RESUMEN
Hippocampal activity represents many behaviorally important variables, including context, an animal's location within a given environmental context, time, and reward. Using longitudinal calcium imaging in mice, multiple large virtual environments, and differing reward contingencies, we derived a unified probabilistic model of CA1 representations centered on a single feature-the field propensity. Each cell's propensity governs how many place fields it has per unit space, predicts its reward-related activity, and is preserved across distinct environments and over months. Propensity is broadly distributed-with many low, and some very high, propensity cells-and thus strongly shapes hippocampal representations. This results in a range of spatial codes, from sparse to dense. Propensity varied â¼10-fold between adjacent cells in salt-and-pepper fashion, indicating substantial functional differences within a presumed cell type. Intracellular recordings linked propensity to cell excitability. The stability of each cell's propensity across conditions suggests this fundamental property has anatomical, transcriptional, and/or developmental origins.
Asunto(s)
Hipocampo/anatomía & histología , Hipocampo/fisiología , Animales , Conducta Animal/fisiología , Fenómenos Biofísicos , Calcio/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Neurológicos , Células Piramidales/fisiología , Recompensa , Análisis y Desempeño de Tareas , Factores de TiempoRESUMEN
Persistent inward currents (PICs) and persistent outward currents (POCs) regulate the excitability and firing behaviours of spinal motoneurons (MNs). Given their potential role in MN excitability dysfunction in amyotrophic lateral sclerosis (ALS), PICs have been previously studied in superoxide dismutase 1 (SOD1)-G93A mice (the standard animal model of ALS); however, conflicting results have been reported on how the net PIC changes during disease progression. Also, individual PICs and POCs have never been examined before in symptomatic ALS. To fill this gap, we measured the net and individual PIC and POC components of wild-type (WT) and SOD MNs in current clamp and voltage clamp during disease progression (assessed by neuroscores). We show that SOD MNs of symptomatic mice experience a much larger net PIC, relative to WT cells from age-matched littermates. Specifically, the Na+ and Ca2+ PICs are larger, whereas the lasting SK-mediated (SKL) POC is smaller than WT (Na+ PIC is the largest and SKL POC is the smallest components in SOD MNs). We also show that PIC dysregulation is present at symptom onset, is sustained throughout advanced disease stages and is proportional to SOD MN cell size (largest dysregulation is in the largest SOD cells, the most vulnerable in ALS). Additionally, we show that studying disease progression using neuroscores is more accurate than using SOD mouse age, which could lead to misleading statistics and age-based trends. Collectively, this study contributes novel PIC and POC data, reveals ionic mechanisms contributing to the vulnerability differential among MN types/sizes, and provides insights on the roles PIC and POC mechanisms play in MN excitability dysfunction in ALS. KEY POINTS: Individual persistent inward currents (PICs) and persistent outward currents (POCs) have never been examined before in spinal motoneurons (MNs) of symptomatic amyotrophic lateral sclerosis (ALS) mice. Thus, we contribute novel PIC and POC data to the ALS literature. Male SOD MNs of symptomatic mice have elevated net PIC, with larger Na+ and Ca2+ PICs but reduced SKL POC vs. wild-type littermates. Na+ PIC is the largest and SKL POC is the smallest current in SOD cells. The PIC/POC dysregulation is present at symptom onset. PIC dysregulation is sustained throughout advanced disease, and is proportional to SOD MN size (largest dysregulation is in the largest cells, the most vulnerable in ALS). Thus, we reveal ionic mechanisms contributing to the vulnerability differential among MN types/sizes in ALS. Studying disease progression using SOD mice neuroscores is more accurate than using age, which could distort the statistical differences between SOD and WT PIC/POC data and the trends during disease progression.
