Extraction of response waveforms of heartbeat and blood pressure to swallowing. Using mixed signal processing of time domain and respiratory phase domain.

BACKGROUND: Evaluating the accurate responses of the cardiovascular system to external stimuli is important for a deeper understanding of cardiovascular homeostasis. However, the responses should be distorted by the conventional time domain analysis when a frequency of the effect of external stimuli matches that of intrinsic fluctuations. OBJECTIVES: The purpose of this study is to propose a mixed signal processing of time domain and respiratory phase domain to extract the response waveforms of heartbeat and blood pressure (BP) to external stimuli and to clarify the physiological mechanisms of swallowing effects on the cardiovascular system. METHODS: Measurements were conducted on 12 healthy humans in the sitting and standing positions, with each subject requested to swallow every 30 s between expiration and inspiration. Waveforms of respiratory sinus arrhythmia (RSA) and respiratory-related BP variations were extracted as functions of the respiratory phase. Then, respiratory effects were subtracted from response waveforms with reference to the respiratory phase in the time domain. RESULTS: As a result, swallowing induced tachycardia, which peaked within 3 s and recovered within 8 s. Tachycardia was greater in the sitting position than during standing. Furthermore, systolic BP and pulse pressure immediately decreased and diastolic BP increased coincident with the occurrence of tachycardia. Subsequently, systolic BP and pulse pressure recovered faster than the R-R interval. CONCLUSIONS: We conclude that swallowing-induced tachycardia arises largely from the decrease of vagal activity and the baroreflex would yield fast oscillatory responses in recovery.

Parallel multipoint recording of aligned and cultured neurons on corresponding Micro Channel Array toward on-chip cell analysis.

This paper describes an advanced Micro Channel Array (MCA) so as to record neuronal network at multiple points simultaneously. Developed MCA is designed for neuronal network analysis which has been studied by co-authors using MEA (Micro Electrode Arrays) system. The MCA employs the principle of the extracellular recording. Presented MCA has the following advantages. First of all, the electrodes integrated around individual micro channels are electrically isolated for parallel multipoint recording. Sucking and clamping of cells through micro channels is expected to improve the cellular selectivity and S/N ratio. In this study, hippocampal neurons were cultured on the developed MCA. As a result, the spontaneous and evoked spike potential could be recorded by sucking and clamping the cells at multiple points. Herein, we describe the successful experimental results together with the design and fabrication of the advanced MCA toward on-chip analysis of neuronal network.

MEA-based recording of neuronal activity in vitro.

Based on the advantages of MEA-based recording, developmental changes of spontaneous activity and tetanus-induced modification of evoked activity were studied. Rat cortical neurons were cultured on MEAs and the spontaneous activity was continuously monitored for two months. The activity started a few days after plating. During the second week, the cultures generated periodic synchronized bursts, which were the characteristic properties of cortical neurons in vitro. In about one month, the cultured networks reached a steady state. Between these two, we found a critical period during which only weak activities were generated. This critical period might reflect the transition from immature networks to mature networks including precisely controlled excitatory and inhibitory synapses. We could elicit clear evoked responses with high reproducibility in mature cultures. A focal tetanic stimulation was applied to the mature cultures and how the tetanus affects 64 kinds of evoked activity was studied. The evoked responses showed bi-directional changes in their propagation patterns, potentiation and depression. These induced changes reflected the correlation properties with the tetanized activity pattern. The next step will be the combination of long-term recording and multi-site stimulation. How long does the induced change last, as well as how additional strong activity affects the previously induced changes, will be studied.

Spatio-temporal cholinergic modulation in cultured networks of rat cortical neurons: spontaneous activity.

Activation of the cholinergic innervation of the cortex has been implicated in sensory processing, learning, and memory. At the cellular level, acetylcholine both increases excitability and depresses synaptic transmission, and its effects on network firing are hard to predict. We studied the effects of carbachol, a cholinergic agonist, on network firing in cultures of rat cortical neurons, using electrode arrays to monitor the activity of large numbers of neurons simultaneously. These cultures show stable spontaneous synchronized burst firing which propagates through dense synaptic connections. Carbachol (10-50 microM), acting through muscarinic receptors, was found to induce a switch to asynchronous single-spike firing and to result in a loss of regularity and fragmentation of the burst structure. To obtain a quantitative measure of cholinergic actions on cortical networks, we applied a cluster Poisson-process model to sets of paralleled spike-trains in the presence and absence of carbachol. This revealed that the time series can be well-characterized by such a simple model, consistent with the observed 1/f(b)-like spectra (0.04

Spatio-temporal cholinergic modulation in cultured networks of rat cortical neurons: evoked activity.

