RESUMO
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.
Assuntos
Potenciais Pós-Sinápticos Excitadores , Neurônios , Bulbo Olfatório , Vasopressinas , Animais , Bulbo Olfatório/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Ratos , Vasopressinas/farmacologia , Vasopressinas/metabolismo , Neurônios/fisiologia , Neurônios/efeitos dos fármacos , Masculino , Técnicas de Patch-Clamp , Potenciais Pós-Sinápticos Inibidores/fisiologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Ratos Wistar , Interneurônios/fisiologia , Interneurônios/efeitos dos fármacosRESUMO
Dysfunction of the Nav1.5, Cav1.2, and Kv channels could interfere with the AP and result in arrhythmias and even heart failure. We herein present a novel library of nuciferine analogs that target ion channels for the treatment of arrhythmias. Patch clamp measurements of ventricular myocytes revealed that 6a dramatically blocked both the INa and ICa without altering the currentvoltage relationship (including the activation potential and peak potential), accelerated the inactivation of Nav and Cav channels and delayed the resurrection of these channels after inactivation. Additionally, 6a significantly decreased the APA and RMP without affecting the APD30 or APD50. The IC50 values of 6a against Nav1.5 and Cav1.2 were 4.98⯵M and 4.62⯵M, respectively. Furthermore, 6a (10⯵M) blocked IKs, IK1, and Ito with values of 17.01â¯%±2.54â¯%, 9.09â¯%±2.78â¯%, and 11.15â¯%±3.52â¯%, respectively. Surprisingly, 6a weakly inhibited hERG channels, suggesting a low risk of proarrhythmia. The cytotoxicity evaluation of 6a with the H9c2 cell line indicated that this compound was noncytotoxic. In vivo studies suggested that these novel nuciferine analogs could shorten the time of arrhythmia continuum induced by BaCl2 and normalize the HR, QRS, QT and QTc interval and the R wave amplitude. Moreover, 6a dose-dependently affected aconitine-induced arrhythmias and notably improved the cumulative dosage of aconitine required to evoke VP, VT, VF and CA in rats with aconitine-induced arrhythmia. In conclusion, nuciferine analogs could be promising ion channel blockers that could be further developed into antiarrhythmic agents.
Assuntos
Potenciais de Ação , Arritmias Cardíacas , Miócitos Cardíacos , Animais , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/induzido quimicamente , Potenciais de Ação/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Masculino , Antiarrítmicos/farmacologia , Aporfinas/farmacologia , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Técnicas de Patch-ClampRESUMO
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.
Assuntos
Acaricidas , Canais de Potássio Cálcio-Ativados , Tetranychidae , Acaricidas/farmacologia , Animais , Tetranychidae/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/metabolismo , Humanos , Técnicas de Patch-ClampRESUMO
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.
Assuntos
Potenciais de Ação , Camundongos Endogâmicos C57BL , Neurônios , Técnicas de Patch-Clamp , Colículos Superiores , Animais , Camundongos , Masculino , Colículos Superiores/fisiologia , Potenciais de Ação/fisiologia , Neurônios/fisiologia , Fenômenos EletrofisiológicosRESUMO
The SARS-CoV-2 E protein is an enigmatic viral structural protein with reported viroporin activity associated with the acute respiratory symptoms of COVID-19, as well as the ability to deform cell membranes for viral budding. Like many viroporins, the E protein is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the structure of the protein complex have yielded inconclusive results, suggesting several possible oligomers, ranging from dimers to pentamers. Here, we combined patch-clamp, confocal fluorescence microscopy on giant unilamellar vesicles, and atomic force microscopy to show that E protein can exhibit two modes of membrane activity depending on membrane lipid composition. In the absence or the presence of a low content of cholesterol, the protein forms short-living transient pores, which are seen as semi-transmembrane defects in a membrane by atomic force microscopy. Approximately 30 mol% cholesterol is a threshold for the transition to the second mode of conductance, which could be a stable pentameric channel penetrating the entire lipid bilayer. Therefore, the E-protein has at least two different types of activity on membrane permeabilization, which are regulated by the amount of cholesterol in the membrane lipid composition and could be associated with different types of protein oligomers.
