RESUMEN
Vasopressin (VP) plays a crucial role in social memory even at the level of the olfactory bulb (OB), where OB VP cells are activated during social interactions. However, it remains unclear how VP modulates olfactory processing to enable enhanced discrimination of very similar odors, e.g., rat body odors. Thus far, it has been shown that VP reduces firing rates in mitral cells (MCs) during odor presentation in vivo and decreases the amplitudes of olfactory nerve-evoked excitatory postsynaptic potentials (ON-evoked EPSPs) in external tufted cells in vitro. We performed whole-cell patch-clamp recordings and population Ca2+ imaging on acute rat OB slices. We recorded ON-evoked EPSPs as well as spontaneous inhibitory postsynaptic currents (IPSCs) from two types of projection neurons: middle tufted cells (mTCs) and MCs. VP bath application reduced the amplitudes of ON-evoked EPSPs and the frequencies of spontaneous IPSCs in mTCs but did not change those in MCs. Therefore, we analyzed ON-evoked EPSPs in inhibitory interneurons, i.e., periglomerular cells (PGCs) and granule cells (GCs), to search for the origin of increased inhibition in mTCs. However, VP did not increase the amplitudes of evoked EPSPs in either type of interneurons. We next performed two-photon population Ca2+ imaging in the glomerular layer and the superficial GC layer of responses to stronger ON stimulation than during patch-clamp experiments that should evoke action potentials in the measured cells. We observed that VP application increased ON-evoked Ca2+ influx in juxtaglomerular cells and GC somata. Thus, our findings indicate inhibition by VP on projection neurons via strong ON input-mediated inhibitory interneuron activity. This neural modulation could improve representation of odors, hence, better discriminability of similar odors, e.g., conspecific body odors.
Asunto(s)
Potenciales Postsinápticos Excitadores , Neuronas , Bulbo Olfatorio , Vasopresinas , Animales , Bulbo Olfatorio/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ratas , Vasopresinas/farmacología , Vasopresinas/metabolismo , Neuronas/fisiología , Neuronas/efectos de los fármacos , Masculino , Técnicas de Placa-Clamp , Potenciales Postsinápticos Inhibidores/fisiología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Ratas Wistar , Interneuronas/fisiología , Interneuronas/efectos de los fármacosRESUMEN
Resistance to insecticides and acaricides is a major impediment to effectively controlling insect pests worldwide. These pests include the two-spotted spider mite Tetranychus urticae (T. urticae), which exists globally. This polyphagous herbivore causes major agricultural problems and can develop resistance to the agents above. Therefore, the continuous development of acaricides with new modes of action is important to circumvent the resistance of insects to pesticides. Acynonapyr is a novel class of acaricides containing an azabicyclo ring. In this study, we determined the activity of acynonapyr and its analogs on calcium-activated potassium (KCa2) channels in two-spotted spider mites using electrophysiological techniques (patch-clamp). We also examined their acaricidal efficacy against mites in the laboratory. The acynonapyr and analogs blocked T. urticae KCa2 (TurKCa2) channels in a concentration-dependent manner. A comparison of acaricidal activity against T. urticae with inhibitory activity against TurKCa2 revealed that TurKCa2 channels are the primary toxicological targets. Finally, we examined the effect of acynonapyr on Homo sapiens KCa2 (HsaKCa2.2) channels and demonstrated that the compound at 10 µM had a limited effect on the activity of this channel.