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Esclerosis Amiotrófica Lateral , Ratones Transgénicos , Neuronas Motoras , Animales , Neuronas Motoras/fisiología , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Ratones , Médula Espinal/fisiología , Superóxido Dismutasa-1/genética , Masculino , Femenino , Ratones Endogámicos C57BL , Potenciales de AcciónRESUMEN
Rhythmic electrical events, termed slow waves, govern the timing and amplitude of phasic contractions of the gastric musculature. Extracellular multielectrode measurement of gastric slow waves can be a biomarker for phenotypes of motility dysfunction. However, a gastric slow-wave conduction pathway for the rat, a common animal model, is unestablished. In this study, the validity of extracellular recording was demonstrated in vitro with simultaneous intracellular and extracellular recordings and by pharmacological inhibition of slow waves. The conduction pathway was determined by in vivo extracellular recordings while considering the effect of motion. Slow-wave characteristics [means (SD)] varied regionally having higher amplitude in the antrum than the distal corpus [1.03 (0.12) mV vs. 0.75 (0.31) mV; n = 7; P = 0.025 paired t test] and faster propagation near the greater curvature than the lesser curvature [1.00 (0.14) mm·s-1 vs. 0.74 (0.14) mm·s-1; n = 9 GC, 7 LC; P = 0.003 unpaired t test]. Notably, in some subjects, separate wavefronts propagated near the lesser and greater curvatures with a loosely coupled region occurring in the area near the distal corpus midline at the interface of the two wavefronts. This region had either the greater or lesser curvature wavefront propagating through it in a time-varying manner. The conduction pattern suggests that slow waves in the rat stomach form annular wavefronts in the antrum and not the corpus. This study has implications for interpretation of the relationship between slow waves, the interstitial cells of Cajal network structure, smooth muscles, and gastric motility.NEW & NOTEWORTHY Mapping of rat gastric slow waves showed regional variations in their organization. In some subjects, separate wavefronts propagated near the lesser and greater curvatures with a loosely coupled region near the midline, between the wavefronts, having a varying slow-wave origin. Furthermore, simultaneous intracellular and extracellular recordings were concordant and independent of movement artifacts, indicating that extracellular recordings can be interpreted in terms of their intracellular counterparts when intracellular recording is not possible.
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Motilidad Gastrointestinal , Músculo Liso , Ratas Sprague-Dawley , Estómago , Animales , Estómago/fisiología , Ratas , Motilidad Gastrointestinal/fisiología , Masculino , Músculo Liso/fisiología , Contracción Muscular/fisiología , Antro Pilórico/fisiología , Células Intersticiales de Cajal/fisiologíaRESUMEN
Electrode arrays are widely used for multipoint recording of electrophysiological activities, and organic electronics have been utilized to achieve both high performance and biocompatibility. However, extracellular electrode arrays record the field potential instead of the membrane potential itself, resulting in the loss of information and signal amplitude. Although much effort has been dedicated to developing intracellular access methods, their three-dimensional structures and advanced protocols prohibited implementation with organic electronics. Here, we show an organic electrochemical transistor (OECT) matrix for the intracellular action potential recording. The driving voltage of sensor matrix simultaneously causes electroporation so that intracellular action potentials are recorded with simple equipment. The amplitude of the recorded peaks was larger than that of an extracellular field potential recording, and it was further enhanced by tuning the driving voltage and geometry of OECTs. The capability of miniaturization and multiplexed recording was demonstrated through a 4 × 4 action potential mapping using a matrix of 5- × 5-µm2 OECTs. Those features are realized using a mild fabrication process and a simple circuit without limiting the potential applications of functional organic electronics.
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Potenciales de Acción , Técnicas Biosensibles/métodos , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Transistores Electrónicos/estadística & datos numéricos , Células Cultivadas , Electroporación , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citologíaRESUMEN
Electroporation is a proven technique that can record action potential of cardiomyocytes and serve for biomolecular delivery. To ensure high cell viability, micro-nanodevices cooperating with low-voltage electroporation are frequently utilized in research, and the effectiveness of delivery for intracellular access is typically assessed using an optical imaging approach like flow cytometry. However, the efficiency of in situ biomedical studies is hampered by the intricacy of these analytical approaches. Here, we develop an integrated cardiomyocyte-based biosensing platform to effectively record action potential and evaluate the electroporation quality in terms of viability, delivery efficiency, and mortality. The ITO-MEA device of the platform possesses sensing/stimulating electrodes which combines with the self-developed system to achieve intracellular action potential recording and delivery by electroporation trigger. Moreover, the image acquisition processing system analyzes various parameters effectively to assess delivery performance. Therefore, this platform has the potential for drug delivery therapy and pathology research for cardiology.
