RESUMEN
Nicotinamide adenine dinucleotide (NAD)+ serves as a crucial coenzyme in numerous essential biological reactions, and its cellular availability relies on the activity of the nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed salvage pathway. Here we show that treatment with saturated fatty acids activates the NAD+ salvage pathway in hypothalamic astrocytes. Furthermore, inhibition of this pathway mitigates hypothalamic inflammation and attenuates the development of obesity in male mice fed a high-fat diet (HFD). Mechanistically, CD38 functions downstream of the NAD+ salvage pathway in hypothalamic astrocytes burdened with excess fat. The activation of the astrocytic NAMPT-NAD+-CD38 axis in response to fat overload induces proinflammatory responses in the hypothalamus. It also leads to aberrantly activated basal Ca2+ signals and compromised Ca2+ responses to metabolic hormones such as insulin, leptin, and glucagon-like peptide 1, ultimately resulting in dysfunctional hypothalamic astrocytes. Our findings highlight the significant contribution of the hypothalamic astrocytic NAD+ salvage pathway, along with its downstream CD38, to HFD-induced obesity.
Asunto(s)
Grasas de la Dieta , NAD , Masculino , Ratones , Animales , NAD/metabolismo , Grasas de la Dieta/metabolismo , Astrocitos/metabolismo , Obesidad/metabolismo , Hipotálamo/metabolismo , Citocinas/metabolismoRESUMEN
Several plant-derived natural products with anti-SARS-CoV-2 activity have been evaluated for the potential to serve as chemotherapeutic agents for the treatment of COVID-19. Codonopsis lanceolata (CL) has long been used as a medicinal herb in East Asian countries to treat inflammatory diseases of the respiratory system but its antiviral activity has not been investigated so far. Here, we showed that CL extract and its active compound lancemaside A (LA) displayed potent inhibitory activity against SARS-CoV-2 infection using a pseudotyped SARS-CoV-2 entry assay system. We demonstrated that this inhibitory effect of LA was due to the alteration of membrane cholesterol and blockade of the membrane fusion between SARS-CoV-2 and host cells by filipin staining and cell-based membrane fusion assays. Our findings also showed that LA, as a membrane fusion blocker, could impede the endosomal entry pathway of SARS-CoV-2 and its variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in Vero cells with similar of IC50 values ranging from 2.23 to 3.37 µM as well as the TMPRSS2-mediated viral entry pathway in A549 cells overexpressing ACE2 and TMPRSS2 with IC50 value of 3.92 µM. We further demonstrated that LA could prevent the formation of multinucleated syncytia arising from SARS-CoV-2 spike protein-mediated membrane fusion. Altogether, the findings reported here suggested that LA could be a broad-spectrum anti-SARS-CoV-2 therapeutic agent by targeting the fusion of viral envelope with the host cell membrane.
Asunto(s)
COVID-19 , Codonopsis , Animales , Chlorocebus aethiops , Humanos , SARS-CoV-2 , Antivirales/farmacología , Células Vero , Codonopsis/metabolismo , Glicoproteína de la Espiga del Coronavirus , Internalización del VirusRESUMEN
Hypothalamus is a brain region that controls food intake and energy expenditure while sensing signals that convey information about energy status. Within the hypothalamus, molecularly and functionally distinct neurons work in concert under physiological conditions. However, under pathological conditions such as in diet-induced obesity (DIO) model, these neurons show dysfunctional firing patterns and distorted regulation by neurotransmitters and neurohormones. Concurrently, resident glial cells including astrocytes dramatically transform into reactive states. In particular, it has been reported that reactive astrogliosis is observed in the hypothalamus, along with various neuroinflammatory signals. However, how the reactive astrocytes control and modulate DIO by influencing neighboring neurons is not well understood. Recently, new lines of evidence have emerged indicating that these reactive astrocytes directly contribute to the pathology of obesity by synthesizing and tonically releasing the major inhibitory transmitter GABA. The released GABA strongly inhibits the neighboring neurons that control energy expenditure. These surprising findings shed light on the interplay between reactive astrocytes and neighboring neurons in the hypothalamus. This review summarizes recent discoveries related to the functions of hypothalamic reactive astrocytes in obesity and raises new potential therapeutic targets against obesity.