Asunto(s)
Acaricidas , Canales de Potasio Calcio-Activados , Tetranychidae , Acaricidas/farmacología , Animales , Tetranychidae/efectos de los fármacos , Canales de Potasio Calcio-Activados/metabolismo , Humanos , Técnicas de Placa-ClampRESUMEN
Objective: To investigate the action potential firing patterns of neurons in the visual sensory layers of the superior colliculus in early postnatal mice and the electrophysiological characteristics of neurons with different firing patterns. Methods: This experimental study utilized whole-cell patch-clamp recordings performed on neurons in the visual sensory layers of the superior colliculus using brain slices from 57 healthy male C57BL/6J mice aged 14 to 20 days (weighing 5.0 to 8.9 g) using brain slices. In current-clamp mode, action potential characteristics were analyzed based on the first action potential generated by depolarizing current, and the firing patterns of neurons were recorded using step depolarizing currents. Neuronal firing patterns were analyzed using hierarchical clustering, and the active electrical properties of neurons with different firing patterns were compared. Results: A total of 135 neurons from the visual sensory layers of the superior colliculus were successfully recorded. Cluster analysis of the neuronal firing patterns identified three types of firing patterns: tonic firing (97, 72%), phasic firing (26, 19%), and single firing (12, 9%). The number of action potentials for each firing pattern was 13.30±7.38, 3.73±3.61, and 0.83±0.39, respectively, with significant differences (P<0.001). There was no significant difference in the membrane potential response to step currents among the three firing pattern types (P>0.05). The action potential amplitudes were (60.45±12.22), (53.67±13.20), and (44.04± 12.92) mV, and the afterhyperpolarization amplitudes were (13.45±13.79), (12.02±13.11), and (20.75±2.85) mV, respectively. The maximum rising slopes were (171.29±77.46), (130.14±61.83), and (78.89±37.08) V/s, and the maximum falling slopes were (-76.33±33.61), (-68.17±31.65), and (-47.97±13.92) V/s, respectively, with all differences being statistically significant (all P<0.05). There were no significant differences in the resting membrane potential, action potential threshold, half-width, and afterhyperpolarization duration among the three firing pattern types (all P>0.05). Conclusions: In the early postnatal mice, neurons in the visual sensory layers of the superior colliculus exhibit three distinct firing patterns: tonic, phasic, and single firing. These firing pattern types show significant differences in action potential amplitude, afterhyperpolarization amplitude, maximum rising slopes, and maximum falling slopes.
Asunto(s)
Potenciales de Acción , Ratones Endogámicos C57BL , Neuronas , Técnicas de Placa-Clamp , Colículos Superiores , Animales , Ratones , Masculino , Colículos Superiores/fisiología , Potenciales de Acción/fisiología , Neuronas/fisiología , Fenómenos ElectrofisiológicosRESUMEN
Inhibitory circuits in the mammalian olfactory bulb (OB) dynamically reformat olfactory information as it propagates from peripheral receptors to downstream cortex. To gain mechanistic insight into how specific OB interneuron types support this sensory processing, we examine unitary synaptic interactions between excitatory mitral and tufted cells (MTCs), the OB projection neurons, and a conserved population of anaxonic external plexiform layer interneurons (EPL-INs) using pair and quartet whole-cell recordings in acute mouse brain slices. Physiological, morphological, neurochemical, and synaptic analyses divide EPL-INs into distinct subtypes and reveal that parvalbumin-expressing fast-spiking EPL-INs (FSIs) perisomatically innervate MTCs with release-competent dendrites and synaptically detonate to mediate fast, short-latency recurrent and lateral inhibition. Sparse MTC synchronization supralinearly increases this high-fidelity inhibition, while sensory afferent activation combined with single-cell silencing reveals that individual FSIs account for a substantial fraction of total network-driven MTC lateral inhibition. OB output is thus powerfully shaped by detonation-driven high-fidelity perisomatic inhibition.
