Astrocytes have been identified to actively contribute to brain functions through Ca2+ signaling, serving as a bridge to communicate with neurons and other brain cells. However, conventional stimulation techniques are hard to apply to delicate investigations on astrocytes. Our group previously reported photostimulation with a femtosecond laser to evoke astrocytic calcium (Ca2+) waves, providing a noninvasive and efficient approach with highly precise targeting. In this work, detailed characteristics of astrocytic Ca2+ signaling induced by photostimulation are presented. In a purified astrocytic culture, after the illumination of a femtosecond laser onto one cell, a Ca2+ wave throughout the network with reduced speed is induced, and intracellular Ca2+ oscillations are observed. The intercellular propagation is pharmacologically confirmed to be mainly mediated by ATP through P2Y receptors. Different patterns of Ca2+ elevations with increased amplitude in the stimulated astrocyte are discovered by varying the femtosecond laser power, which is correspondingly followed by broader intercellular waves. These indicate that the strength of photogenerated Ca2+ signaling in astrocytes has a positive relationship with the stimulating laser power. Therefore, distinct Ca2+ signaling is feasibly available for specific studies on astrocytes by employing precisely controlled photostimulation.
Both neuroscience and nonlinear science have focused attention on the dynamics of the neural network. However, litter
is known concerning the electrical activity of the cultured neuronal network because of the high complexity and moment
change. Instead of traditional methods, we use chaotic time series analysis and temporal coding to analyze the
spontaneous firing spike train recorded from hippocampal neuronal network cultured on multi-electrode array. When
analyzing interspike interval series of different firing patterns, we found when single spike and burst alternate, the largest
Lyapunov exponent of interspike interval (ISI) series is positive. It suggests that chaos should exist. Furthermore, a
nonlinear phenomenon of bifurcation is found in the ISI vs. number histogram. It determined that this complex firing
pattern of neuron and the irregular ISI series were resulted from deterministic factors and chaos should exist in cultured
term.These results suggest that chaotic time series analysis and temporal coding provide us effective methods to
investigate the role played by deterministic and stochastic component in neuron information coding, but further research
should be carried out because of the high complexity and remarkable noise of the electric activity.
In this paper, we study the modulation of low frequency closed-loop electric stimulation on spontaneous activity in cultured hippocampal neuronal networks. First, we plated monolayer cultures of hippocampal neurons from rat embryos (E18) on multi-electrode arrays and the experiments were performed in the networks from the second week to the sixth week continuously. During the experiments, we detected the spontaneous spikes of the networks firstly, and then stimulated the networks at low frequency (0.2 Hz or 1 Hz) stimulation respectively until a desired response was observed 20-80msec after a stimulus. The protocol was closed-loop. After that, we detected the spontaneous spikes of the networks. It is observed that the spontaneous activity in the developing networks is developing, which is oscillatory and periodic. Low frequency (0.2 Hz or 1 Hz) stimulation enhanced the spontaneous synchronous burst activity of the developing networks. These results implicated that activity-dependent mechanism in the modulation of plasticity of synaptic transmission in the cultured neuronal networks. Closed-loop stimulation will give a better view on the functional significance of networks activities. Besides, close-loop stimulation could set up the stimulus-reward system in the neuronal networks, which is of great benefit to the plasticity of synaptic transmission in the cultured neuronal networks.
Burst as a unit of information coding is widely investigated in the developing central nervous system. However the
mechanism underling the bursts generate and disappear is unclear at present. Neurons cultured on the multi-electrode
arrays, are spontaneously active, and show complex pattern with random spikes and bursts firing. With long-term
recording, the course of bursts generation and disappearance was detected. The results showed that the firing pattern
could transform from random spikes to bursts firing. In the beginning, the random spikes rate decreased, accompanied
with bursts occurred once in a while. It appeared both single spikes and bursts at the same time. After that, the random
spikes disappeared. Spontaneous activity displayed a regular occurrence of bursts with shorter interspike interval. During
such bursts the firing rate at the active sites was increased dramatically. After several seconds, firing rate decreased,
interburst interval extended, accompanied with the occurrence of random spikes, opposite to the beginning. At last,
bursts disappeared and the networks just fired in random spikes. The observation showed that the complex
electrophysiological activities of the cultured neuronal networks could implicate the spontaneous generation of burst
firing. Understanding how bursts generate and disappear might be significant for deeply investigating the function and
mechanism of bursts information coding.