We studied the effects of carbachol, a cholinergic agonist, on extracellularly evoked firing of networks in mature cultures of rat cortical neurons, using multi-electrode arrays to monitor the activity of large numbers of neurons simultaneously. These cultures show evoked burst firing which propagates through dense synaptic connections. When a brief voltage pulse was applied to one extracellular electrode, spiking electrical responses were evoked in neurons throughout the network. The response had two components: an early phase, terminating within 30-80 ms, and a late phase which could last several hundreds of milliseconds. Action potentials evoked during the early phase were precisely timed, with only small jitter. In contrast, the late phase characteristically showed clusters of electrical activity with significant spatio-temporal fluctuations. The late phase was suppressed by applying a relatively small amount of carbachol (5 microM) in the external solution, even though the spontaneous firing rate was not significantly changed. Carbachol increased both the spike-timing precision and the speed of propagation of population spikes, and selectively increased the firing coincidence in a subset of neuron pairs in the network, while suppressing late variable firing in responses. Hence, the results give quantitative support for the idea that cholinergic activation in the cortex has a general role of focusing or enhancing significant associative firing of neurons.

Pattern modification of a neuronal network for individual-cell-based electrophysiological measurement using photothermal etching of an agarose architecture with a multielectrode array.

A new type of individual-cell-based on-chip multielectrode array (MEA) cell-cultivation system with an agarose microchamber (AMC) array for topographical control of the network patterns of a living neuronal network has been developed. The advantages of this system are that it allows control of the cell positions and numbers for cultivation using AMCs, as well as easy and flexible control of the pattern of connections between the AMCs through photothermal etching where a portion of the agarose layer is melted with a 1480 nm infrared laser beam. With adequate laser power, narrow micrometer-order grooves (microchannels) can easily be fabricated that can be used to combine neighbouring AMCs to enable topographical control of the neural network pattern. Using this system, an individual-cell-based neural network pattern was formed of rat hippocampal cells within the AMC array without cells escaping from the electrode positions in the microchamber during an eight-day cultivation, and could record cell firing in response to 1.5 V, 500 kHz stimulation through an electrode. This demonstrated the potential of the on-chip AMCMEA cell cultivation system for long-term single-cell-based electrophysiological measurement of a neural network system.

Real-time multisite observation of glutamate release in rat hippocampal slices.

A multichannel glutamate sensor was fabricated that consists of enzyme modified electrodes and has a high sensitivity and selectivity to glutamate. We placed a rat hippocampal slice on the sensor and monitored the current at four electrodes resulting from the stimulation with muscimol, a gamma-aminobutyric acid(A) (GABA(A)) receptor agonist. We obtained different glutamate concentration increases at the different positions, suppressed by bicuculline, a GABA(A) receptor antagonist. This demonstrated that the sensor can monitor the glutamate released via GABA(A) receptors pathways, and the difference in the concentrations may indicate differences in the distribution of GABA(A) receptor as well as diverse receptor functions. This multichannel sensor may be useful for non-invasive, real-time monitoring of glutamate distribution, which would make it a valuable tool for pharmacological analysis.

The dynamics of a neuronal culture of dissociated cortical neurons of neonatal rats.

Neuronal networks of dissociated cortical neurons from neonatal rats were cultured over a multielectrode dish with 64 active sites, which were used both for recording the electrical activity and for stimulation. After about 4 weeks of culture, a dense network of neurons had developed and their electrical activity was studied. When a brief voltage pulse was applied to one extracellular electrode, a clear electrical response was evoked over almost the entire network. When a strong voltage pulse was used, the response was composed of an early phase, terminating within 25 ms, and a late phase which could last several hundreds of milliseconds. Action potentials evoked during the early phase occurred with a precise timing with a small jitter and the electrical activity initiated by a localized stimulation diffused significantly over the network. In contrast, the late phase was characterized by the occurrence of clusters of electrical activity with significant spatio-temporal fluctuations. The late phase was suppressed by adding small amounts of D(-)-2-amino-5-phosphonovaleric acid to the extracellular medium, or by increasing the amount of extracellular Mg2+. The electrical activity of the network was substantially increased by the addition of bicuculline to the extracellular medium. The results presented here show that the neuronal network may exist in two different dynamical states: one state in which the neuronal network behaves as a non-chaotic deterministic system and another state where the system exhibits large spatio-temporal fluctuations, characteristic of stochastic or chaotic systems.

