RESUMEN
Paramecium exhibits responsive behavior to environmental changes, moving either closer to or further away from stimuli. Electrophysiological experiments have revealed that these behavioral responses are controlled by membrane potentials. Anoctamin, a Ca2+-activated Cl- channel, is involved in the regulation of membrane potential in mammals. However, it remains uncertain whether Cl- channels like anoctamin regulate Paramecium behavior. Herein, replacement of external Cl- ions with acetate ion and application of Cl- channel blocker niflumic acid (NFA, 0.1 µM) increased spontaneous avoiding reactions (sARs). Hence, we hypothesized that anoctamin is involved in the stabilization of membrane potential fluctuation. Paramecium cells in which the anoctamin-like protein 1 gene was knocked down displayed frequent sARs in the culture medium without external stimulation. Treatment of anoctamin-like protein 1-knockdown cells with the Ca2+ chelator BAPTA or Ca-channel blocker nicardipine reversed the increase in sARs. Electrophysiological experiments revealed extension of membrane depolarization when positive currents were applied to anoctamin-like protein 1-knockdown cells. We concluded that anoctamin-like protein 1 works as a Cl-channel and stabilizes the membrane potential oscillation, reducing sARs.
Asunto(s)
Potenciales de la Membrana , Paramecium , Proteínas Protozoarias , Paramecium/fisiología , Paramecium/genética , Potenciales de la Membrana/efectos de los fármacos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Canales de Cloruro/metabolismo , Canales de Cloruro/genética , Calcio/metabolismo , Ácido Niflúmico/farmacología , Técnicas de Silenciamiento del GenRESUMEN
Neonicotinoids are insecticides widely used in the world. Although neonicotinoids are believed to be toxic only to insects, their developmental neurotoxicity in mammals is a concern. Therefore, we examined the effects of developmental exposure to neonicotinoids on immune system in the brain and post-developmental behaviors in this study. Imidacloprid or clothianidin was orally administered to dams at a dosage of 0.1 mg/kg/day from embryonic day 11 to postnatal day 21. Imidacloprid decreased sociability, and both imidacloprid and clothianidin decreased locomotor activity and induced anxiety, depression and abnormal repetitive behaviors after the developmental period. There was no change in the number of neurons in the hippocampus of mice exposed to imidacloprid. However, the number and activity of microglia during development were significantly decreased by imidacloprid exposure. Imidacloprid also induced neural circuit dysfunction in the CA1 and CA3 regions of the hippocampus during the early postnatal period. Exposure to imidacloprid suppressed the expression of csf1r during development. Collectively, these results suggest that developmental exposure to imidacloprid decreases the number and activity of microglia, which can cause neural circuit dysfunction and abnormal behaviors after the developmental period. Care must be taken to avoid exposure to neonicotinoids, especially during development.
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Insecticidas , Microglía , Neonicotinoides , Nitrocompuestos , Animales , Neonicotinoides/toxicidad , Microglía/efectos de los fármacos , Nitrocompuestos/toxicidad , Ratones , Insecticidas/toxicidad , Femenino , Guanidinas/toxicidad , Tiazoles/toxicidad , Conducta Animal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Masculino , Embarazo , Neuronas/efectos de los fármacosRESUMEN
In several experimental conditions, neuronal excitation at the perirhinal cortex (PC) does not propagate to the entorhinal cortex (EC) due to a "wall" of inhibition, which may help to create functional coupling and un-coupling of the PC and EC in the medial temporal lobe. However, little is known regarding the coupling control process. Herein, we propose that the deep layer of area 35 in the PC plays a pivotal role in opening the gate for coupling, thus allowing the activity in the PC to propagate to the EC. Using voltage-sensitive dye imaging for the brain slices of rodents, we show that a slowly inactivating potassium conductance in this area is essential to induce excitation overtaking the inhibitory control. This coupling between the distinct neural circuits persists for at least 1 h. We elucidate further implications of this network-level plastic behavior and its mechanism.