KEYWORDS: Neurons, Signal processing, Signal detection, Brain, Electrodes, In vitro testing, Action potentials, Biomedical optics, Photonics, In vivo imaging
The neuronal network cultured in virto used as a important tool for brain study have been realized by more and more people owing to its non-invasive nature. But till now, there isn't a parameter that conveniently describes the changing states of neuronal network from the whole. In this paper, the synchrony calculation acted as the reactive results of the neuronal network to electrical stimulation (used for learning training) or bicuculine is analyzed and the variety of the synchrony of the network is tried as a important value to depict the diversification of the neuronal network. These experimental results processed in this way were given out in the end of the paper.
Changes in the intracellular Ca2+ concentration ([Ca2+]i) play a crucial role involved in the modulation of signal transduction, development, and plasticity in the CNS. Glial cells can respond to various stimuli with an increase in [Ca2+]i. In this paper, we used confocal microscopy to study calcium transient induced by glutamate in cultured astrocytes. Firstly, 100 μM glutamate induced long-time intracellular calcium oscillations in astrocytes and only a single spike under calcium-free solution. When the concentration of glutamate decreased to 1 μM, only a single spike could be induced. It shows that intracellular calcium oscillations depend on agonist concentration and extracellular Ca2+. Secondly, we investigated amplitude of responses under different stimulation. The amplitude of initial peak induced by 100 μM glutamate decreased in Ca2+-free condition, whereas the duration of kinetics was prolonged. But both the amplitude and area of a single spike induced by 1 μM Glu decreased in Ca2+-free condition. The results show that areaof peak is more accurate than amplitude to display transients of [Ca2+]i. All results above suggest that astrocytes are not passive, they display diverse temporal and spatial increases in [Ca2+]i in response to a variety of stimuli. These [Ca2+]i increases provide a possible means for information coding.
Intracellular calcium, as an important second messenger, plays a significant role in cell signaling transduction and metabolism. Glutamate can induce the intracellular calcium transient through triggering diverse signaling pathways. To test the effect of glutamate to neurons, we loaded Fluo-3/Am in cultured rat hippocampal neurons, and then acquired two-dimensional fluorescent image by confocal microscopy and the analyzed fluorescent intensity. In cultured neurons, we observed two types of neurons that have different morphology: bipolar-type and pyramidal-type. Inducing [Ca2+]i transient by glutamate, we found the amplitude and time constant of the response curves of bipolar neurons are larger than those of pyramidal neurons. Further, we induced [Ca2+]ii transient under different concentrations of glutamate. Two different types of kinetic of the [Ca2+]i transient have been found, corresponded to the two kinds of neuron. The amplitude of [Ca2+]i transient increased when applying higher concentration of glutamate in pyramidal neurons; while it decreased in bipolar ones. Responses of neurons bathing in calcium-free extracellular solution to glutamate were different from those bathing in normal solution. [Ca2+]i transient of pyramidal neurons caused by any concentration were totally blocked; while [Ca2+]i transient in bipolar neurons caused by high concentration of glutamate (500μM) were partly inhibited. All of the phenomena suggest that different types of cultured hippocampal neurons may have different mechanism of the response to glutamate.
Transient and substantial elevation of postsynaptic calcium was important for hippocampal long-term potentiation (LTP), so detection of calcium changes in spine was necessary to understand the mechanisms underlying synaptic plasticity. Unfortunately most recent calcium fluorescence indicators severely perturbed calcium transients, and traditional cameleons’ poor dynamic ranges prevented detection of changes of calcium. We presented a new method to monitor quantificationally free calcium concentration in dendritic spines with a new yellow cameleon (YC3.60) basing on culture of hippocampal neurons and calcium phosphate transfection technique and confocal microscopy with 458nm laser. In transiently transfected hippocampal neurons, the ratio of YFP to CFP was detected as FRET level. In our study, we got the parameters of YC3.60 excited with 458nm laser. Under control conditions, FRET levels in different dendritic spines of cultured hippocampal neurons were diverse but showed robust increases upon treatment with potassium chloride. FRET levels in different parts of hippocampal neurons were also different, the calcium concentration decreased with the distance from soma. These results suggested that the FRET methodology with YC3.60 could monitor calcium concentration in spines and it might be useful in analyzing mechanisms underlying synaptic plasticity.
