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5- HT6, and 5-HT7 receptors have shown antidepressant properties in clinical and preclinical studies. However, paradoxical antidepressant-like effects of both agonists and antagonists at particular 5-HT receptors suggest the need to consider the neurochemical mechanisms of each 5-HT receptor subtype. Therefore, better knowledge of the involvement of individual 5-HT receptors in the mechanisms of action of currently used antidepressants as well as antidepressant effects of selective ligands of 5- HT receptor subtypes will provide opportunities for the development of future antidepressants with more rapid onset of action, fewer side effects, and better efficacy than SSRIs.

Asunto(s)

Antidepresivos/química , Antidepresivos/farmacología , Trastorno Depresivo Mayor/tratamiento farmacológico , Receptores de Serotonina/clasificación , Receptores de Serotonina/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Descubrimiento de Drogas , Humanos , Receptores de Serotonina/genética

RESUMEN

Adult neurogenesis is necessary for proper cognition and behavior, however, the mechanisms that underlie the integration and maturation of newborn neurons into the pre-existing hippocampal circuit are not entirely known. In this study, we sought to determine the role of action potential (AP)-dependent synaptic transmission by adult-generated dentate granule cells (DGCs) in their survival and function within the existing circuitry. We used a triple transgenic mouse (NestinCreERT2 :Snap25fl/fl : tdTomato) to inducibly inactivate AP-dependent synaptic transmission within adult hippocampal progenitors and their progeny. Behavioral testing in a hippocampal-dependent A/B contextual fear-discrimination task revealed impaired discrimination learning in mice harboring SNAP-25-deficient adult-generated dentate granule cells (DGCs). Despite poor performance on this neurogenesis-dependent task, the production and survival of newborn DGCs was quantitatively unaltered in tamoxifen-treated NestinCreERT2 :Snap25fl/fl : tdTomato SNAP compared to tamoxifen-treated NestinCreERT2 :Snap25wt/wt : tdTomato control mice. Although SNAP-25-deficient adult DGCs displayed a small but statistically significant enhancement in proximal dendritic branching, their overall dendritic length and distal branching complexity was unchanged. SNAP-25-deficient newborn DGCs also displayed robust efferent mossy fiber output to CA3, with normal linear density of large mossy fiber terminals (LMTs). These studies suggest that AP-dependent neurotransmitter release by newborn DGCs is not essential for their survival or rudimentary structural maturation within the adult hippocampus.

Asunto(s)

Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Discapacidades para el Aprendizaje/genética , Neurogénesis/fisiología , Neuronas/fisiología , Proteína 25 Asociada a Sinaptosomas/deficiencia , Animales , Animales Recién Nacidos , Células Cultivadas , Aprendizaje Discriminativo/efectos de los fármacos , Aprendizaje Discriminativo/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Miedo/fisiología , Ácido Glutámico/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Discapacidades para el Aprendizaje/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nestina/genética , Nestina/metabolismo , Neuronas/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , Proteína 25 Asociada a Sinaptosomas/genética , Transfección

RESUMEN

Ohtahara syndrome, also known as type 4 of Early Infantile Epileptic Encephalopathy with suppression bursts (EIEE-4) is currently an untreatable disorder that presents with seizures and impaired cognition. EIEE-4 patients have mutations most frequently in the STXBP1 gene encoding a Sec protein, munc18-1. The exact molecular mechanism of how these munc18-1 mutations cause impaired cognition, remains elusive. The leading haploinsufficiency hypothesis posits that mutations in munc18-1 render the protein unstable leading to its degradation. Expression driven by the healthy allele is not sufficient to maintain the physiological function resulting in haploinsufficiency. The aim of this study has been to understand how munc18-1 haploinsufficiency causes cognitive impairment seen in EIEE-4. Here we present results from behavioral to cellular effects from a mouse model of munc18-1 haploinsufficiency. Munc18-1 heterozygous knock-out mice showed impaired spatial learning and memory in behavior tests as well as reduced synaptic plasticity in hippocampal CA1 long-term potentiation. Cultured munc18-1 heterozygous hippocampal neurons had significantly slower rate of synaptic vesicle release and decreased readily releasable vesicle pool compared to wild-type control neurons in fluorescent FM dye assays. These results demonstrate that reduced munc18-1 levels are sufficient to impair learning and memory by reducing neurotransmitter release. Therefore, our study implicates munc18-1 haploinsufficiency as a primary cause of cognitive impairment seen in EIEE-4 patients.