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Corteza Perirrinal , Corteza Entorrinal , Hipocampo , Canales de PotasioRESUMEN
BACKGROUND: Valproic acid (VPA) is a clinically used antiepileptic drug, but it is associated with a significant risk of a low verbal intelligence quotient (IQ) score, attention-deficit hyperactivity disorder and autism spectrum disorder in children when it is administered during pregnancy. Prenatal VPA exposure has been reported to affect neurogenesis and neuronal migration and differentiation. In addition, growing evidence has shown that microglia and brain immune cells are activated by VPA treatment. However, the role of VPA-activated microglia remains unclear. METHODS: Pregnant female mice received sodium valproate on E11.5. A microglial activation inhibitor, minocycline or a CCR5 antagonist, maraviroc was dissolved in drinking water and administered to dams from P1 to P21. Measurement of microglial activity, evaluation of neural circuit function and expression analysis were performed on P10. Behavioral tests were performed in the order of open field test, Y-maze test, social affiliation test and marble burying test from the age of 6 weeks. RESULTS: Prenatal exposure of mice to VPA induced microglial activation and neural circuit dysfunction in the CA1 region of the hippocampus during the early postnatal periods and post-developmental defects in working memory and social interaction and repetitive behaviors. Minocycline, a microglial activation inhibitor, clearly suppressed the above effects, suggesting that microglia elicit neural dysfunction and behavioral disorders. Next-generation sequencing analysis revealed that the expression of a chemokine, C-C motif chemokine ligand 3 (CCL3), was upregulated in the hippocampi of VPA-treated mice. CCL3 expression increased in microglia during the early postnatal periods via an epigenetic mechanism. The CCR5 antagonist maraviroc significantly suppressed neural circuit dysfunction and post-developmental behavioral disorders induced by prenatal VPA exposure. CONCLUSION: These findings suggest that microglial CCL3 might act during development to contribute to VPA-induced post-developmental behavioral abnormalities. CCR5-targeting compounds such as maraviroc might alleviate behavioral disorders when administered early.
Asunto(s)
Trastorno del Espectro Autista , Efectos Tardíos de la Exposición Prenatal , Animales , Trastorno del Espectro Autista/inducido químicamente , Conducta Animal , Modelos Animales de Enfermedad , Femenino , Maraviroc/uso terapéutico , Maraviroc/toxicidad , Ratones , Minociclina/farmacología , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Receptores CCR5/genética , Ácido Valproico/toxicidadRESUMEN
In a ciliate Paramecium, the presence of water channels on the membrane of contractile vacuole has long been predicted by both morphological and physiological data, however, to date either the biochemical or the molecular biological data have not been provided. In the present study, to examine the presence of aquaporin in Paramecium, we carried out RT-PCR with degenerated primers designed based on the ParameciumDB, and an aquaporin cDNA (aquaporin 1, aqp1) with a full-length ORF encoding 251 amino acids was obtained from Paramecium multimicronucleatum by using RACE. The deduced amino acid sequence of AQP1 had NPA-NPG motifs, and the prediction of protein secondary structure by CNR5000 and hydropathy plot showed the presence of six putative transmembrane domains and five connecting loops. Phylogenetic analysis results showed that the amino acid sequence of AQP1 was close to that of the Super-aquaporin group. The AQP1-GFP fusion protein clearly demonstrated the subcellular localization of AQP1 on the contractile vacuole complex, except for the decorated spongiome membrane. The functional analyses of aqp1 were done by RNA interference-based gene silencing, using an established feeding method. The aqp1 was found to be crucial for the total fluid output of the cell, the function of contractile vacuole membranes.