Asunto(s)
Astrocitos , Hipotálamo , Dieta Alta en Grasa , Metabolismo Energético , Humanos , Obesidad/patologíaRESUMEN
BACKGROUND: NMDA receptor (NMDAR) hypofunction has been implicated in several psychiatric disorders with impairment of cognitive flexibility. However, the molecular mechanism of how NMDAR hypofunction with decreased NMDAR tone causes the impairment of cognitive flexibility has been minimally understood. Furthermore, it has been unclear whether hippocampal astrocytes regulate NMDAR tone and cognitive flexibility. METHODS: We employed cell type-specific genetic manipulations, ex vivo electrophysiological recordings, sniffer patch recordings, cutting-edge biosensor for norepinephrine, and behavioral assays to investigate whether astrocytes can regulate NMDAR tone by releasing D-serine and glutamate. Subsequently, we further investigated the role of NMDAR tone in heterosynaptic long-term depression, metaplasticity, and cognitive flexibility. RESULTS: We found that hippocampal astrocytes regulate NMDAR tone via BEST1-mediated corelease of D-serine and glutamate. Best1 knockout mice exhibited reduced NMDAR tone and impairments of homosynaptic and α1 adrenergic receptor-dependent heterosynaptic long-term depression, which leads to defects in metaplasticity and cognitive flexibility. These impairments in Best1 knockout mice can be rescued by hippocampal astrocyte-specific BEST1 expression or enhanced NMDAR tone through D-serine supplement. D-serine injection in Best1 knockout mice during initial learning rescues subsequent reversal learning. CONCLUSIONS: These findings indicate that NMDAR tone during initial learning is important for subsequent learning, and hippocampal NMDAR tone regulated by astrocytic BEST1 is critical for heterosynaptic long-term depression, metaplasticity, and cognitive flexibility.
Asunto(s)
Astrocitos , Receptores de N-Metil-D-Aspartato , Animales , Astrocitos/metabolismo , Bestrofinas/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Humanos , Ratones , Receptores de N-Metil-D-Aspartato/fisiología , Serina/metabolismoRESUMEN
ABSTRACT: Nociceptors are known to directly recognize bacterial cell wall components or secreted toxins, thereby leading to pain induced by bacterial infection. However, direct activation of nociceptors by bacterial metabolites remains unclear although bacteria produce numerous metabolites related to health and disease. In this study, we investigated whether and how a common bacterial metabolite, indole, which is produced by normal microflora of the gastrointestinal tract and oral cavity, can directly activate nociceptive sensory neurons. We found that indole elicits calcium response and evokes inward currents in subsets of dorsal root ganglia (DRG) neurons. Intraplantar (i.pl.) injection of indole produced nocifensive behaviors in adult mice, which were enhanced in complete Freund's adjuvant-induced chronic inflammatory condition. Indole increased calcitonin gene-related peptide release in DRG neurons, and i.pl. injection of indole increased hind paw thickness, suggesting its role in generation of neurogenic inflammation. These in vitro and in vivo indole-induced responses were pharmacologically blocked by transient receptor potential ankyrin 1 (TRPA1) antagonist, HC-030031, and significantly abolished in TRPA1 knockout (KO) mice, indicating that indole targets TRPA1 for its action in DRG neurons. Nocifensive licking behavior induced by the injection of live Escherichia coli was significantly decreased in tryptophanase mutant (TnaA KO) E. coli- injected mice that lack indole production, further supporting the idea that bacteria-derived indole can induce pain during infection. Identifying the mechanism of action of indole through TRPA1 provides insights into bacteria-neuron interactions and the role of bacterial metabolites in pain signaling, especially in inflammation-accompanied bacterial infection.