Asunto(s)
Interneuronas , Bulbo Olfatorio , Animales , Interneuronas/fisiología , Interneuronas/metabolismo , Bulbo Olfatorio/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Ratones , Potenciales de Acción/fisiología , Inhibición Neural/fisiología , Ratones Endogámicos C57BL , Masculino , Sinapsis/fisiología , Sinapsis/metabolismo , Técnicas de Placa-Clamp , Dendritas/fisiología , Dendritas/metabolismo , Parvalbúminas/metabolismo , FemeninoRESUMEN
Previous studies have suggested a role for selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac®) in the treatment of dizziness and inner ear vestibular dysfunction. The potential mechanism of action within the vestibular system remains unclear; however, fluoxetine has been reported to block certain types of K+ channel in other systems. Here, we investigated the direct actions of fluoxetine on membrane currents in presynaptic hair cells and postsynaptic calyx afferents of the gerbil peripheral vestibular system using whole cell patch clamp recordings in crista slices. We explored differences in K+ currents in peripheral zone (PZ) and central zone (CZ) calyces of the crista and their response to fluoxetine application. Outward K+ currents in PZ calyces showed greater inactivation at depolarized membrane potentials compared to CZ calyces. The application of 100 µM fluoxetine notably reduced K+ currents in calyx terminals within both zones of the crista, and the remaining currents exhibited distinct traits. In PZ cells, fluoxetine inhibited a non-inactivating K+ current and revealed a rapidly activating and inactivating K+ current, which was sensitive to blocking by 4-aminopyridine. This was in contrast to CZ calyces, where low-voltage-activated and non-inactivating K+ currents persisted following application of 100 µM fluoxetine. Additionally, marked inhibition of transient inward Na+ currents by fluoxetine was observed in calyces from both crista zones. Different concentrations of fluoxetine were tested, and the EC50 values were found to be 40 µM and 32 µM for K+ and Na+ currents, respectively. In contrast, 100 µM fluoxetine had no impact on voltage-dependent K+ currents in mechanosensory type I and type II vestibular hair cells. In summary, micromolar concentrations of fluoxetine are expected to strongly reduce both Na+ and K+ conductance in afferent neurons of the peripheral vestibular system in vivo. This would lead to inhibition of action potential firing in vestibular sensory neurons and has therapeutic implications for disorders of balance.
Asunto(s)
Fluoxetina , Gerbillinae , Fluoxetina/farmacología , Animales , Potenciales de la Membrana/efectos de los fármacos , Vestíbulo del Laberinto/efectos de los fármacos , Vestíbulo del Laberinto/metabolismo , Técnicas de Placa-Clamp , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Canales de Potasio/metabolismo , Masculino , Células Ciliadas Vestibulares/efectos de los fármacos , Células Ciliadas Vestibulares/metabolismoRESUMEN
The medial prefrontal cortex (mPFC) plays a pivotal role in regulating working memory, executive function, and self-regulatory behaviors. Dysfunction in the mPFC circuits is a characteristic feature of several neuropsychiatric disorders including schizophrenia, depression, and post-traumatic stress disorder. Chronic stress (CS) is widely recognized as a major triggering factor for the onset of these disorders. Although evidence suggests synaptic dysfunction in mPFC circuits following CS exposure, it remains unclear how different neuronal populations in the infralimbic (IL) and prelimbic (PL) cortices are affected in terms of synaptic inhibition/excitation balance (I/E ratio). Here, using neuroproteomic analysis and whole-cell patch-clamp recordings in pyramidal neurons (PNs) and parvalbumin (PV) interneurons within the PL and IL cortices, we examined the synaptic changes after 21â d of chronic unpredictable stress, in male mice. Our results reveal distinct impacts of CS on PL and IL PNs, resulting in an increased I/E ratio in both subregions but through different mechanisms: CS increases inhibitory synaptic drive in the PL while decreasing excitatory synaptic drive in the IL. Notably, the I/E ratio and excitatory and inhibitory synaptic drive of PV interneurons remained unaffected in both PL and IL circuits following CS exposure. These findings offer novel mechanistic insights into the influence of CS on mPFC circuits and support the hypothesis of stress-induced mPFC hypofunction.
Asunto(s)
Interneuronas , Ratones Endogámicos C57BL , Parvalbúminas , Corteza Prefrontal , Células Piramidales , Estrés Psicológico , Animales , Interneuronas/fisiología , Interneuronas/metabolismo , Células Piramidales/fisiología , Masculino , Estrés Psicológico/fisiopatología , Parvalbúminas/metabolismo , Inhibición Neural/fisiología , Ratones , Técnicas de Placa-Clamp , Potenciales Postsinápticos Excitadores/fisiología , Sinapsis/fisiología , Potenciales Postsinápticos Inhibidores/fisiologíaRESUMEN
Focal cortical dysplasia type I (FCD I) is the most common cause of pharmaco-resistant epilepsy with the poorest prognosis. To understand the epileptogenic mechanisms of FCD I, we obtained tissue resected from patients with FCD I epilepsy, and from tumor patients as control. Using whole-cell patch clamp in acute human brain slices, we investigated the cellular properties of fast-spiking interneurons (FSINs) and pyramidal neurons (PNs) within the ictal onset zone. In FCD I epilepsy, FSINs exhibited lower firing rates from slower repolarization and action potential broadening, while PNs had increased firing. Importantly, excitatory synaptic drive of FSINs increased progressively with the scale of cortical activation as a general property across species, but this relationship was inverted towards net inhibition in FCD I epilepsy. Further comparison with intracranial electroencephalography (iEEG) from the same patients revealed that the spatial extent of pathological high-frequency oscillations (pHFO) was associated with synaptic events at FSINs.