Asunto(s)
Paramecium , Secuencia de Aminoácidos , Acuaporina 1/genética , Paramecium/genética , Filogenia , VacuolasRESUMEN
Intrinsic optical signal (IOS) imaging has been widely used to map the patterns of brain activity in vivo in a label-free manner. Traditional IOS refers to changes in light transmission, absorption, reflectance, and scattering of the brain tissue. Here, we use polarized light for IOS imaging to monitor structural changes of cellular and subcellular architectures due to their neuronal activity in isolated brain slices. To reveal fast spatiotemporal changes of subcellular structures associated with neuronal activity, we developed the instantaneous polarized light microscope (PolScope), which allows us to observe birefringence changes in neuronal cells and tissues while stimulating neuronal activity. The instantaneous PolScope records changes in transmission, birefringence, and slow axis orientation in tissue at a high spatial and temporal resolution using a single camera exposure. These capabilities enabled us to correlate polarization-sensitive IOS with traditional IOS on the same preparations. We detected reproducible spatiotemporal changes in both IOSs at the stratum radiatum in mouse hippocampal slices evoked by electrical stimulation at Schaffer collaterals. Upon stimulation, changes in traditional IOS signals were broadly uniform across the area, whereas birefringence imaging revealed local variations not seen in traditional IOS. Locations with high resting birefringence produced larger stimulation-evoked birefringence changes than those produced at low resting birefringence. Local application of glutamate to the synaptic region in CA1 induced an increase in both transmittance and birefringence signals. Blocking synaptic transmission with inhibitors CNQX (for AMPA-type glutamate receptor) and D-APV (for NMDA-type glutamate receptor) reduced the peak amplitude of the optical signals. Changes in both IOSs were enhanced by an inhibitor of the membranous glutamate transporter, DL-TBOA. Our results indicate that the detection of activity-induced structural changes of the subcellular architecture in dendrites is possible in a label-free manner.
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Hipocampo , Microscopía , Animales , Birrefringencia , Dendritas , Técnicas In Vitro , RatonesRESUMEN
Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K+ -outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K+ channels in the membrane. Using gene-specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus-induced hyperpolarization-coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)-gene and 2 (ac2)-gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8-Br-cAMP and dilution of extracellular [K+ ] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization-activated inward K+ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus-induced acceleration of ciliary beat in Paramecium.
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Adenilil Ciclasas/genética , Cilios/fisiología , Paramecium/fisiología , Proteínas Protozoarias/genética , Adenilil Ciclasas/metabolismo , Fenómenos Biomecánicos , Paramecium/enzimología , Paramecium/genética , Filogenia , Proteínas Protozoarias/metabolismoRESUMEN
The type IX secretion system (T9SS) was originally discovered in Porphyromonas gingivalis, one of the pathogenic bacteria associated with periodontal disease and is now known to be present in many members of the phylum Bacteroidetes. The T9SS secretes a number of potent virulence factors, including the highly hydrolytic proteases called gingipains, across the outer membrane in P. gingivalis. To understand the entire machinery of T9SS, an exhaustive search for T9SS-related genes in P. gingivalis using the mariner family transposon (Tn) and Tn-seq analysis was performed. Seven hundred and two Tn insertion sites in Tn mutants with no colony pigmentation that is associated with Lys-gingipain (Kgp) defectiveness were determined, and it was found that the Tn was inserted in the kgp gene and 54 T9SS-related candidate genes. Thirty-three out of the 54 genes were already known as T9SS-related genes. Furthermore, deletion mutant analysis of the remaining 21 genes revealed that they were not related to the T9SS. The 33 T9SS-related genes include a gene for PGN_0297, which was found to be associated with the T9SS components PorK and PorN. A PGN_0297 gene deletion mutant was constructed, and it was found that the mutant showed no colony pigmentation, hemagglutination or gingipain activities, indicating that PGN_0297 was an essential component of the T9SS. The 33 genes did not include the six genes (gppX, omp17, porY, rfa, sigP and wzx) that were also reported as T9SS-related genes. gppX deletion and insertion mutants were constructed, and it was found that they did not show deficiency in the T9SS.