Assuntos
Colesterol , Proteínas do Envelope de Coronavírus , Microscopia de Força Atômica , SARS-CoV-2 , Colesterol/metabolismo , Colesterol/química , SARS-CoV-2/metabolismo , Humanos , Proteínas do Envelope de Coronavírus/metabolismo , Proteínas do Envelope de Coronavírus/química , Membrana Celular/metabolismo , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química , COVID-19/metabolismo , COVID-19/virologia , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/química , Proteínas Viroporinas/metabolismo , Técnicas de Patch-Clamp , Multimerização Proteica , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/químicaRESUMO
Treatment with melatonin is routinely prescribed for its potent antioxidant and cognitive-promoting effects, nevertheless, it has yet to find neuromodulatory effects in normal and disease conditions. Therefore, to investigate its neuromodulatory mechanisms, melatonin was systemically administered over 10 consecutive days to both intracortical normal saline- and amyloid-ß 1-42 (Aß) peptide-injected rats. At the behavioral level, treatment with melatonin was associated with reduced efficacy in restoring Aß-induced deficit in passive-avoidance memory. Whole-cell patch-clamp recordings from CA1 pyramidal neurons revealed that melatonin treatment reduced spontaneous and evoked intrinsic excitability in control rats while exerting a reduction of spontaneous, but not evoked activity, in the Aß-injected group. Interestingly, treatment with melatonin enhances after-hyperpolarization in control, but not Aß-injected rats. In contrast, our voltage-clamp study showed that Ih current is significantly enhanced by Aß injection, and this effect is further strengthened by treatment with melatonin in Aß-injected rats. Finally, we discovered that the transcription of melatonin receptors 1 (MT1) and 2 (MT2) is significantly upregulated in the hippocampi of Aß-injected rats. Collectively, our study demonstrates that systemic treatment with melatonin has differential neuromodulation on CA1 neuronal excitability, at least in part, via differential effects on after-hyperpolarization and Ih currents due to Aß-induced neurotoxicity.
Assuntos
Peptídeos beta-Amiloides , Hipocampo , Melatonina , Animais , Melatonina/farmacologia , Peptídeos beta-Amiloides/metabolismo , Ratos , Masculino , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Receptores de Melatonina/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptor MT2 de Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Ratos Sprague-Dawley , Técnicas de Patch-ClampRESUMO
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.
Assuntos
Trifosfato de Adenosina , Canais de Cloreto , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Doenças Neurodegenerativas , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/química , Humanos , Trifosfato de Adenosina/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Nucleotídeos de Adenina/metabolismo , Técnicas de Patch-Clamp , Mutação , Difosfato de Adenosina/metabolismo , Células HEK293 , Monofosfato de Adenosina/metabolismo , Animais , Conformação ProteicaRESUMO
Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca2+-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca2+-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.
Assuntos
Células Cromafins , Dinamina II , Endocitose , Exocitose , Mutação , Miopatias Congênitas Estruturais , Células Cromafins/metabolismo , Endocitose/fisiologia , Endocitose/genética , Dinamina II/genética , Dinamina II/metabolismo , Animais , Exocitose/fisiologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/metabolismo , Mutação/genética , Bovinos , Humanos , Actinas/metabolismo , Actinas/genética , Células Cultivadas , Técnicas de Patch-Clamp , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologiaRESUMO
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.
Assuntos
Membrana Celular , Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositol 4,5-Difosfato/metabolismo , Humanos , Membrana Celular/metabolismo , Células HEK293 , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Prótons , Sítios de Ligação , Animais , Técnicas de Patch-Clamp , Anoctaminas/metabolismo , Anoctaminas/genética , Anoctaminas/química , Proteínas de Transferência de FosfolipídeosRESUMO
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.
Assuntos
Interneurônios , Camundongos Endogâmicos C57BL , Parvalbuminas , Córtex Pré-Frontal , Células Piramidais , Estresse Psicológico , Animais , Interneurônios/fisiologia , Interneurônios/metabolismo , Células Piramidais/fisiologia , Masculino , Estresse Psicológico/fisiopatologia , Parvalbuminas/metabolismo , Inibição Neural/fisiologia , Camundongos , Técnicas de Patch-Clamp , Potenciais Pós-Sinápticos Excitadores/fisiologia , Sinapses/fisiologia , Potenciais Pós-Sinápticos Inibidores/fisiologiaRESUMO
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.
Assuntos
Canais de Cloreto , Inseticidas , Mariposas , Neurônios , Piretrinas , Animais , Inseticidas/toxicidade , Inseticidas/farmacologia , Piretrinas/toxicidade , Piretrinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Canais de Cloreto/metabolismo , Canais de Cloreto/antagonistas & inibidores , Mariposas/efeitos dos fármacos , Anoctamina-1/metabolismo , Anoctamina-1/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Nitrobenzoatos/farmacologia , Helicoverpa armigera , Ciclopropanos , Hidrocarbonetos FluoradosRESUMO
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.
Assuntos
Fluoxetina , Gerbillinae , Fluoxetina/farmacologia , Animais , Potenciais da Membrana/efeitos dos fármacos , Vestíbulo do Labirinto/efeitos dos fármacos , Vestíbulo do Labirinto/metabolismo , Técnicas de Patch-Clamp , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Canais de Potássio/metabolismo , Masculino , Células Ciliadas Vestibulares/efeitos dos fármacos , Células Ciliadas Vestibulares/metabolismoRESUMO
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.
Assuntos
Tonsila do Cerebelo , Potenciais Pós-Sinápticos Excitadores , Depressão Sináptica de Longo Prazo , Técnicas de Patch-Clamp , Depressão Sináptica de Longo Prazo/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Tonsila do Cerebelo/fisiologia , Masculino , Células Piramidais/fisiologia , Estimulação Elétrica , Ratos , Ratos Wistar , Córtex Cerebral/fisiologia , Vias Neurais/fisiologia , Técnicas In VitroRESUMO
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.