Asunto(s)
Potenciales de Acción , Epilepsia , Interneuronas , Células Piramidales , Humanos , Interneuronas/fisiología , Femenino , Masculino , Células Piramidales/fisiología , Potenciales de Acción/fisiología , Epilepsia/fisiopatología , Adulto , Malformaciones del Desarrollo Cortical/fisiopatología , Adolescente , Adulto Joven , Niño , Técnicas de Placa-Clamp , Sinapsis/fisiología , Preescolar , Epilepsia Refractaria/fisiopatología , Epilepsia Refractaria/cirugía , ElectrocorticografíaRESUMEN
Measurement of cellular resting membrane potential (RMP) is important in understanding ion channels and their role in regulation of cell function across a wide range of cell types. However, methods available for the measurement of RMP (including patch clamp, microelectrodes, and potential-sensitive fluorophores) are expensive, slow, open to operator bias, and often result in cell destruction. We present non-contact, label-free membrane potential estimation which uses dielectrophoresis to determine the cytoplasm conductivity slope as a function of medium conductivity. By comparing this to patch clamp data available in the literature, we have demonstratet the accuracy of this approach using seven different cell types, including primary suspension cells (red blood cells, platelets), cultured suspension cells (THP-1), primary adherent cells (chondrocytes, human umbilical mesenchymal stem cells), and adherent (HeLa) and suspension (Jurkat) cancer cell lines. Analysis of the effect of ion channel inhibitors suggests the effects of pharmaceutical agents (TEA on HeLa; DMSO and neuraminidase on red blood cells) can also be measured. Comparison with published values of membrane potential suggest that the differences between our estimates and values recorded by patch clamp are accurate to within published margins of error. The method is low-cost, non-destructive, operator-independent and label-free, and has previously been shown to allow cells to be recovered after measurement.
Asunto(s)
Electroforesis , Potenciales de la Membrana , Humanos , Potenciales de la Membrana/fisiología , Electroforesis/métodos , Células HeLa , Células Jurkat , Técnicas de Placa-Clamp/métodos , Eritrocitos/citología , Eritrocitos/metabolismoRESUMEN
TMEM16A, a member of the Transmembrane protein 16 family, serves as the molecular basis for calcium activated chloride channels (CaCCs). We use RT-PCR to demonstrate the expression of TMEM16A in the neurons of Helicoverpa armigera, and record the CaCCs current of acute isolated neurons of H. armigera for the first time using patch clamp technology. In order to screen effective inhibitors of calcium-activated chloride channels, the inhibitory effects of four chloride channel inhibitors, CaCCinh-A01, NPPB, DIDS, and SITS, on CaCCs were compared. The inhibitory effects of the four inhibitors on the outward current of CaCCs were CaCCinh-A01 (10 µM, 56.31 %), NPPB (200 µM, 43.69 %), SITS (1 mM, 12.41 %) and DIDS (1 mM, 13.29 %). Among these inhibitors, CaCCinh-A01 demonstrated the highest efficacy as a blocker. To further explore whether calcium channel proteins can serve as potential targets of pyrethroids, we compared the effects of (type I) tefluthrin and (type II) deltamethrin on CaCCs. 10 µM and 100 µM tefluthrin can stimulate a large tail current in CaCCs, prolonging their deactivation time by 10.44 ms and 31.49 ms, and the V0.5 shifted in the hyperpolarization by 2-8 mV. Then, deltamethrin had no obvious effect on the deactivation and activation of CaCCs. Therefore, CaCCs of H. armigera can be used as a potential target of pyrethroids, but type I and type II pyrethroids have different effects on CaCCs.