Asunto(s)
Sistemas de Secreción Bacterianos/genética , Sistemas de Secreción Bacterianos/metabolismo , Genes Bacterianos/genética , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/metabolismo , Adhesinas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Cisteína Endopeptidasas/metabolismo , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Cisteína-Endopeptidasas Gingipaínas , Hemaglutinación , Péptido Hidrolasas/metabolismo , Pigmentación/genética , Pigmentación/fisiología , Eliminación de Secuencia , Factores de Virulencia/genéticaRESUMEN
Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are unclear. In this study, we investigated the effects of low-level, chronic permethrin exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to the acceptable daily intake level of permethrin (0.3 ppm) in drinking water during the prenatal and postnatal periods. We then examined the effects on the central nervous system in adult male offspring. In the permethrin group, we detected behavior that displayed incomplete adaptation to a novel environment, as well as an impairment in learning and memory. In addition, immunohistochemical analysis revealed an increase in doublecortin- (an immature neuron marker) positive cells in the hippocampal dentate gyrus in the permethrin exposure group compared with the control group. Additionally, in the permethrin exposure group there was a decrease in astrocyte number in the hilus of the dentate gyrus, and remaining astrocytes were often irregularly shaped. These results suggest that exposure to permethrin at low levels in early life affects the formation of the neural circuit base and behavior after maturation. Therefore, in the central nervous system of male mice, low-level, chronic permethrin exposure during the prenatal and postnatal periods has effects that were not expected based on the known effects of permethrin exposure in mature animals.
Asunto(s)
Insecticidas/toxicidad , Aprendizaje/efectos de los fármacos , Memoria/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Permetrina/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Animales Recién Nacidos , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Hipocampo/patología , Masculino , Ratones Endogámicos C57BL , Neuroglía/patología , Neuronas/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/fisiopatologíaRESUMEN
Hypothalamic insulin receptor signaling regulates energy balance and glucose homeostasis via agouti-related protein (AgRP). While protein tyrosine phosphatase 1B (PTP1B) is classically known to be a negative regulator of peripheral insulin signaling by dephosphorylating both insulin receptor ß (IRß) and insulin receptor substrate, the role of PTP1B in hypothalamic insulin signaling remains to be fully elucidated. In the present study, we investigated the role of PTP1B in hypothalamic insulin signaling using PTP1B deficient (KO) mice in vivo and ex vivo. For the in vivo study, hypothalamic insulin resistance induced by a high-fat diet (HFD) improved in KO mice compared to wild-type (WT) mice. Hypothalamic AgRP mRNA expression levels were also significantly decreased in KO mice independent of body weight changes. In an ex vivo study using hypothalamic organotypic cultures, insulin treatment significantly increased the phosphorylation of both IRß and Akt in the hypothalamus of KO mice compared to WT mice, and also significantly decreased AgRP mRNA expression levels in KO mice. While incubation with inhibitors of phosphatidylinositol-3 kinase (PI3K) had no effect on basal levels of Akt phosphorylation, these suppressed insulin induction of Akt phosphorylation to almost basal levels in WT and KO mice. The inhibition of the PI3K-Akt pathway blocked the downregulation of AgRP mRNA expression in KO mice treated with insulin. These data suggest that PTP1B acts on the hypothalamic insulin signaling via the PI3K-Akt pathway. Together, our results suggest a deficiency of PTP1B improves hypothalamic insulin sensitivity resulting in the attenuation of AgRP mRNA expression under HFD conditions.
Asunto(s)
Proteína Relacionada con Agouti/genética , Dieta Alta en Grasa , Hipotálamo/metabolismo , Resistencia a la Insulina/genética , Insulina/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , ARN Mensajero/genética , Proteína Relacionada con Agouti/metabolismo , Animales , Perfilación de la Expresión Génica , Insulina/sangre , Ratones , Ratones Noqueados , ARN Mensajero/metabolismoRESUMEN
The Internet of Things (IoT) allows collecting vast amounts of health-relevant data such as daily activity, body weight (BW), and blood pressure (BP) automatically. The use of IoT devices to monitor diabetic patients has been studied, but could not evaluate IoT-dependent effects because health data were not measured in control groups. This multicenter, open-label, randomized, parallel group study will compare the impact of intensive health guidance using IoT and conventional medical guidance on glucose control. It will be conducted in outpatients with type 2 diabetes for a period of 6 months. IoT devices to measure amount of daily activity, BW, and BP will be provided to IoT group patients. Healthcare professionals (HCPs) will provide appropriate feedback according to the data. Non-IoT control, patients will be given measurement devices that do not have a feedback function. The primary outcome is glycated hemoglobin at 6 months. The study has already enrolled 101 patients, 50 in the IoT group and 51 in the non-IoT group, at the two participating outpatient clinics. The baseline characteristics of two groups did not differ, except for triglycerides. This will be the first randomized, controlled study to evaluate IoT-dependent effects of intensive feedback from HCPs. The results will validate a new method of health-data collection and provision of feedback suitable for diabetes support with increased effectiveness and low cost.