Asunto(s)
Indoles , Nociceptores , Canal Catiónico TRPA1 , Animales , Escherichia coli/metabolismo , Ganglios Espinales , Indoles/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nociceptores/metabolismo , Dolor/inducido químicamente , Dolor/metabolismo , Células Receptoras Sensoriales/metabolismo , Canal Catiónico TRPA1/antagonistas & inhibidores , Canal Catiónico TRPA1/genéticaRESUMEN
Many studies have focussed on modulating the activity of γ-aminobutyric acid transaminase (GABA-T), a GABA-catabolizing enzyme, for treating neurological diseases, such as epilepsy and drug addiction. Nevertheless, human GABA-T synthesis and purification have not been established. Thus, biochemical and drug design studies on GABA-T have been performed by using porcine GABA-T mostly and even bacterial GABA-T. Here we report an optimised protocol for overexpression of 6xHis-tagged human GABA-T in human cells followed by a two-step protein purification. Then, we established an optimised human GABA-T (0.5 U/mg) activity assay. Finally, we compared the difference between human and bacterial GABA-T in sensitivity to two irreversible GABA-T inhibitors, gabaculine and vigabatrin. Human GABA-T in homodimeric form showed 70-fold higher sensitivity to vigabatrin than bacterial GABA-T in multimeric form, indicating the importance of using human GABA-T. In summary, our newly developed protocol can be an important first step in developing more effective human GABA-T modulators.
Asunto(s)
4-Aminobutirato Transaminasa/biosíntesis , 4-Aminobutirato Transaminasa/aislamiento & purificación , 4-Aminobutirato Transaminasa/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/efectos de los fármacos , Saponinas/farmacología , Serina Endopeptidasas/metabolismo , Triterpenos/farmacología , Internalización del Virus/efectos de los fármacos , Antivirales/química , COVID-19/metabolismo , Línea Celular , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Modelos Moleculares , Platycodon/química , SARS-CoV-2/fisiología , Saponinas/química , Triterpenos/químicaRESUMEN
Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release.
Asunto(s)
Astrocitos/fisiología , Inhibición Neural/fisiología , Tálamo/fisiología , Percepción del Tacto/fisiología , Ácido gamma-Aminobutírico/fisiología , Familia de Aldehído Deshidrogenasa 1/metabolismo , Amina Oxidasa (conteniendo Cobre)/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Bestrofinas/biosíntesis , Bestrofinas/genética , Femenino , Antagonistas del GABA , Inmunohistoquímica , Potenciales Postsinápticos Inhibidores/fisiología , Macrólidos/farmacología , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Cultivo Primario de Células , Piridazinas/farmacología , ARN Interferente Pequeño/farmacología , Retinal-Deshidrogenasa/metabolismo , Ácido gamma-Aminobutírico/biosíntesis , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
In the descending analgesia pathway, opioids are known to disinhibit the projections from the periaqueductal gray (PAG) to the rostral ventromedial medulla (RVM), leading to suppression of pain signals at the spinal cord level. The locus coeruleus (LC) has been proposed to engage in the descending pathway through noradrenergic inputs to the spinal cord. Nevertheless, how the LC is integrated in the descending analgesia circuit has remained unknown. Here, we show that the opioidergic analgesia pathway is bifurcated in structure and function at the PAG. A knockout as well as a PAG-specific knockdown of phospholipase C ß4 (PLCß4), a signaling molecule for G protein-coupled receptors, enhanced swim stress-induced and morphine-induced analgesia in mice. Immunostaining after simultaneous retrograde labeling from the RVM and the LC revealed two mutually exclusive neuronal populations at the PAG, each projecting either to the LC or the RVM, with PLCß4 expression only in the PAG-LC projecting cells that provide a direct synaptic input to LC-spinal cord (SC) projection neurons. The PAG-LC projection neurons in wild-type mice turned quiescent in response to opiates, but remained active in the PLCß4 mutant, suggesting a possibility that an increased adrenergic function induced by the persistent PAG-LC activity underlies the enhanced opioid analgesia in the mutant. Indeed, the enhanced analgesia in the mutant was reversed by blocking α2-noradrenergic receptors. These findings indicate that opioids suppress descending analgesia through the PAG-LC pathway, while enhancing it through the PAG-RVM pathway, i.e., two distinct pathways with opposing effects on opioid analgesia. These results point to a therapeutic target in pain control.