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Canales de Cloruro , Insecticidas , Mariposas Nocturnas , Neuronas , Piretrinas , Animales , Insecticidas/toxicidad , Insecticidas/farmacología , Piretrinas/toxicidad , Piretrinas/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Canales de Cloruro/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Mariposas Nocturnas/efectos de los fármacos , Anoctamina-1/metabolismo , Anoctamina-1/antagonistas & inhibidores , Proteínas de Insectos/metabolismo , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Nitrobenzoatos/farmacología , Helicoverpa armigera , Ciclopropanos , Hidrocarburos FluoradosRESUMEN
The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.
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Calcio , Receptores Nicotínicos , Animales , Ratas , Acetilcolina/metabolismo , Acetilcolina/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Simulación de Dinámica Molecular , Técnicas de Placa-Clamp , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Xenopus laevisRESUMEN
The convergence of conditioned and unconditioned stimuli (CS and US) into the lateral amygdala (LA) serves as a substrate for an adequate fear response in vivo. This well-known Pavlovian paradigm modulates the synaptic plasticity of neurons, as can be proved by the long-term potentiation (LTP) phenomenon in vitro. Although there is an increasing body of evidence for the existence of LTP in the amygdala, only a few studies were able to show a reliable long-term depression (LTD) of excitation in this structure. We have used coronal brain slices and conducted patch-clamp recordings in pyramidal neurons of the lateral amygdala (LA). After obtaining a stable baseline excitatory postsynaptic current (EPSC) response at a holding potential of -70 mV, we employed a paired-pulse paradigm at 1 Hz at the same membrane potential and could observe a reliable LTD. The different durations of stimulation (ranging between 1.5-24 min) were tested first in the same neuron, but the intensity was kept constant. The latter paradigm resulted in a step-wise LTD with a gradually increasing magnitude under these conditions.
Asunto(s)
Amígdala del Cerebelo , Potenciales Postsinápticos Excitadores , Depresión Sináptica a Largo Plazo , Técnicas de Placa-Clamp , Depresión Sináptica a Largo Plazo/fisiología , Animales , Potenciales Postsinápticos Excitadores/fisiología , Amígdala del Cerebelo/fisiología , Masculino , Células Piramidales/fisiología , Estimulación Eléctrica , Ratas , Ratas Wistar , Corteza Cerebral/fisiología , Vías Nerviosas/fisiología , Técnicas In VitroRESUMEN
BACKGROUND: The conventional "whole-cell patch-clamp" recording technique is widely used to measure the resting membrane potential (VM) and to dissect the underlying membrane ionic conductances in isolated vascular endothelial cells. NEW METHOD: Herein, we assessed whether the automated patch-clamp (APC) technology, which replaces the traditional patch-pipette with a planar substrate to permit researchers lacking formal training in electrophysiology to generate large amounts of data in a relatively short time, can be used to characterize the bioelectrical activity of vascular endothelial cells. We assessed whether the Port-a-Patch planar patch-clamp system, which is regarded as the smallest electrophysiological rig available on the market, can be used to measure the VM and resting membrane currents in the human cerebrovascular endothelial cell line, hCMEC/D3. COMPARISON WITH EXISTING METHODS: We demonstrated that the Port-a-Patch planar patch-clamp system provides the same values of the resting VM as those provided by the conventional patch-clamp technique. Furthermore, the APC technology provides preliminary data demonstrating that the resting VM of hCMEC/D3 cells is primarily contributed by Cl- and Na+, as demonstrated with the patch-clamp technique for many other endothelial cell types. CONCLUSIONS: The Port-a-Patch planar patch-clamp system can be successfully used to measure the resting VM and the underlying membrane ionic conductances in hCMEC/D3 cells. We envisage that this easy-to-use APC system could also be extremely useful for the investigation of the membrane currents that can be activated by chemical, thermal, optical, and mechanical stimuli in this cell line as well as in other types of isolated vascular endothelial cells.