Fluorescence recovery after photobleaching (FRAP) has become a popular technique to investigate the behavior of protein in living cells. There are various mathematical models for the processing of FRAP data. Among them, Compartmental modeling enables researchers to extract information such as the association and dissociation constants, distribution of a protein between mobile and immobilized pools, and the effective diffusion transfer coefficient of the molecule under study. This model is a simple system of linear ordinary differential equations, and its solution used to fit the FRAP data is a simple two exponential function. Therefore, Gustavo Carrero and some other scientists suggest the use of this model. However we find that the length of FRAP data affects the stabilization of data processing. We believe that it is the two-exponential fitting function that causes the instabilization. This paper attempts the study of fitting FRAP data using three exponential sum function and gets better and more stable fitting. As researchers begin to focus on the relative influence of protein domains within individual protein, this approach will allow a quantitative assessment of the relative effect of different molecular interactions on the steady-state distribution of protein in vivo.
As a ubiquitous intracellular signal, calcium is responsible for controlling numerous cellular processes, such as neuronal signal transmission. In this paper we show that calcium imaging of cultured neuronal network from hippocampus of newborn rats, revealed synchronized calcium wave in intracellular Ca2+ concentration and induced calcium oscillation by stimulating embedded-microelectrode on glass substrate.
Learning is crucial for brain functioning, especially for hippocampus. To understand the basis of physiological information processing of hippocampus, we developed the networks of purified-cultured rat hippocampus neurons on a planar 60-microelectrode array MEA. We describe how these networks are prepared, their structure, and the forms of spontaneous activity they exhibit. We stimulate the network at a low frequency until a desired predefined response is observed 50- 10 msec after a stimulus, at which point the stimulus is stopped for 5 min. Repeated cycles of this procedure ultimately lead to the desired response being directly elicited by the stimulus. These results demonstrate that there is learning in networks composed of purified-cultured hippocampus neurons.
The two-photon excitation (TPE) microscopy has become an important tool of noninvasive imaging due to the better penetration and relative harmlessness of the longer wavelength. However, the high photon flux in two-photon excitation can potentially lead to higher-order photobleaching within the focal volume. This paper measured the relationship between the photobleaching rate and the excitation power for Chemical dyes and green fluorescence proteins (GFPs) in vivo at biological imaging level. As expected, the photobleaching rate increased near-linearly with the excitation power for one-photon excitation. However, the two-photon photobleaching rate increased with high-order power (≥3.5) of excitation power, indicating the presence of high-order photon interaction in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study may be limited by increased photobleaching.
The autofluorescence of 5-Hydroxytryptamine (5-HT) loaded rat mucosal mast cells (RBL-2H3 cells) is imaged with multiphoton excitation laser scanning microscope (MPELSM). 5-HT correlative visible fluorescence (Fco-vis) excited with 740-nm multiphoton excitation is observed in live cells for the first time, and the generating mechanism of 5-HT Fco-vis is studied. The spatial distribution of 5-HT in live cells is imaged at high spatial resolution in our experiment, which provides a new way to study the correlation between 5-HT spatial distribution and content, and the cellular functional state in live tissue or cells.
Green fluorescent protein (GFP) transfected Hela cell was planted in naked mice, to construct an in vivo model for monitoring the therapeutic effect of laser hyperthermia in real time. A cooled CCD fluorescence imaging system was used to record the tumor fluorescence image during the hyperthermia process. Primary experimental results were presented in this paper. To make sure the fluorescent probe GFP does not have strong effect on the biologic function of the host tumor cell (Hela cell), several conventional biological processes were observed in real time. First, neurons, which are much more tender than tumor cells, were transfected with GFP (cameleons). No morphological inhomogenities were observed, and normal functional responses of the neurons were observed when stimulated with histamine. In the second step, the mitosis process of cultured Hela cell was monitored. The features observed during mitosis confirmed that the transfection does not ruin the mitosis process of the tumor cell. At last, naked mice with tumor cell was constructed, which emit fluorescence in the tumor region when excited with faint laser. This presentation provides an in vivo biological model for quick monitoring of the therapeutic results of tumor hyperthermia.