Asunto(s)
Glucemia/análisis , Diabetes Mellitus Tipo 2/sangre , Internet , Monitoreo Fisiológico/métodos , Dispositivos Electrónicos Vestibles , Anciano , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
We report a rare case of a patient with spinal epidural hematoma who presented with transient hemiplegia. A 90-year-old man awakened from sleep due to sudden neck pain. Fifteen minutes later, the man experienced progressively worsening weakness in his left hand, and was transported in an ambulance to our hospital. At the hospital, he presented with hemiplegia, and we suspected intracranial disease. Therefore, we performed magnetic resonance imaging (MRI), which revealed no intracranial lesions. Shortly after the MRI, the patient showed no signs of hemiplegia. However, since the severe neck pain persisted, we performed cervical MRI, which showed a high-intensity area at the C2-C5 level, predominantly on the left side. Despite recovery from hemiplegia, we performed a laminectomy of C3-C5 with evacuation of a hematoma at the C2-C6 level. After the surgery, the patient had no neck pain.
Asunto(s)
Hematoma Espinal Epidural/cirugía , Hemiplejía/etiología , Dolor de Cuello/etiología , Anciano de 80 o más Años , Terapia Combinada , Hematoma Espinal Epidural/complicaciones , Humanos , Laminectomía , Imagen por Resonancia Magnética , Masculino , Tomografía Computarizada por Rayos XRESUMEN
A woman in her 60s visited our hospital because of frequent hypoglycemia and episodes of unconsciousness over the last 6 years. A 4 cm tumor was detected on the pancreatic tail using abdominal computed tomography and ultrasonography. An insulinoma was strongly suspected from the results of the fasting test and glucagon load test, and a distal pancreatectomy with splenectomy was performed. Pathological examination indicated an insulinoma and neuroendocrine tumor(NET)G2 based on the WHO 2010 classification. The patient's blood sugar and insulin levels returned to normal, and hypoglycemic attacks disappeared postoperatively. Six months later, a total parathyroidectomy was performed because of primary hyperparathyroidism with hypertrophy of the parathyroid glands. Furthermore, pituitary swellingwas also detected usinghead MRI. However, the patient has been under observation because the tumor was non-functional without any associated symptoms. Thus, we diagnosed the patient with multiple endocrine neoplasia type 1(MEN1). In patients with pancreatic NET, it is necessary to consider the possibility of MEN1.
Asunto(s)
Hipoglucemia/etiología , Neoplasia Endocrina Múltiple Tipo 1 , Neoplasias Pancreáticas/patología , Femenino , Humanos , Neoplasia Endocrina Múltiple Tipo 1/complicaciones , Neoplasia Endocrina Múltiple Tipo 1/diagnóstico por imagen , Neoplasia Endocrina Múltiple Tipo 1/cirugía , Pancreatectomía , Neoplasias Pancreáticas/complicaciones , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/cirugía , Pronóstico , Tomografía Computarizada por Rayos XRESUMEN
Extracorporeal life support (ECLS) is used after congenital heart surgery for several indications, including failure to separate from cardiopulmonary bypass, postoperative low cardiac output syndrome, and extracorporeal cardiopulmonary resuscitation. Here, we assessed the outcomes of ECLS in children after cardiac surgery at our institution. Medical records of all children who required postoperative ECLS at our institution were reviewed. Between 2003 and 2011, 36 (1.4%) of 2541 pediatric cardiac surgical cases required postoperative ECLS. Median age of patients was 64 days (range: 0 days-4.1 years). ECLS was in the form of either extracorporeal membrane oxygenation (ECMO; n = 24) or ventricular assist system (VAS; n = 12). Mean duration of ECLS was 4.9 ± 4.2 days. Overall, 21 patients (58%) were weaned off ECLS, and 17 patients (47%) were successfully discharged from the hospital. Patients with biventricular heart (BVH) had higher survival-to-hospital discharge rates compared with those with univentricular heart (UVH) (P = 0.019). Regarding ECLS type, UVH patients who received VAS showed higher rates of device discontinuation than UVH patients who received ECMO (P = 0.012). However, rates of hospital discharge were not significantly different between UVH patients who received VAS or ECMO. Surgical interventions, such as banding of Blalock-Taussig shunt to reduce pulmonary blood flow or placing bidirectional cavopulmonary shunt to minimize ventricular volume overload, were effective for weaning off ECLS in patients with UVH. ECLS is beneficial to children with low cardiac output after cardiac surgery. Rates of survival-to-hospital discharge were higher in BVH patients than UVH patients. Additional interventions to reduce ventricular volume load may be effective for discontinuing ECLS in patients with UVH.