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Células Endoteliales , Potenciales de la Membrana , Técnicas de Placa-Clamp , Humanos , Técnicas de Placa-Clamp/métodos , Técnicas de Placa-Clamp/instrumentación , Células Endoteliales/fisiología , Células Endoteliales/citología , Potenciales de la Membrana/fisiología , Línea CelularRESUMEN
Chronic ethanol exposure produces neuroadaptations in the medial prefrontal cortex (mPFC) that are thought to facilitate maladaptive behaviors that interfere with recovery from alcohol use disorder. Despite evidence that different cortico-subcortical projections play distinct roles in behavior, few studies have examined the physiological effects of chronic ethanol at the circuit level. The rostromedial tegmental nucleus (RMTg) is functionally altered by chronic ethanol exposure. Our recent work identified dense input from the mPFC to the RMTg, yet the effects of chronic ethanol exposure on this circuitry is unknown. In the current study, we examined physiological changes after chronic ethanol exposure in prelimbic (PL) and infralimbic (IL) mPFC neurons projecting to the RMTg. Adult male Long-Evans rats were injected with fluorescent retrobeads into the RMTg and rendered dependent using a 14-day chronic intermittent ethanol (CIE) vapor exposure paradigm. Whole-cell patch-clamp electrophysiological recordings were performed in fluorescently-labeled (RMTg-projecting) and -unlabeled (projection-undefined) layer 5 pyramidal neurons 7-10 days following ethanol exposure. CIE exposure significantly increased intrinsic excitability as well as spontaneous excitatory and inhibitory postsynaptic currents (sE/IPSCs) in RMTg-projecting IL neurons. In contrast, no lasting changes in excitability were observed in RMTg-projecting PL neurons, although a CIE-induced reduction in excitability was observed in projection-undefined PL neurons. CIE exposure also increased the frequency of sEPSCs in RMTg-projecting PL neurons. These data uncover novel subregion- and circuit-specific neuroadaptations in the mPFC following chronic ethanol exposure and reveal that the IL mPFC-RMTg projection is uniquely vulnerable to long-lasting effects of chronic ethanol exposure. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".
Asunto(s)
Etanol , Corteza Prefrontal , Ratas Long-Evans , Animales , Etanol/farmacología , Etanol/administración & dosificación , Masculino , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/fisiología , Ratas , Neuronas/efectos de los fármacos , Neuronas/fisiología , Depresores del Sistema Nervioso Central/farmacología , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/fisiología , Vías Nerviosas/efectos de los fármacos , Técnicas de Placa-Clamp , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiologíaRESUMEN
The ClC-3 chloride/proton exchanger is both physiologically and pathologically critical, as it is potentiated by ATP to detect metabolic energy level and point mutations in ClC-3 lead to severe neurodegenerative diseases in human. However, why this exchanger is differentially modulated by ATP, ADP or AMP and how mutations caused gain-of-function remains largely unknow. Here we determine the high-resolution structures of dimeric wildtype ClC-3 in the apo state and in complex with ATP, ADP and AMP, and the disease-causing I607T mutant in the apo and ATP-bounded state by cryo-electron microscopy. In combination with patch-clamp recordings and molecular dynamic simulations, we reveal how the adenine nucleotides binds to ClC-3 and changes in ion occupancy between apo and ATP-bounded state. We further observe I607T mutation induced conformational changes and augments in current. Therefore, our study not only lays the structural basis of adenine nucleotides regulation in ClC-3, but also clearly indicates the target region for drug discovery against ClC-3 mediated neurodegenerative diseases.
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Adenosina Trifosfato , Canales de Cloruro , Microscopía por Crioelectrón , Simulación de Dinámica Molecular , Enfermedades Neurodegenerativas , Canales de Cloruro/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/química , Humanos , Adenosina Trifosfato/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Nucleótidos de Adenina/metabolismo , Técnicas de Placa-Clamp , Mutación , Adenosina Difosfato/metabolismo , Células HEK293 , Adenosina Monofosfato/metabolismo , Animales , Conformación ProteicaRESUMEN
Nicotinic acetylcholine receptors (nAChRs) are expressed in excitable and non-excitable cells of the organism. Extensive studies suggest that nAChR ligands have therapeutic potential, notably for neurological and psychiatric disorders. Organometallic ruthenium complexes are known to inhibit several medically important enzymes such as cholinesterases. In addition, they can also interact with muscle- and neuronal-subtype nAChRs. The present study aimed to investigate the direct effects of three organometallic ruthenium complexes, [(η6-p-cymene)Ru(II)(5-nitro-1,10-phenanthroline)Cl]Cl (C1-Cl), [(η6-p-cymene)Ru(II)(1-hydroxypyridine-2(1H)-thionato)Cl] (C1a) and [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1), on muscle-subtype (Torpedo) nAChRs and on the two most abundant human neuronal-subtype nAChRs in the CNS (α4ß2 and α7) expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp. The results show that none of the three compounds had agonistic activity on any of the nAChR subtypes studied. In contrast, C1-Cl reversibly blocked Torpedo nAChR (half-reduction of ACh-evoked peak current amplitude by 332 nM of compound). When tested at 10 µM, C1-Cl was statistically more potent to inhibit TorpedonAChR than α4ß2 and α7 nAChRs. Similar results of C1 effects were obtained on Torpedo and α4ß2 nAChRs, while no action of the compound was detected on α7 nAChRs. Finally, the effects of C1a were statistically similar on the three nAChR subtypes but, in contrast to C1-Cl and C1, the inhibition was hardly reversible. These results, together with our previous studies on isolated mouse neuromuscular preparations, strongly suggest that C1-Cl is, among the three compounds studied, the only molecule that could be used as a potential myorelaxant drug.