KEYWORDS: Phosphorescence, Confocal microscopy, Oxygen, Molecules, Argon ion lasers, Quenching (fluorescence), Gas lasers, Ion lasers, Optical choppers, Imaging systems
The continuous light coming from the Argon Gas Ion Laser can be converted to pulse light when an optical chopper is equipped. The sample is excited by the pulse light in the confocal microscopy system. The light intensity of the excitation light and the phosphorescence is simultaneously recorded in terms of gray value by the confocal microscopy system. The phosphorescence lifetime measurement can be realized with time-resolved analysis for the phosphorescence intensity decay. The different decay lifetimes of the Oxy- Phor R2 sample under different oxygen concentration are measured with the reconstructed confocal microscopy system.
Ca2+ is the most common signal transduction element in cells and plays critical rolls in neuronal development and plasticity. Ca2+ signals encode information in their oscillation frequency or amplitude and response time to regular cellular function. In this study, in order to reveal the spatio-temporal characterization of Ca2+ oscillations in rat hippocampal neurons, two kinds of Ca2+ fluorescent probes, yellow cameleons 2.1 (YC2.1) and Fluo-3, were used to monitor the change of the intracellular free Ca2+ concentration (]Ca2+[i). Spontaneous Ca2+ oscillations and glutamate elicited Ca2+ oscillations were observed with multi-photon excitation laser scan microscope (MPELSM) and confocal laser scan microscope (CLSM). The observation showed that the spatio- temporal characterization of either spontaneous or glutamate provoked Ca2+ oscillations had difference between the neurites and somata in individual nerons, especially in some distal end of neurites. The result indicated that Ca2+ oscillations were most important signal transduction pattern in neuronal development and activation. The spatio-temporal characterization of difference of Ca2+ signals between the distal endo of neurites and the somata might be associated with the distribution of ionotropic receptor and metabotropic glutamate receptors, and Ca2+ response mechanism mediated by two kinds of glutamate receptor. Ca2+ signal elicited by glutamate in the distal end of neurites appeared more complex and generated faster than that in the somata. It was suggested that Ca2+ signal in glutamate stimulated hippacamal neurons first generated from the distal end of neurites and then transduted to the somata. The complicated Ca2+ signal characterization in the distal end of neurites might be associated with neuronal activitation, neurotransmitter releasing, and other functions of neurons.
A novel photobleaching model was proposed, in which a D-P photobleaching mechanism was developed. By combining with the conventional D-D and D-O photobleaching mechanisms, the dependence of photobleaching rate on the excitation power is well illustrated. The validity of this model was demonstrated with GFP photobleaching experiments in cases of one-photon excitation (1PE) and two-photon excitation (2PE) respectively. Previously inexplicable experimental results published in literature were also illustrated with this new model.
Green fluorescent protein based fluorescence resonance energy transfer is widely used in studies of inter-and intra-molecular dynamic interactions in alive cells. But it is a trade off between quantitative measurement and real time measurement because of the emission cross talk. In stead of using more than one excitation wavelength and several sets of filters, we proposed a more efficient correction scheme which needs only a single excitation and one filter set. This method makes use of the known emission spectra of the fluorophores and can be run in real time. The validity and advantages were demonstrated by experiments with chamaeleons.
The whole process of neutrophilic granulocyte phagocytosis of E.Coli BL21-expressed green fluorescent protein gene was recorded with CCD coupled fluorescence microscopy in a real-time. This process contains of three stages: adhesion, ingestion, and exocytosis. The properties of each stages of phagocytosis were analyzed. The method opened new perspective in continuously observing the specific and non-specific immunity mechanism on alive cell.
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