Asunto(s)
Analgesia/métodos , Mesencéfalo/fisiopatología , Manejo del Dolor/métodos , Analgésicos Opioides/farmacología , Animales , Masculino , Mesencéfalo/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Morfina/farmacología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Dolor/fisiopatología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiología , Yin-YangRESUMEN
Repositioning of the antipsychotic drug trifluoperazine for treatment of glioblastoma, an aggressive brain tumor, has been previously suggested. However, trifluoperazine did not increase the survival time in mice models of glioblastoma. In attempt to identify an effective trifluoperazine analog, fourteen compounds have been synthesized and biologically in vitro and in vivo assessed. Using MTT assay, compounds 3dc and 3dd elicited 4-5 times more potent inhibitory activity than trifluoperazine with IC50â¯=â¯2.3 and 2.2⯵M against U87MG glioblastoma cells, as well as, IC50â¯=â¯2.2 and 2.1⯵M against GBL28 human glioblastoma patient derived primary cells, respectively. Furthermore, they have shown a reasonable selectivity for glioblastoma cells over NSC normal neural cell. In vivo evaluation of analog 3dc confirmed its advantageous effect on reduction of tumor size and increasing the survival time in brain xenograft mouse model of glioblastoma. Molecular modeling simulation provided a reasonable explanation for the observed variation in the capability of the synthesized analogs to increase the intracellular Ca2+ levels. In summary, this study presents compound 3dc as a proposed new tool for the adjuvant chemotherapy of glioblastoma.
Asunto(s)
Antineoplásicos/uso terapéutico , Antipsicóticos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Trifluoperazina/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antipsicóticos/química , Antipsicóticos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Calcio/metabolismo , Línea Celular Tumoral , Reposicionamiento de Medicamentos , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Simulación del Acoplamiento Molecular , Trifluoperazina/análogos & derivados , Trifluoperazina/farmacología , Células Tumorales CultivadasRESUMEN
Neuronal firing patterns, which are crucial for determining the nature of encoded information, have been widely studied; however, the molecular identity and cellular mechanisms of spike-frequency adaptation are still not fully understood. Here we show that spike-frequency adaptation in thalamocortical (TC) neurons is mediated by the Ca2+-activated Cl- channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in TC neurons results in significantly reduced spike-frequency adaptation along with increased tonic spiking. Moreover, thalamus-specific knockdown of ANO2 increases visceral pain responses. These results indicate that ANO2 contributes to reductions in spike generation in highly activated TC neurons and thereby restricts persistent information transmission.