Asunto(s)

Haploinsuficiencia/genética , Aprendizaje/fisiología , Memoria/fisiología , Proteínas Munc18/genética , Espasmos Infantiles/genética , Animales , Encéfalo/fisiopatología , Heterocigoto , Ratones Noqueados , Mutación/genética , Neuronas/metabolismo , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismo

RESUMEN

In this study, the dopamine-lipid bilayer interactions were probed with three physiologically relevant ion compositions using atomistic molecular dynamics simulations and free energy calculations. The in silico results indicate that calcium is able to decrease significantly the binding of dopamine to a neutral (zwitterionic) phosphatidylcholine lipid bilayer model mimicking the inner leaflet of a presynaptic vesicle. We argue that the observed calcium-induced effect is likely in crucial role in the neurotransmitter release from the presynaptic vesicles docked in the active zone of nerve terminals. The inner leaflets of presynaptic vesicles, which are responsible for releasing neurotransmitters into the synaptic cleft, are mainly composed of neutral lipids such as phosphatidylcholine and phosphatidylethanolamine. The neutrality of the lipid head group region, enhanced by a low pH level, should limit membrane aggregation of transmitters. In addition, the simulations suggest that the high calcium levels inside presynaptic vesicles prevent even the most lipophilic transmitters such as dopamine from adhering to the inner leaflet surface, thus rendering unhindered neurotransmitter release feasible.

Asunto(s)

Calcio/metabolismo , Dopamina/metabolismo , Terminales Presinápticos/metabolismo , Transmisión Sináptica/fisiología , Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular

RESUMEN

The effects of the intravenous anesthetic, propofol, on glycinergic transmission and on glycine receptor-mediated whole-cell currents (IGly) were examined in the substantia gelatinosa (SG) neuronal cell body, mechanically dissociated from the rat spinal cord. This "synaptic bouton" preparation, which retains functional native nerve endings, allowed us to evaluate glycinergic inhibitory postsynaptic currents (IPSCs) and whole-cell currents in a preparation in which experimental solution could rapidly access synaptic terminals. Synaptic IPSCs were measured as spontaneous (s) and evoked (e) IPSCs. The eIPSCs were elicited by applying paired-pulse focal electrical stimulation, while IGly was evoked by a bath application of glycine. A concentration-dependent enhancement of IGly was observed for ≥10µM propofol. Propofol (≥3µM) significantly increased the frequency of sIPSCs and prolonged the decay time without altering the current amplitude. However, propofol (≥3µM) also significantly increased the mean amplitude of eIPSCs and decreased the failure rate (Rf). A decrease in the paired-pulse ratio (PPR) was noted at higher concentrations (≥10µM). The decay time of eIPSCs was prolonged only at the maximum concentration tested (30µM). Propofol thus acts at both presynaptic glycine release machinery and postsynaptic glycine receptors. At clinically relevant concentrations (<1µM) there was no effect on IGly, sIPSCs or eIPSCs suggesting that at anesthetic doses propofol does not affect inhibitory glycinergic synapses in the spinal cord.

Asunto(s)

Glicinérgicos/farmacología , Neuronas/efectos de los fármacos , Propofol/farmacología , Sinapsis/efectos de los fármacos , Anestésicos Intravenosos/farmacología , Animales , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Glicina/metabolismo , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Neuronas/metabolismo , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Glicina/metabolismo , Médula Espinal/efectos de los fármacos , Sinapsis/metabolismo , Transmisión Sináptica/fisiología