Asunto(s)
Electroporación , Miocitos Cardíacos , Terapia de Electroporación , Electrodos , Supervivencia CelularRESUMEN
The pond snail Lymnaea stagnalis exhibits clear photoperiodism in egg laying; it lays more eggs in long-day conditions than in medium-day conditions. A key regulator of egg laying is neurosecretory caudo-dorsal cells (CDCs) producing an ovulation hormone in the cerebral ganglia. Paired small budding structures of the cerebral ganglia (viz. the lateral lobe) also promote egg laying in addition to spermatogenesis and maturation of female accessory sex organs. However, it remains unknown which cells in the lateral lobe are responsible for these. Previous anatomical and physiological studies prompted us to hypothesize that canopy cells in the lateral lobe modulate activity of CDCs. However, double labeling of the canopy cell and CDCs revealed no sign of direct neural connections, suggesting that activity of CDCs is regulated either humorally or through a neural pathway independent of canopy cells. In addition, our detailed anatomical re-evaluation confirmed previous observations that the canopy cell bears fine neurites along the ipsilateral axon and extensions from the plasma membrane of the cell body, although the function of these extensions remains unexplored. Furthermore, comparison of electrophysiological properties between long-day and medium-day conditions indicated that the canopy cell's activity is moderately under photoperiodic regulation: resting membrane potentials of long-day snails are shallower than those of medium-day snails, and spontaneously spiking neurons are only observed in long-day conditions. Thus, canopy cells appear to receive photoperiodic information and regulate photoperiod-dependent phenomena, but not provide direct neural inputs to CDCs.
Asunto(s)
Lymnaea , Sistemas Neurosecretores , Animales , Masculino , Femenino , Lymnaea/fisiología , Neuronas/fisiología , Caracoles , Axones/fisiologíaRESUMEN
The reliable identification of chronic cardiotoxic effects in in vitro screenings is fundamental for filtering out toxic molecular entities before in vivo animal experimentation and clinical trials. Present techniques such as patch-clamp, voltage indicators, and standard microelectrode arrays do not offer at the same time high sensitivity for measuring transmembrane ion currents and low-invasiveness for monitoring cells over long time. Here, we show that optoporation applied to microelectrode arrays enables measuring action potentials from human-derived cardiac syncytia for more than 1 continuous month and provides reliable data on chronic cardiotoxic effects caused by known compounds such as pentamidine. The technique has high potential for detecting chronic cardiotoxicity in the early phases of drug development.
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Cardiotoxicidad , Miocitos Cardíacos , Animales , Humanos , Potenciales de Acción , MicroelectrodosRESUMEN
The effects of aging on the nervous system are well documented. However, most previous studies on this topic were performed on the central nervous system. The present study was carried out on the dorsal root ganglia (DRGs) of mice, and focused on age-related changes in DRG neurons and satellite glial cells (SGCs). Intracellular electrodes were used for dye injection to examine the gap junction-mediated coupling between neurons and SGCs, and for intracellular electrical recordings from the neurons. Tactile sensitivity was assessed with von Frey hairs. We found that 3-23% of DRG neurons were dye-coupled to SGCs surrounding neighboring neurons in 8-24-month (Mo)-old mice, whereas in young adult (3 Mo) mice, the figure was 0%. The threshold current for firing an action potential in sensory neurons was significantly lower in DRGs from 12 Mo mice compared with those from 3 Mo mice. The percentage of neurons with spontaneous subthreshold membrane potential oscillation was greater by two-fold in 12 Mo mice. The withdrawal threshold was lower by 22% in 12 Mo mice compared with 3 Mo ones. These results show that in the aged mice, a proportion of DRG neurons is coupled to SGCs, and that the membrane excitability of the DRG neurons increases with age. We propose that augmented neuron-SGC communications via gap junctions are caused by low-grade inflammation associated with aging, and this may contribute to pain behavior.