Asunto(s)
Anoctaminas/metabolismo , Calcio/farmacología , Células Receptoras Sensoriales/fisiología , Tálamo/fisiología , Adenoviridae , Animales , Anoctaminas/genética , Bestrofinas/genética , Bestrofinas/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Técnicas de Placa-Clamp , ortoaminobenzoatos/farmacologíaRESUMEN
α-Pinene is a major monoterpene of the pine tree essential oils. It has been reported that α-pinene shows anxiolytic and hypnotic effects upon inhaled administration. However, hypnotic effect by oral supplementation and the molecular mechanism of α-pinene have not been determined yet. By combining in vivo sleep behavior, ex vivo electrophysiological recording from brain slices, and in silico molecular modeling, we demonstrate that (-)-α-pinene shows sleep enhancing property through a direct binding to GABAA-benzodiazepine (BZD) receptors by acting as a partial modulator at the BZD binding site. The effect of (-)-α-pinene on sleep-wake profiles was evaluated by recording electroencephalogram and electromyogram. The molecular mechanism of (-)-α-pinene was investigated by electrophysiology and molecular docking study. (-)-α-pinene significantly increased the duration of non-rapid eye movement sleep (NREMS) and reduced the sleep latency by oral administration without affecting duration of rapid eye movement sleep and delta activity. (-)-α-pinene potentiated the GABAA receptor-mediated synaptic response by increasing the decay time constant of sIPSCs in hippocampal CA1 pyramidal neurons. These effects of (-)-α-pinene on sleep and inhibitory synaptic response were mimicked by zolpidem, acting as a modulator for GABAA-BZD receptors, and fully antagonized by flumazenil, an antagonist for GABAA-BZD receptor. (-)-α-pinene was found to bind to aromatic residues of α1- and -γ2 subunits of GABAA-BZD receptors in the molecular model. We conclude that (-)-α-pinene enhances the quantity of NREMS without affecting the intensity of NREMS by prolonging GABAergic synaptic transmission, acting as a partial modulator of GABAA-BZD receptors and directly binding to the BZD binding site of GABAA receptor.
Asunto(s)
Benzodiazepinas/metabolismo , Movimientos Oculares/efectos de los fármacos , Monoterpenos/farmacología , Pinus/química , Aceites de Plantas/farmacología , Receptores de GABA-A/metabolismo , Sueño/efectos de los fármacos , Animales , Monoterpenos Bicíclicos , Sitios de Unión , Flumazenil/química , Flumazenil/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Modelos Moleculares , Monoterpenos/química , Pentobarbital , Piridinas/química , Piridinas/farmacología , Sueño REM/efectos de los fármacos , Factores de Tiempo , Vigilia/efectos de los fármacos , ZolpidemRESUMEN
Transmembrane protein with unknown function 16/anoctamin-1 (ANO1) is a protein widely expressed in mammalian tissues, and it has the properties of the classic calcium-activated chloride channel (CaCC). This protein has been implicated in numerous major physiological functions. However, the lack of effective and selective blockers has hindered a detailed study of the physiological functions of this channel. In this study, we have developed a potent and selective blocker for endogenous ANO1 in Xenopus laevis oocytes (xANO1) using a drug screening method we previously established (Oh et al., 2008). We have synthesized a number of anthranilic acid derivatives and have determined the correlation between biological activity and the nature and position of substituents in these derived compounds. A structure-activity relationship revealed novel chemical classes of xANO1 blockers. The derivatives contain a --NO2 group on position 5 of a naphthyl group-substituted anthranilic acid, and they fully blocked xANO1 chloride currents with an IC50 < 10 µM. The most potent blocker, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA), had an IC50 of 0.08 µM for xANO1. Selectivity tests revealed that other chloride channels such as bestrophin-1, chloride channel protein 2, and cystic fibrosis transmembrane conductance regulator were not appreciably blocked by 10â¼30 µM MONNA. The potent and selective blockers for ANO1 identified here should permit pharmacological dissection of ANO1/CaCC function and serve as potential candidates for drug therapy of related diseases such as hypertension, cystic fibrosis, bronchitis, asthma, and hyperalgesia.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , ortoaminobenzoatos/farmacología , Animales , Anoctamina-1 , Evaluación Preclínica de Medicamentos , Femenino , Células HEK293 , Humanos , Relación Estructura-Actividad , Xenopus laevisRESUMEN
AIM OF THE STUDY: Many medicinal plants have been used for treatment of insomnia in Asia. However, scientific evidence and precise mechanism for their sedative-hypnotic activity have not been fully investigated. Thus, we investigated the binding activity of the oriental plant extracts (mainly from Korea and Japan) to the well-known molecular targets for sleep regulation, GABA(A) and 5-HT(2C) receptors. Following the binding assay, sedative-hypnotic effects of the extracts with high affinity were examined in an animal model of sleep. MATERIALS AND METHODS: Aqueous and ethanol extracts of 15 medicinal plants were tested for binding at the benzodiazepine site of GABA(A) receptor and 5-HT site of 5-HT(2C) receptor. The sedative-hypnotic effects of selected extracts were evaluated by measuring the sleep latency and sleep duration during pentobarbital-induced sleep in mice after oral administration of extracts. RESULTS: In the GABA(A) assay, the ethanol extracts of licorice and danshen displayed concentration-dependent, high affinity binding, whereas in the 5-HT(2C) assay, the ethanol extracts of ginseng and silk tree showed high affinity. Among these extracts we tested previously uncharacterized licorice and silk tree for hypnotic effects. We found the ethanol extracts of licorice and silk tree significantly decreased sleep latency and increased sleep duration in pentobarbital-induced sleep. CONCLUSIONS: We demonstrate for the first time that licorice and silk tree have the sedative-hypnotic activity possibly by modulating GABA(A) and 5-HT(2C) receptors. We propose that licorice and silk tree might be effective candidates for treatment of insomnia.