Asunto(s)
Oxigenación por Membrana Extracorpórea/instrumentación , Cardiopatías Congénitas/cirugía , Corazón Auxiliar , Sistemas de Manutención de la Vida/instrumentación , Preescolar , Femenino , Cardiopatías Congénitas/terapia , Humanos , Lactante , Recién Nacido , Masculino , Resultado del TratamientoRESUMEN
The balance of activity between glutamatergic and GABAergic networks is particularly important for oscillatory neural activities in the brain. Here, we investigated the roles of GABAB receptors in network oscillation in the oral somatosensory cortex (OSC), focusing on NMDA receptors. Neural oscillation at the frequency of 8-10 Hz was elicited in rat brain slices after caffeine application. Oscillations comprised a non-NMDA receptor-dependent initial phase and a later NMDA receptor-dependent oscillatory phase, with the oscillator located in the upper layer of the OSC. Baclofen was applied to investigate the actions of GABAB receptors. The later NMDA receptor-dependent oscillatory phase completely disappeared, but the initial phase did not. These results suggest that GABAB receptors mainly act on NMDA receptor, in which metabotropic actions of GABAB receptors may contribute to the attenuation of NMDA receptor activities. A regulatory system for network oscillation involving GABAB receptors may be present in the OSC.
Asunto(s)
Receptores de GABA-B , Receptores de N-Metil-D-Aspartato , Ratas , Animales , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de GABA-B/metabolismo , Corteza Somatosensorial/metabolismo , BaclofenoRESUMEN
Recent advances in fluorescent confocal microscopy and voltage-sensitive and Ca(2+) dyes have vastly improved our ability to image neuronal circuits. However, existing confocal systems are not fast enough or too noisy for many live-cell functional imaging studies. Here, we describe and demonstrate the function of a novel, nonscanning confocal microscopy module. The optics, which are designed to fit the standard camera port of the Olympus BX51WI epifluorescent microscope, achieve a high signal-to-noise ratio (SNR) at high temporal resolution, making this configuration ideal for functional imaging of neuronal activities such as the voltage-sensitive dye (VSD) imaging. The optics employ fixed 100- × 100-pinhole arrays at the back focal plane (optical conjugation plane), above the tube lens of a usual upright microscope. The excitation light travels through these pinholes, and the fluorescence signal, emitted from subject, passes through corresponding pinholes before exciting the photodiodes of the imager: a 100- × 100-pixel metal-oxide semiconductor (MOS)-type pixel imager with each pixel corresponding to a single 100- × 100-µm photodiode. This design eliminated the need for a scanning device; therefore, acquisition rate of the imager (maximum rate of 10 kHz) is the only factor limiting acquisition speed. We tested the application of the system for VSD and Ca(2+) imaging of evoked neuronal responses on electrical stimuli in rat hippocampal slices. The results indicate that, at least for these applications, the new microscope maintains a high SNR at image acquisition rates of ≤0.3 ms per frame.
Asunto(s)
Calcio/análisis , Microscopía Confocal/instrumentación , Imagen de Colorante Sensible al Voltaje/instrumentación , Animales , Hipocampo/química , Masculino , RatasRESUMEN
The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca(2+) and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥ 1 mM Mg(2+)-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.