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Ganglios Espinales , Neuroglía , Ratones , Animales , Potenciales de la Membrana , Células Receptoras Sensoriales , Ratones Endogámicos BALB CRESUMEN
Retinal horizontal cells form a broad receptive field, which contributes to generating antagonistic surround responses in retinal bipolar cells. Here, I report that certain horizontal cells themselves have center-surround antagonistic receptive fields. The receptive fields of yellow/red, blue-type horizontal cells (Y/RB HCs) in the carp retina were measured by the response to the slit of light stimulus using the conventional intracellular electrode. A center stimulus of monochromatic light of 500 nm hyperpolarized Y/RB HCs, whereas the peripheral light depolarized the cells, suggesting that these cells exhibit an antagonistic receptive field at 500 nm light. The length constant of Y/RB HC's depolarizing responses to 600 nm light was 1.22 ± 0.08 mm, which was larger than that (0.61 ± 0.06 mm) of hyperpolarizing responses to 500 nm light. Thus, depolarizing responses of Y/RB HCs exhibit a larger receptive field than hyperpolarizing responses. The length constant of hyperpolarizing responses of luminosity-type HCs (LHCs) was 1.19 ± 0.07 mm, which was similar to that of 500 nm depolarizing responses of Y/RB HCs (1.34 ± 0.11 mm). Depolarizing response of Y/RB HCs was decreased by bath application of GABA and picrotoxin, a GABA receptor antagonist, suggesting that GABAergic signaling may modulate center-surround antagonistic mechanisms in Y/RB HCs. Bipolar cells display center-surround antagonistic receptive fields that play important roles to improve visual contrast. Wide receptive fields of HCs contribute to generating surround responses in bipolar cells. Therefore, the response polarity of Y/RB HCs may affect the width of the surround receptive field in bipolar cells.NEW & NOTEWORTHY Retinal horizontal cells form a broad receptive field, which contributes to generating antagonistic surround responses in retinal bipolar cells. Here, I found that depolarizing responses of yellow/red, blue-type horizontal cells (Y/RB HCs) exhibit a larger receptive field than hyperpolarizing responses at monochromatic lights between 480 nm and 520 nm. Because bipolar cells play a key role in the detection of visual contrast, depolarization or hyperpolarization of Y/RB HCs may regulate the size of the surround receptive field in the bipolar cells.
Asunto(s)
Retina , Células Horizontales de la Retina , Estimulación Luminosa , Retina/fisiología , Células Bipolares de la Retina/fisiología , Antagonistas del GABA/farmacologíaRESUMEN
The progress in optogenetics largely depends on the development of light-activated proteins as new molecular tools. Using cultured hippocampal neurons, we compared the properties of two light-activated cation channels - classical channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) and recently described channelrhodopsin isolated from the alga Platymonas subcordiformis (PsChR2). PsChR2 ensured generation of action potentials by neurons when activated by the pulsed light stimulation with the frequencies up to 40-50 Hz, while the upper limit for CrChR2 was 20-30 Hz. An important advantage of PsChR2 compared to classical channelrhodopsin CrChR2 is the blue shift of its excitation spectrum, which opens the possibility for its application in all-optical electrophysiology experiments that require the separation of the maxima of the spectra of channelrhodopsins used for the stimulation of neurons and the maxima of the excitation spectra of various red fluorescent probes. We compared the response (generation of action potentials) of neurons expressing CrChR2 and PsChR2 to light stimuli at 530 and 550 nm commonly used for the excitation of red fluorescent probes. The 530-nm light was significantly (3.7 times) less efficient in the activation of neurons expressing PsChR2 vs. CrChR2-expressing neurons. The light at 550 nm, even at the maximal used intensity, failed to stimulate neurons expressing either of the studied opsins. This indicates that the PsChR2 channelrhodopsin from the alga P. subcordiformis is a promising optogenetic tool, both in terms of its frequency characteristics and possibility of its application for neuronal stimulation with a short-wavelength (blue, 470 nm) light accompanied by simultaneous recording of various physiological processes using fluorescent probes.