Assuntos
Células Endoteliais , Potenciais da Membrana , Técnicas de Patch-Clamp , Humanos , Técnicas de Patch-Clamp/métodos , Técnicas de Patch-Clamp/instrumentação , Células Endoteliais/fisiologia , Células Endoteliais/citologia , Potenciais da Membrana/fisiologia , Linhagem CelularRESUMO
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".
Assuntos
Etanol , Córtex Pré-Frontal , Ratos Long-Evans , Animais , Etanol/farmacologia , Etanol/administração & dosagem , Masculino , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiologia , Ratos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Depressores do Sistema Nervoso Central/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Vias Neurais/efeitos dos fármacos , Técnicas de Patch-Clamp , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologiaRESUMO
Many types of neurons exhibit a daily rhythm of intrinsic excitability. Here, we present a protocol for assessing circadian regulation of dentate granule cell excitability using a mouse model for conditional knockout of the molecular clock protein BMAL1. We describe steps for obtaining healthy oblique horizontal slices that contain the hippocampus and measuring intrinsic excitability and synaptic potentials by combining whole-cell patch-clamp recordings and perforant-path electric stimulation. We then detail procedures for validating single-cell genetic deletion of Bmal1 by immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Gonzalez et al.1.
Assuntos
Ritmo Circadiano , Giro Denteado , Animais , Camundongos , Giro Denteado/citologia , Giro Denteado/metabolismo , Giro Denteado/fisiologia , Ritmo Circadiano/fisiologia , Técnicas de Patch-Clamp/métodos , Neurônios/metabolismo , Neurônios/fisiologia , Neurônios/citologia , Fatores de Transcrição ARNTL/metabolismo , Fatores de Transcrição ARNTL/genética , Camundongos Knockout , MasculinoRESUMO
Pufferfish is one of the most poisonous marine organisms, responsible for numerous poisoning incidents and some human fatalities due to its capability to accumulate potent neurotoxins such as tetrodotoxins (TTXs) and paralytic shellfish toxins (PSTs). In this study, tissue extracts (muscle, skin, liver, intestinal tract and gonads) obtained from sixteen pufferfish specimens of the Lagocephalus lagocephalus and Sphoeroides pachygaster species, collected along the Spanish Mediterranean coast, were analysed for the presence of voltage-gated sodium channel (also known as Nav channel) blockers using cell-based assay (CBA) and automated patch clamp (APC). No toxicity was observed in any of the S. pachygaster specimens, but toxicity was detected in the liver of most L. lagocephalus specimens. Instrumental analysis of these specimens, as well as in one Lagocephalus sceleratus specimen, by high-performance liquid chromatography coupled to fluorescence detection (HPLC-FLD) was performed, which confirmed the presence of PSTs only in L. lagocephalus specimens. This analysis reported the presence of saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) in all positive samples, being dcSTX the major analogue. These results demonstrate the ability of this species to accumulate PSTs, being the first report of the presence of PSTs in Mediterranean L.lagocephalus specimens. Furthermore, the presence of high PSTs contents in all five tested tissues of one L. lagocephalus specimen pointed the risk that the presence of this toxic fish in the Mediterranean Sea may represent for seafood safety and human health in case of accidental consumption.
Assuntos
Toxinas Marinhas , Saxitoxina , Tetraodontiformes , Animais , Toxinas Marinhas/toxicidade , Toxinas Marinhas/análise , Cromatografia Líquida de Alta Pressão , Saxitoxina/análogos & derivados , Saxitoxina/análise , Saxitoxina/toxicidade , Espanha , Intoxicação por Frutos do Mar , Mar Mediterrâneo , Técnicas de Patch-Clamp , Tetrodotoxina/toxicidade , Tetrodotoxina/análiseRESUMO
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.
Assuntos
Oócitos , Receptores Nicotínicos , Xenopus laevis , Animais , Receptores Nicotínicos/metabolismo , Humanos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Rutênio/química , Rutênio/farmacologia , Torpedo , Compostos Organometálicos/farmacologia , Compostos Organometálicos/química , Técnicas de Patch-Clamp , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Piridinas/farmacologia , Piridinas/químicaRESUMO
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.
Assuntos
Interneurônios , Bulbo Olfatório , Animais , Interneurônios/fisiologia , Interneurônios/metabolismo , Bulbo Olfatório/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Camundongos , Potenciais de Ação/fisiologia , Inibição Neural/fisiologia , Camundongos Endogâmicos C57BL , Masculino , Sinapses/fisiologia , Sinapses/metabolismo , Técnicas de Patch-Clamp , Dendritos/fisiologia , Dendritos/metabolismo , Parvalbuminas/metabolismo , FemininoRESUMO
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.