Asunto(s)
Hipnóticos y Sedantes/uso terapéutico , Medicina Tradicional de Asia Oriental , Extractos Vegetales/uso terapéutico , Plantas Medicinales/química , Receptor de Serotonina 5-HT2C/metabolismo , Receptores de GABA-A/metabolismo , Trastornos del Inicio y del Mantenimiento del Sueño/tratamiento farmacológico , Animales , Asia , Hipnóticos y Sedantes/aislamiento & purificación , Hipnóticos y Sedantes/farmacología , Masculino , Ratones , Ratones Endogámicos ICR , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Plantas Medicinales/crecimiento & desarrollo , Unión Proteica , Ensayo de Unión Radioligante , Sueño/efectos de los fármacos , Trastornos del Inicio y del Mantenimiento del Sueño/metabolismoRESUMEN
It is believed that memory reactivation transiently renders consolidated memory labile and that this labile or deconsolidated memory is reconsolidated in a protein synthesis-dependent manner. The synaptic correlate of memory deconsolidation upon reactivation, however, has not been fully characterized. Here, we show that 3,5-dihydroxyphenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRI), induces synaptic depotentiation only at thalamic input synapses onto the lateral amygdala (T-LA synapses) where synaptic potentiation is consolidated, but not at synapses where synaptic potentiation is not consolidated. Using this mGluRI-induced synaptic depotentiation (mGluRI-depotentiation) as a marker of consolidated synapses, we found that mGluRI-depotentiation correlated well with the state of memory deconsolidation and reconsolidation in a predictable manner. DHPG failed to induce mGluRI-depotentiation in slices prepared immediately after reactivation when the reactivated memory was deconsolidated. DHPG induced mGluRI-depotentiation 1 h after reactivation when the reactivated memory was reconsolidated, but it failed to do so when reconsolidation was blocked by a protein synthesis inhibitor. To test the memory-specificity of mGluRI-depotentiation, conditioned fear was acquired twice using two discriminative tones (2.8 and 20 kHz). Under this condition, mGluRI-depotentiation was fully impaired in slices prepared immediately after reactivation with both tones, whereas mGluRI-depotentiation was partially impaired immediately after reactivation with the 20 kHz tone. Consistently, microinjection of DHPG into the LA 1 h after reactivation reduced fear memory retention, whereas DHPG injection immediately after reactivation failed to do so. Our findings suggest that, upon memory reactivation, consolidated T-LA synapses enter a temporary labile state, displaying insensitivity to mGluRI-depotentiation.