Asunto(s)
Chlorophyta , Colorantes Fluorescentes , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Optogenética , CationesRESUMEN
Drug-induced cardiotoxicity is a major barrier to drug development and a main cause of withdrawal of marketed drugs. Drugs can strongly alter the spontaneous functioning of the heart by interacting with the cardiac membrane ion channels. If these effects only surface during in vivo preclinical tests, clinical trials or worse after commercialization, the societal and economic burden will be significant and seriously hinder the efficient drug development process. Hence, cardiac safety pharmacology requires in vitro electrophysiological screening assays of all drug candidates to predict cardiotoxic effects before clinical trials. In the past 10 years, microelectrode array (MEA) technology began to be considered a valuable approach in pharmaceutical applications. However, an effective tool for high-throughput intracellular measurements, compatible with pharmaceutical standards, is not yet available. Here, we propose laser-induced optoacoustic poration combined with CMOS-MEA technology as a reliable and effective platform to detect cardiotoxicity. This approach enables the acquisition of high-quality action potential recordings from large numbers of cardiomyocytes within the same culture well, providing reliable data using single-well MEA devices and single cardiac syncytia per each drug. Thus, this technology could be applied in drug safety screening platforms reducing times and costs of cardiotoxicity assessments, while simultaneously improving the data reliability.
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Potenciales de Acción/efectos de los fármacos , Arritmias Cardíacas/inducido químicamente , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Rayos Láser , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Técnicas Fotoacústicas/instrumentación , Pruebas de Toxicidad/instrumentación , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Cardiotoxicidad , Ahorro de Costo , Análisis Costo-Beneficio , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Microelectrodos/economía , Miocitos Cardíacos/metabolismo , Técnicas Fotoacústicas/economía , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de Tiempo , Pruebas de Toxicidad/economía , Flujo de TrabajoRESUMEN
The patch-clamp technique is one of the best approaches to investigate neural excitability. Impressive improvements towards the automation of the patch-clamp technique have been made, but obvious limitations and hurdles still exist, such as parallelization, volume displacement in vivo, and long-term recording. Nanotechnologies have provided opportunities to overcome these hurdles by applying electrical devices on the nanoscale. Electrodes based on nanowires, nanotubes, and nanoscale field-effect transistors (FETs) are confirmed to be robust and less invasive tools for intracellular electrophysiological recording. Research on the interface between the nanoelectrode and cell membrane aims to reduce the seal conductance and further improve the recording quality. Many novel recording approaches advance the parallelization, and precision with reduced invasiveness, thus improving the overall intracellular recording system. The combination of nanotechnology and the present intracellular recording framework is a revolutionary and promising orientation, potentially becoming the next generation electrophysiological recording technique and replacing the conventional patch-clamp technique. Here, this paper reviews the recent advances in intracellular electrophysiological recording techniques using nanotechnology, focusing on the design of noninvasive and greatly parallelized recording systems based on nanoelectronics.
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Electrodos , Electrofisiología/métodos , Nanotecnología/métodos , Animales , Membrana Celular , Citoplasma , Humanos , Nanotubos , Nanocables , NeuronasRESUMEN
Anoxia induces a reversible coma in insects. Coma onset is triggered by the arrest of mechanisms responsible for maintaining membrane ion homeostasis in the CNS, resulting in a wave of neuronal and glial depolarization known as spreading depolarization (SD). Different methods of anoxia influence the behavioural response but their effects on SD are unknown. We investigated the effects of CO2, N2, and H2O on the characteristics of coma induction and recovery in Locusta migratoria. Water immersion delayed coma onset and recovery, likely due to involvement of the tracheal system and the nature of asphyxiation but otherwise resembled N2 delivery. The main difference between N2 and CO2 was that CO2 hastened onset of neural failure and SD and delayed recovery. In the CNS, this was associated with CO2 inducing an abrupt and immediate decrease of interstitial pH and increase of extracellular [K+]. Recording of the transperineurial potential showed that SD propagation and a postanoxic negativity (PAN) were similar with both gases. The PAN increased with ouabain treatment, likely due to removal of the counteracting electrogenic effect of Na+/K+-ATPase, and was inhibited by bafilomycin, a proton pump inhibitor, suggesting that it was generated by the electrogenic effect of a Vacuolar-type ATPase (VA). Muscle fibres depolarized by ~20 mV, which happened more rapidly with CO2 compared with N2. Wing muscle motoneurons depolarized nearly completely in two stages, with CO2 causing more rapid onset and slower recovery than N2. Other parameters of SD onset and recovery were similar with the two gases. Electrical resistance across the ganglion sheath increased during anoxia and at SD onset. We provisionally attribute this to cell swelling reducing the dimensions of the interstitial pathway from neuropil to the bathing saline. Neuronal membrane resistance decreased abruptly at SD onset indicating opening of an unidentified membrane conductance. Consideration of the intracellular recording relative to the saline suggests that the apical membrane of perineurial glia depolarizes prior to neuron depolarization. We propose that SD is triggered by events at the perineurial sheath and then propagates laterally and more deeply into the neuropil. We conclude that the fundamental nature of SD is not dependent on the method of anoxia however the timing of onset and recovery are influenced; water immersion is complicated by the tracheal system and CO2 delivery has more rapid and longer lasting effects, associated with severe interstitial acidosis.