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Oocitos , Receptores Nicotínicos , Xenopus laevis , Animales , Receptores Nicotínicos/metabolismo , Humanos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Rutenio/química , Rutenio/farmacología , Torpedo , Compuestos Organometálicos/farmacología , Compuestos Organometálicos/química , Técnicas de Placa-Clamp , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Piridinas/farmacología , Piridinas/químicaRESUMEN
The processing of synaptic signals in somatodendritic compartments determines neuronal computation. Although the amplification of excitatory signals by local voltage-dependent cation channels has been extensively studied, their spatiotemporal dynamics in elaborate dendritic branches remain obscure owing to technical limitations. Using fluorescent voltage imaging throughout dendritic arborizations in hippocampal pyramidal neurons, we demonstrate a unique chloride ion (Cl-)-dependent remote computation mechanism in the distal branches. Excitatory postsynaptic potentials triggered by local laser photolysis of caged glutamate spread along dendrites, with gradual amplification toward the distal end while attenuation toward the soma. Tour de force subcellular patch-clamp recordings from thin branches complemented by biophysical model simulations revealed that the asymmetric augmentation of excitation relies on tetrodotoxin-resistant sodium ion (Na+) channels and Cl- conductance accompanied by a more hyperpolarized dendritic resting potential. Together, this study reveals the cooperative voltage-dependent actions of cation and anion conductance for dendritic supralinear computation, which can locally decode the spatiotemporal context of synaptic inputs.
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Cloruros , Dendritas , Potenciales Postsinápticos Excitadores , Dendritas/fisiología , Dendritas/metabolismo , Animales , Potenciales Postsinápticos Excitadores/fisiología , Cloruros/metabolismo , Células Piramidales/fisiología , Células Piramidales/metabolismo , Ratas , Técnicas de Placa-Clamp , Hipocampo/fisiología , Hipocampo/metabolismo , Sinapsis/fisiología , Sinapsis/metabolismoRESUMEN
BACKGROUND: Cardiovascular disease remains one of the leading causes of death globally. Myocardial ischemia and infarction, in particular, frequently cause disturbances in cardiac electrical activity that can trigger ventricular arrhythmias. We aimed to investigate whether catestatin, an endogenous catecholamine-inhibiting peptide, ameliorates myocardial ischemia-induced ventricular arrhythmias in rats and the underlying ionic mechanisms. METHODS AND RESULTS: Adult male Sprague-Dawley rats were randomly divided into control and catestatin groups. Ventricular arrhythmias were induced by ligation of the left anterior descending coronary artery and electrical stimulation. Action potential, transient outward potassium current, delayed rectifier potassium current, inward rectifying potassium current, and L-type calcium current (ICa-L) of rat ventricular myocytes were recorded using a patch-clamp technique. Catestatin notably reduced ventricular arrhythmia caused by myocardial ischemia/reperfusion and electrical stimulation of rats. In ventricular myocytes, catestatin markedly shortened the action potential duration of ventricular myocytes, which was counteracted by potassium channel antagonists TEACl and 4-AP, and ICa-L current channel agonist Bay K8644. In addition, catestatin significantly increased transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current density in a concentration-dependent manner. Catestatin accelerated the activation and decelerated the inactivation of the transient outward potassium current channel. Furthermore, catestatin decreased ICa-L current density in a concentration-dependent manner. Catestatin also accelerated the inactivation of the ICa-L channel and slowed down the recovery of ICa-L from inactivation. CONCLUSIONS: Catestatin enhances the activity of transient outward potassium current, delayed rectifier potassium current, and inward rectifying potassium current, while suppressing the ICa-L in ventricular myocytes, leading to shortened action potential duration and ultimately reducing the ventricular arrhythmia in rats.