Asunto(s)
Amígdala del Cerebelo/fisiología , Miedo/fisiología , Memoria/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Tálamo/fisiología , Amígdala del Cerebelo/efectos de los fármacos , Análisis de Varianza , Animales , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Agonistas de Aminoácidos Excitadores/farmacología , Miedo/efectos de los fármacos , Glicina/análogos & derivados , Glicina/farmacología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/fisiología , Masculino , Memoria/efectos de los fármacos , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/fisiología , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/agonistas , Resorcinoles/farmacología , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Tálamo/efectos de los fármacosRESUMEN
Despite considerable evidence for a critical role of neuroligin-1 in the specification of excitatory synapses, the cellular mechanisms and physiological roles of neuroligin-1 in mature neural circuits are poorly understood. In mutant mice deficient in neuroligin-1, or adult rats in which neuroligin-1 was depleted, we have found that neuroligin-1 stabilizes the NMDA receptors residing in the postsynaptic membrane of amygdala principal neurons, which allows for a normal range of NMDA receptor-mediated synaptic transmission. We observed marked decreases in NMDA receptor-mediated synaptic currents at afferent inputs to the amygdala of neuroligin-1 knockout mice. However, the knockout mice exhibited a significant impairment in spike-timing-dependent long-term potentiation (STD-LTP) at the thalamic but not the cortical inputs to the amygdala. Subsequent electrophysiological analyses indicated that STD-LTP in the cortical pathway is largely independent of activation of postsynaptic NMDA receptors. These findings suggest that neuroligin-1 can modulate, in a pathway-specific manner, synaptic plasticity in the amygdala circuits of adult animals, likely by regulating the abundance of postsynaptic NMDA receptors.
Asunto(s)
Amígdala del Cerebelo/fisiología , Moléculas de Adhesión Celular Neuronal/fisiología , Plasticidad Neuronal/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Potenciales de Acción , Amígdala del Cerebelo/metabolismo , Animales , Western Blotting , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores , Humanos , Potenciación a Largo Plazo , Ratones , Ratones Noqueados , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Tálamo/metabolismo , Tálamo/fisiologíaRESUMEN
Two firing modes of thalamocortical (TC) neurons, tonic and burst firings, are thought to reflect the divergent states of sensory signal transmission from the thalamus to the cortex. However, the behavioral consequences of changes in the thalamic firing between the two modes have not been well demonstrated. Moreover, although the firing modes of TC neurons are known to be affected by corticothalamic inputs via thalamic metabotropic glutamate receptor type 1 (mGluR1)-phospholipase C beta4 (PLCbeta4) pathway, its molecular mechanisms have not been well elucidated. We addressed these questions using PLCbeta4-deficient mice, which show decreased visceral pain responses. We demonstrate that burst and tonic firings of TC neurons are concomitantly regulated by PLCbeta4 pathway. Blocking of this pathway by the mutation simultaneously increases bursting and decreases tonic firing of TC neurons through concurrent upregulation of T- and L-type Ca(2+) currents. The mice with increased bursting and decreased tonic firing of TC neurons showed reduced visceral pain responses. Furthermore, we show that modulation of the Ca(2+) channels or protein kinase C (PKC), a downstream molecule of PLCbeta4, altered the firing modes of TC neurons and pain responses in the predicted ways. Our data demonstrate the molecular mechanism and behavioral consequences of altered firing modes of TC neurons in relaying the visceral pain signals. Our study also highlights the thalamic PLCbeta4-PKC pathway as a "molecular switch" for the firing modes of TC neurons and thus for pain sensory gating.