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Dióxido de Carbono/metabolismo , Sistema Nervioso Central/fisiología , Hipoxia/metabolismo , Locusta migratoria/fisiología , Neuroglía/metabolismo , Ouabaína/farmacología , Animales , Membrana Celular/metabolismo , Electromiografía , Electrofisiología , Femenino , Homeostasis/efectos de los fármacos , Iones/metabolismo , Locusta migratoria/metabolismo , Macrólidos/farmacología , Masculino , Neuronas/metabolismo , Potasio/metabolismo , Tráquea/metabolismoRESUMEN
Although the bell-shaped nectophores of the siphonophore Nanomia bijuga are clearly specialized for locomotion, their complex neuroanatomy described here testifies to multiple subsidiary functions. These include secretion, by the extensively innervated 'flask cells' located around the bell margin, and protection, by the numerous nematocytes that line the nectophore's exposed ridges. The main nerve complex consists of a nerve ring at the base of the bell, an adjacent column-shaped matrix plus two associated nerve projections. At the top of the nectophore the upper nerve tract appears to have a sensory role; on the lower surface a second nerve tract provides a motor input connecting the nectophore with the rest of the colony via a cluster of nerve cells at the stem. N. bijuga is capable of both forward and backward jet-propelled swimming. During backwards swimming the water jet is redirected by the contraction of the Claus' muscle system, part of the muscular velum that fringes the bell aperture. Contractions can be elicited by electrical stimulation of the nectophore surface, even when both upper and lower nerve tracts have been destroyed. Epithelial impulses elicited there, generate slow potentials and action potentials in the velum musculature. Slow potentials arise at different sites around the bell margin and give rise to action potentials in contracting Claus' muscle fibres. A synaptic rather than an electrotonic model more readily accounts for the time course of the slow potentials. During backward swimming, isometrically contracting muscle fibres in the endoderm provide the Claus' fibres with an immobile base.
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Hidrozoos , Potenciales de Acción , Animales , Potenciales de la Membrana , Sistema Nervioso , NataciónRESUMEN
CMOS microelectrode arrays (MEAs) can record electrophysiological activities of a large number of neurons in parallel but only extracellularly with low signal-to-noise ratio. Patch clamp electrodes can perform intracellular recording with high signal-to-noise ratio but only from a few neurons in parallel. Recently we have developed and reported a neuroelectronic interface that combines the parallelism of the CMOS MEA and the intracellular sensitivity of the patch clamp. Here, we report the design and characterization of the CMOS integrated circuit (IC), a critical component of the neuroelectronic interface. Fabricated in 0.18-µm technology, the IC features an array of 4,096 platinum black (PtB) nanoelectrodes spaced at a 20 µm pitch on its surface and contains 4,096 active pixel circuits. Each active pixel circuit, consisting of a new switched-capacitor current injector--capable of injecting from ±15 pA to ±0.7 µA with a 5 pA resolution--and an operational amplifier, is highly configurable. When configured into current-clamp mode, the pixel intracellularly records membrane potentials including subthreshold activities with â¼23 µVrms input referred noise while injecting a current for simultaneous stimulation. When configured into voltage-clamp mode, the pixel becomes a switched-capacitor transimpedance amplifier with â¼1 pArms input referred noise, and intracellularly records ion channel currents while applying a voltage for simultaneous stimulation. Such voltage/current-clamp intracellular recording/stimulation is a feat only previously possible with the patch clamp method. At the same time, as an array, the IC overcomes the lack of parallelism of the patch clamp method, measuring thousands of mammalian neurons in parallel, with full-frame intracellular recording/stimulation at 9.4 kHz.