Asunto(s)
Cilios/metabolismo , AMP Cíclico/metabolismo , Dineínas/metabolismo , Locomoción , Paramecium tetraurelia/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Axonema/genética , Axonema/metabolismo , Cloruro de Calcio/farmacología , Cilios/efectos de los fármacos , Técnicas de Cultivo , Dineínas/genética , Electroforesis en Gel de Poliacrilamida , Genes Protozoarios , Datos de Secuencia Molecular , Paramecium tetraurelia/genética , Fenotipo , Fosforilación , Proteínas Protozoarias/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Plasticity is the key feature of our brain function. Specifically, plasticity of hippocampal synapses is critical for learning and memory. The functional properties of the neuronal circuit change as a result of synaptic plasticity. This review summarizes the use of voltage-sensitive dyes (VSDs) to examine neuronal circuit plasticity. We will discuss the significance of plastic changes in circuit function as well as the technical issue of using VSDs. Further, we will discuss the neural circuit level plasticity of the hippocampus caused by long-term potentiation and the entorhinal-perirhinal connection. This review article is an extended version of the Japanese article, Membrane Potential Imaging with Voltage-sensitive Dye (VSD) for Long-term Recording, published in SEIBUTSU BUTSURI Vol. 61, p. 404-408 (2021).
RESUMEN
In Paramecium, a mechanical stimulus applied to the posterior portion of the cell causes a transient increase in membrane permeability to potassium ions, transiently rendering the membrane in a hyperpolarized state. Hyperpolarization causes a transient increase in Cyclic adenosine monophosphate (cAMP) concentration in the cilia, resulting in a transient fast-forward swimming of the cell. Schultz and coworkers (1992) reported that a unique adenylate cyclase (AC)-coupled potassium channel is involved in the reaction underlying this response, which is known as the "escape response." However, the AC responsible for this reaction remains to be identified. Moreover, the molecular linkage between mechanoreception and AC activation has not been elucidated adequately. Currently, we can perform an efficient and simple gene-knockdown technique in Paramecium using RNA interference (RNAi). Paramecium is one of the several model organisms for which whole-genome sequences have been elucidated. The RNAi technique can be applied to whole genome sequences derived from the Paramecium database (ParameciumDB) to investigate the types of proteins that elicit specific biological responses and compare them with those of other model organisms. In this review, we describe the applications of the RNAi technique in elucidating the molecular mechanism underlying the escape response and identifying the AC involved in this reaction. The findings of this study highlight the advantages of the RNAi technique and ParameciumDB.
RESUMEN
Prefrontal cortex (PFC) intrahemispheric activity and the interhemispheric connection have a significant impact on neuropsychiatric disorder pathology. This study aimed to generate a functional map of FC intrahemispheric and interhemispheric connections. Functional dissection of mouse PFCs was performed using the voltage-sensitive dye (VSD) imaging method with high speed (1 ms/frame), high resolution (256 × 256 pixels), and a large field of view (â¼10 mm). Acute serial 350 µm slices were prepared from the bregma covering the PFC and numbered 1-5 based on their distance from the bregma (i.e., 1.70, 1.34, 0.98, 0.62, and 0.26 mm) with reference to the Mouse Brain Atlas (Paxinos and Franklin, 2008). The neural response to electrical stimulation was measured at nine sites and then averaged, and a functional map of the propagation patterns was created. Intracortical propagation was observed in slices 3-5, encompassing the anterior cingulate cortex (ACC) and corpus callosum (CC). The activity reached area 33 of the ACC. Direct white matter stimulation activated area 33 in both hemispheres. Similar findings were obtained via DiI staining of the CC. Imaging analysis revealed directional biases in neural signals traveling within the ACC, whereby the signal transmission speed and probability varied based on the signal direction. Specifically, the spread of neural signals from cg2 to cg1 was stronger than that from cingulate cortex area 1(cg1) to cingulate cortex area 2(cg2), which has implications for interhemispheric functional connections. These findings highlight the importance of understanding the PFC functional anatomy in evaluating neuromodulators like serotonin and dopamine, as well as other factors related to neuropsychiatric diseases.