Asunto(s)
Potenciales de Acción/genética , Canales de Calcio/metabolismo , Neuronas/metabolismo , Dolor/metabolismo , Fosfolipasa C beta/genética , Tálamo/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/genética , Dolor/genética , Dolor/fisiopatología , Umbral del Dolor/fisiología , Proteína Quinasa C/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/genética , Transmisión Sináptica/genéticaRESUMEN
BACKGROUND: Ca²(+)-activated Clâ» channels (CaCCs) participate in many important physiological processes. However, the lack of effective and selective blockers has hindered the study of these channels, mostly due to the lack of good assay system. Here, we have developed a reliable drug screening method for better blockers of CaCCs, using the endogeneous CaCCs in Xenopus laevis oocytes and two-electrode voltage-clamp (TEVC) technique. RESULTS: Oocytes were prepared with a treatment of Ca²(+) ionophore, which was followed by a treatment of thapsigargin which depletes Ca²(+) stores to eliminate any contribution of Ca²(+) release. TEVC was performed with micropipette containing chelerythrine to prevent PKC dependent run-up or run-down. Under these conditions, Ca²(+)-activated Clâ» currents induced by bath application of Ca²(+) to oocytes showed stable peak amplitude when repetitively activated, allowing us to test several concentrations of a test compound from one oocyte. Inhibitory activities of commercially available blockers and synthesized anthranilic acid derivatives were tested using this method. As a result, newly synthesized N-(4-trifluoromethylphenyl)anthranilic acid with trifluoromethyl group (-CF3) at para position on the benzene ring showed the lowest IC50. CONCLUSION: Our results provide an optimal drug screening strategy suitable for high throughput screening, and propose N-(4-trifluoromethylphenyl)anthranilic acid as an improved CaCC blocker.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Moduladores del Transporte de Membrana/análisis , Moduladores del Transporte de Membrana/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Xenopus laevis/metabolismo , Animales , Benceno/química , Calcio/metabolismo , Canales de Cloruro/metabolismo , Concentración 50 Inhibidora , Activación del Canal Iónico/efectos de los fármacos , Ionomicina/farmacología , Permeabilidad/efectos de los fármacos , ortoaminobenzoatos/síntesis química , ortoaminobenzoatos/farmacologíaRESUMEN
Neuroligin-1 is a potent trigger for the de novo formation of synaptic connections, and it has recently been suggested that it is required for the maturation of functionally competent excitatory synapses. Despite evidence for the role of neuroligin-1 in specifying excitatory synapses, the underlying molecular mechanisms and physiological consequences that neuroligin-1 may have at mature synapses of normal adult animals remain unknown. By silencing endogenous neuroligin-1 acutely in the amygdala of live behaving animals, we have found that neuroligin-1 is required for the storage of associative fear memory. Subsequent cellular physiological studies showed that suppression of neuroligin-1 reduces NMDA receptor-mediated currents and prevents the expression of long-term potentiation without affecting basal synaptic connectivity at the thalamo-amygdala pathway. These results indicate that persistent expression of neuroligin-1 is required for the maintenance of NMDAR-mediated synaptic transmission, which enables normal development of synaptic plasticity and long-term memory in the amygdala of adult animals.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Miedo/fisiología , Potenciación a Largo Plazo , Proteínas de la Membrana/metabolismo , Memoria/fisiología , Proteínas del Tejido Nervioso/metabolismo , Amígdala del Cerebelo/citología , Animales , Moléculas de Adhesión Celular Neuronal , Activación del Canal Iónico , Masculino , Proteínas de la Membrana/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Células Piramidales/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica , Tálamo/metabolismoRESUMEN
Heteromeric NMDARs are composed of coagonist glycine-binding NR1 subunits and glutamate-binding NR2 subunits. The majority of functional NMDARs in the mammalian central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there are four types (A-D). We show that the potency of a variety of endogenous and synthetic glycine-site coagonists varies between recombinant NMDARs such that the highest potency is seen at NR2D-containing and the lowest at NR2A-containing NMDARs. This heterogeneity is specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged the S1 and S2 regions that form the ligand-binding domains and coexpressed these with NR1 subunits in Xenopus laevis oocytes. Glycine concentration-response curves for NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC(50) values similar to NR2A(WT)-containing NMDARs. However, receptors containing NR2A subunits including the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen in NR2D(WT)-containing NMDARs. In particular, two residues in the S2 region of the NR2A subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D subunit influence glycine potency. We conclude that the variation in glycine potency is caused by interactions between the NR1 and NR2 ligand-binding domains that occur following agonist binding and which may be involved in the initial conformation changes that determine channel gating.