RESUMEN
Animals use binocular information to guide many behaviors. In highly visual arthropods, complex binocular computations involved in processing panoramic optic flow generated during self-motion occur in the optic neuropils. However, the extent to which binocular processing of object motion occurs in these neuropils remains unknown. We investigated this in a crab, where the distance between the eyes and the extensive overlapping of their visual fields advocate for the use of binocular processing. By performing in vivo intracellular recordings from the lobula (third optic neuropil) of male crabs, we assessed responses of object-motion-sensitive neurons to ipsilateral or contralateral moving objects under binocular and monocular conditions. Most recorded neurons responded to stimuli seen independently with either eye, proving that each lobula receives profuse visual information from both eyes. The contribution of each eye to the binocular response varies among neurons, from those receiving comparable inputs from both eyes to those with mainly ipsilateral or contralateral components, some including contralateral inhibition. Electrophysiological profiles indicated that a similar number of neurons were recorded from their input or their output side. In monocular conditions, the first group showed shorter response delays to ipsilateral than to contralateral stimulation, whereas the second group showed the opposite. These results fit well with neurons conveying centripetal and centrifugal information from and toward the lobula, respectively. Intracellular and massive stainings provided anatomical support for this and for direct connections between the two lobulae, but simultaneous recordings failed to reveal such connections. Simplified model circuits of interocular connections are discussed.SIGNIFICANCE STATEMENT Most active animals became equipped with two eyes, which contributes to functions like depth perception, objects spatial location, and motion processing, all used for guiding behaviors. In visually active arthropods, binocular neural processing of the panoramic optic flow generated during self-motion happens already in the optic neuropils. However, whether binocular processing of single-object motion occurs in these neuropils remained unknown. We investigated this in a crab, where motion-sensitive neurons from the lobula can be recorded in the intact animal. Here we demonstrate that different classes of neurons from the lobula compute binocular information. Our results provide new insight into where and how the visual information acquired by the two eyes is first combined in the brain of an arthropod.
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Braquiuros/fisiología , Percepción de Movimiento/fisiología , Neuronas/fisiología , Neurópilo/fisiología , Visión Binocular/fisiología , Vías Visuales/fisiología , Animales , Dominancia Cerebral , Masculino , Neurópilo/ultraestructura , Técnicas de Placa-Clamp , Estimulación Luminosa , Tiempo de Reacción , Visión Monocular/fisiologíaRESUMEN
Activation of GABAA receptors on sensory axons produces a primary afferent depolarization (PAD) that modulates sensory transmission in the spinal cord. While axoaxonic synaptic contacts of GABAergic interneurons onto afferent terminals have been extensively studied, less is known about the function of extrasynaptic GABA receptors on afferents. Thus, we examined extrasynaptic α5GABAA receptors on low-threshold proprioceptive (group Ia) and cutaneous afferents. Afferents were impaled with intracellular electrodes and filled with neurobiotin in the sacrocaudal spinal cord of rats. Confocal microscopy was used to reconstruct the afferents and locate immunolabelled α5GABAA receptors. In all afferents α5GABAA receptors were found throughout the extensive central axon arbors. They were most densely located at branch points near sodium channel nodes, including in the dorsal horn. Unexpectedly, proprioceptive afferent terminals on motoneurons had a relative lack of α5GABAA receptors. When recording intracellularly from these afferents, blocking α5GABAA receptors (with L655708, gabazine, or bicuculline) hyperpolarized the afferents, as did blocking neuronal activity with tetrodotoxin, indicating a tonic GABA tone and tonic PAD. This tonic PAD was increased by repeatedly stimulating the dorsal root at low rates and remained elevated for many seconds after the stimulation. It is puzzling that tonic PAD arises from α5GABAA receptors located far from the afferent terminal where they can have relatively little effect on terminal presynaptic inhibition. However, consistent with the nodal location of α5GABAA receptors, we find tonic PAD helps produce sodium spikes that propagate antidromically out the dorsal roots, and we suggest that it may well be involved in assisting spike transmission in general. NEW & NOTEWORTHY GABAergic neurons are well known to form synaptic contacts on proprioceptive afferent terminals innervating motoneurons and to cause presynaptic inhibition. However, the particular GABA receptors involved are unknown. Here, we examined the distribution of extrasynaptic α5GABAA receptors on proprioceptive Ia afferents. Unexpectedly, these receptors were found preferentially near nodal sodium channels throughout the afferent and were largely absent from afferent terminals. These receptors produced a tonic afferent depolarization that modulated sodium spikes, consistent with their location.