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Potenciales de Acción , Cromogranina A , Miocitos Cardíacos , Fragmentos de Péptidos , Ratas Sprague-Dawley , Animales , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Cromogranina A/farmacología , Cromogranina A/metabolismo , Potenciales de Acción/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/prevención & control , Arritmias Cardíacas/metabolismo , Antiarrítmicos/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Modelos Animales de Enfermedad , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Técnicas de Placa-Clamp , Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Canales de Potasio de Tipo Rectificador Tardío/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio/efectos de los fármacosRESUMEN
The master control of mammalian circadian rhythms is the suprachiasmatic nucleus (SCN), which is formed by the ventral and dorsal regions. In SCN neurons, GABA has an important function and even excitatory actions in adulthood. However, the physiological role of this neurotransmitter in the developing SCN is unknown. Here, we recorded GABAergic postsynaptic currents (in the perforated-patch configuration using gramicidin) to determine the chloride reversal potential (ECl) and also assessed the immunological expression of the Na-K-Cl cotransporter 1 (NKCC1) at early ages of the rat (postnatal days (P) 3 to 25), during the day and night, in the two SCN regions. We detected that ECl greatly varied with age and depending on the SCN region and time of day. Broadly speaking, ECl was more hyperpolarized with age, except for the oldest age studied (P20-25) in both day and night in the ventral SCN, where it was less negative. Likewise, ECl was more hyperpolarized in the dorsal SCN both during the day and at night; while ECl was more negative at night both in the ventral and the dorsal SCN. Moreover, the total NKCC1 fluorescent expression was higher during the day than at night. These results imply that NKCC1 regulates the circadian and developmental fluctuations in the [Cl-]i to fine-tune ECl, which is crucial for either excitatory or inhibitory GABAergic actions to occur in the SCN.
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Cloruros , Ritmo Circadiano , Miembro 2 de la Familia de Transportadores de Soluto 12 , Núcleo Supraquiasmático , Animales , Núcleo Supraquiasmático/metabolismo , Ritmo Circadiano/fisiología , Ratas , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Masculino , Cloruros/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Ratas Wistar , Técnicas de Placa-Clamp , Envejecimiento/fisiologíaRESUMEN
Tetrodotoxin (TTX) is a potent marine neurotoxin, responsible for numerous poisoning incidents and some human fatalities. To date, more than 30 TTX analogues have been identified, but their individual toxicities and roles in poisoning remain largely unknown. In this work, the toxicity equivalency factors (TEFs) of five TTX analogues were determined by assessing the blockade of voltage-gated sodium channels in Neuro-2a cells using automated patch clamp (APC). All TTX analogues were less toxic than TTX. The derived TEFs were applied to the individual TTX analogues concentrations measured in pufferfish samples, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A comparison of these results with those obtained from APC analysis demonstrated that TEFs can be effectively used to translate LC-MS/MS analytical data into meaningful toxicological information. This is the first study to utilize APC device for the toxicological assessment of TTX analogues, highlighting its potential as a bioanalytical tool for seafood safety management and human health protection.
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Técnicas de Placa-Clamp , Espectrometría de Masas en Tándem , Tetrodotoxina , Canales de Sodio Activados por Voltaje , Tetrodotoxina/toxicidad , Tetrodotoxina/química , Tetrodotoxina/análogos & derivados , Animales , Canales de Sodio Activados por Voltaje/metabolismo , Humanos , Ratones , Tetraodontiformes , Alimentos Marinos/análisis , Línea Celular , Cromatografía LiquidaRESUMEN
Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl- levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P2 depletion by activating phosphatidylinositol 5-phosphatases or Gq protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P2-binding site is localized within the cytosol in resting state but shifts to the cell membrane's inner surface in an activated state and interacts with inner leaflet PI(4,5)P2. Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P2.