RESUMEN

We evaluated the effects of N2O on synaptic transmission using a preparation of mechanically dissociated rat hippocampal CA3 neurons that allowed assays of single bouton responses evoked from native functional nerve endings. We studied the effects of N2O on GABAA, glutamate, AMPA and NMDA receptor-mediated currents (IGABA, IGlu, IAMPA and INMDA) elicited by exogenous application of GABA, glutamate, (S)-AMPA, and NMDA and spontaneous, miniature, and evoked GABAergic inhibitory and glutamatergic excitatory postsynaptic current (sIPSC, mIPSC, eIPSC, sEPSC, mEPSC and eEPSC) in mechanically dissociated CA3 neurons. eIPSC and eEPSC were evoked by focal electrical stimulation of a single bouton. Administration of 70% N2O altered neither IGABA nor the frequency and amplitude of both sIPSCs and mIPSCs. In contrast, N2O decreased the amplitude of eIPSCs, while increasing failure rates (Rf) and paired-pulse ratios (PPR) in a concentration-dependent manner. On the other hand, N2O decreased IGlu, IAMPA and INMDA. Again N2O did not change the frequency and amplitude of either sEPSCs of mEPSCs. N2O also decreased amplitudes of eEPSCs with increased Rf and PPR. The decay phases of all synaptic responses were unchanged. The present results indicated that N2O inhibits the activation of AMPA/KA and NMDA receptors and also that N2O preferentially depress the action potential-dependent GABA and glutamate releases but had little effects on spontaneous and miniature releases.

Asunto(s)

Región CA3 Hipocampal/efectos de los fármacos , Óxido Nitroso/farmacología , Terminales Presinápticos/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Estimulación Eléctrica , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Ácido Glutámico/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , N-Metilaspartato/farmacología , Ratas , Ratas Wistar , Transmisión Sináptica/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Ácido gamma-Aminobutírico/farmacología

RESUMEN

BACKGROUND: Tourette syndrome is a disorder characterized by persistent motor and vocal tics, and frequently accompanied by the comorbidities attention deficit hyperactivity disorder and obsessive-compulsive disorder. Impaired synaptic neurotransmission has been implicated in its pathogenesis. Our aim was to investigate the association of 28 candidate genes, including genes related to synaptic neurotransmission and neurotrophic factors, with Tourette syndrome. METHODS: We genotyped 506 polymorphisms in a discovery cohort from the United States composed of 112 families and 47 unrelated singletons with Tourette syndrome (201 cases and 253 controls). Genes containing significant polymorphisms were imputed to fine-map the signal(s) to potential causal variants. Allelic analyses in Tourette syndrome cases were performed to check the role in attention deficit hyperactivity disorder and obsessive-compulsive disorder comorbidities. Target polymorphisms were further studied in a replication cohort from southern Spain composed of 37 families and three unrelated singletons (44 cases and 73 controls). RESULTS: The polymorphism rs3096140 in glial cell line-derived neurotrophic factor gene (GDNF) was significant in the discovery cohort after correction (P = 1.5 × 10(-4) ). No linkage disequilibrium was found between rs3096140 and other functional variants in the gene. We selected rs3096140 as target polymorphism, and the association was confirmed in the replication cohort (P = 0.01). No association with any comorbidity was found. CONCLUSIONS: As a conclusion, a common genetic variant in GDNF is associated with Tourette syndrome. A defect in the production of GDNF could compromise the survival of parvalbumin interneurons, thus altering the excitatory/inhibitory balance in the corticostriatal circuitry. Validation of this variant in other family cohorts is necessary.

Asunto(s)

Estudios de Asociación Genética , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Transmisión Sináptica/genética , Síndrome de Tourette/genética , Adolescente , Adulto , Niño , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo Genético , Estados Unidos , Adulto Joven

RESUMEN

BACKGROUND AND PURPOSE: Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. EXPERIMENTAL APPROACH: Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. KEY RESULTS: Menthol (150-750 µM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 µM), bicuculline (30 µM) and Zn(2+) (100 µM), but unaffected by gabazine (10 µM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 µM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 µM). CONCLUSIONS AND IMPLICATIONS: These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA -mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA -related pharmacotherapies.

Asunto(s)

Mentol/farmacología , Neuronas/efectos de los fármacos , Sustancia Gris Periacueductal/efectos de los fármacos , Receptores de GABA-A/fisiología , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Neuronas/fisiología , Sustancia Gris Periacueductal/fisiología , Ratas Sprague-Dawley