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Potenciales de la Membrana , Neuronas Aferentes/metabolismo , Propiocepción , Receptores de GABA-A/metabolismo , Canales de Sodio/metabolismo , Médula Espinal/metabolismo , Animales , Femenino , Antagonistas de Receptores de GABA-A/farmacología , Inhibición Neural , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Médula Espinal/fisiología , Sinapsis/metabolismo , Sinapsis/fisiologíaRESUMEN
Sex pheromones orient male moths toward conspecific female moths; the presence of visual information modulates this behavior. In the current study, we explore candidate neuronal pathways for the interaction between vision and the locomotor signal for pheromone orientation. We describe the connectivity between visual neuropils and brain premotor centers, the posterior slope (PS) and the lateral accessory lobe (LAL), in the silkmoth Bombyx mori. Using a single-cell labeling technique, we analyze visual projection neurons supplying these areas. Neurons from both the medulla and lobula complex projected to the PS but only the neurons originating in the lobula complex had additional processes to the LAL. Further, we identified populations of putative feedback neurons from the premotor centers to the optic lobe. Neurons originating in the PS were likely to project to the medulla, whereas those originating in the LAL were likely to project to the lobula complex. The anatomical study contributes to further understanding of integration of visual information on the locomotor control in the insect brain.
Asunto(s)
Bombyx/anatomía & histología , Corteza Motora/anatomía & histología , Corteza Motora/citología , Neuronas/citología , Vías Visuales/anatomía & histología , Animales , Retroalimentación , Lóbulo Óptico de Animales no Mamíferos/anatomía & histologíaRESUMEN
During hunting, the duration and amplitude of bat's echolocation sounds co-vary. Our previous studies showed the inferior collicular neurons of constant frequency-frequency modulation (CF-FM) bat discharged as single-on (SO) or double-on (DO) responders when stimulated with behavior related CF-FM sounds. However, how the co-varied sound duration and amplitude modulate the response properties of SO and DO neurons were understudied. Therefore, we investigated amplitude- and duration-sensitivity in 121 neurons isolated in the inferior colliculus of CF-FM bat, Pratt's roundleaf bat (Hipposideros pratti). Responses of SO and DO neurons were obtained by in vivo intracellular recordings and examined for different stimulus amplitudes and durations. Our results revealed that response patterns of SO neurons were unaffected by changes in amplitude and duration of CF-FM stimuli. However, the excitability of DO neurons increased with prolonged CF duration and higher amplitude of CF-FM stimuli. These data suggested that the invariance of SO neurons play a key role in detection of Doppler shift and glint-like changes of frequency and amplitude induced by wingbeats of insects. In contrast, amplitude- and duration-sensitivity of DO neurons to CF-FM stimuli is consistent with the systematic changes in these signal parameters during sequential phases of foraging in CF-FM bats.
Asunto(s)
Quirópteros/fisiología , Ecolocación/fisiología , Audición/fisiología , Colículos Inferiores/fisiología , Neuronas/fisiología , Animales , Femenino , Masculino , Potenciales de la MembranaRESUMEN
Three-dimensional vertical micro- and nanostructures can enhance the signal quality of multielectrode arrays and promise to become the prime methodology for the investigation of large networks of electrogenic cells. So far, access to the intracellular environment has been obtained via spontaneous poration, electroporation, or by surface functionalization of the micro/nanostructures; however, these methods still suffer from some limitations due to their intrinsic characteristics that limit their widespread use. Here, we demonstrate the ability to continuously record both extracellular and intracellular-like action potentials at each electrode site in spontaneously active mammalian neurons and HL-1 cardiac-derived cells via the combination of vertical nanoelectrodes with plasmonic optoporation. We demonstrate long-term and stable recordings with a very good signal-to-noise ratio. Additionally, plasmonic optoporation does not perturb the spontaneous electrical activity; it permits continuous recording even during the poration process and can regulate extracellular and intracellular contributions by means of partial cellular poration.