Propagation of spontaneous synchronized activity in cortical slice cultures recorded by planar electrode arrays.

The spatial propagation of synchronized activity in cortical slice cultures was characterized by multi-site extracellular recording. Spontaneous activity was studied in normal culture medium, and in bicuculline- or kainic acid-containing media. A common feature in all these conditions was that activity was generated first in superficial layers (i.e., layer I/II) before spreading over the whole area of the slice. In culture medium or bicuculline-containing medium, the initiation site of the activity was not constant and showed a large variety of patterns of horizontal propagation. Kainic acid induced epileptiform activity, consisting of intense initial bursts followed by repetitive after-discharges. Though the patterns of spatial propagation of the bursts were variable as in the other conditions, the after-discharges followed a constant path. Cross-correlation analysis indicated that the network moved in a graded fashion to a steady state during the sequence of after-discharges.

Properties of the evoked spatio-temporal electrical activity in neuronal assemblies.

Properties of neural computation were studied in two types of neuronal networks: isolated leech ganglia and neuronal cultures of dissociated cortical neurons from neonatal rats. With appropriate experimental set-ups it was possible to obtain a precise description of the spread of excitation induced by specific inputs. The evoked spatio-temporal electrical activity was characterized by large variability and the electrical activity of neurons activated by the same stimulation was found to be statistically independent to a high degree. The variability presumably originates from basic properties of synaptic transmission, which is stochastic in nature. As a consequence, the large variability of the evoked spatio-temporal electrical activity appears to be a general property of neural computation and a typical feature of neuronal assemblies. It is shown, however, that the observed statistical independence of co-activated neurons may be used to reduce the effects of variability by appropriately averaging or pooling the electrical activity.

Activity-dependent enhancement in the reliability of correlated spike timings in cultured cortical neurons.

To study the use-dependent modification of activity in neural networks, we investigated the spike timing by simultaneously recording activity at multiple sites in a network of cultured cortical neurons. We used dynamical analysis to study the temporal structure of spike trains and the activity-dependent changes in the reliability and reproducibility of spike patterns evoked by a stimulus. We also used cross-correlation analysis to evaluate the interactions of neuron pairs. Our main conclusions are that even when no obvious change in spike numbers can be seen, use-dependent modification occurs, either enhancing or reducing in the reliability and reproducibility of spike trains evoked by a stimulus, and the fine temporal structure of stimulus-evoked spike trains and interactions between neurons are also modified by tetanic stimulation.

Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons.

Activity-dependent modification of synaptic efficacy is widely recognized as a cellular basis of learning, memory, and developmental plasticity. Little is known, however, of the consequences of such modification on network activity. Using electrode arrays, we examined how a single, localized tetanic stimulus affects the firing of up to 72 neurons recorded simultaneously in cultured networks of cortical neurons, in response to activation through 64 different test stimulus pathways. The same tetanus produced potentiated transmission in some stimulus pathways and depressed transmission in others. Unexpectedly, responses were homogeneous: for any one stimulus pathway, neuronal responses were either all enhanced or all depressed. Cross-correlation of responses with the responses elicited through the tetanized site revealed that both enhanced and depressed responses followed a common principle: activity that was closely correlated before tetanus with spikes elicited through the tetanized pathway was enhanced, whereas activity outside a 40-ms time window of correlation to tetanic pathway spikes was depressed. Response homogeneity could result from pathway-specific recurrently excitatory circuits, whose gain is increased or decreased by the tetanus, according to its cross-correlation with the tetanized pathway response. The results show how spatial responses following localized tetanic stimuli, although complex, can be accounted for by a simple rule for activity-dependent modification.

Strengthening of synchronized activity by tetanic stimulation in cortical cultures: application of planar electrode arrays.

Rat cortical neurons were cultured on planar electrode arrays with 64 embedded electrodes. Whole-cell recording from single neurons and multisite extracellular recording were carried out simultaneously in the cultured cortical networks, and the effects of focal tetanic stimulation of the culture were studied. Both the number of action potentials and the propagation velocity of stimulated bursts were increased after tetanic stimulation. These changes were associated with a marked increase in the number of late components in the synaptic current, but with little or no increase in the early peak synaptic current. The effects of tetanic stimulation were consistent with a widespread increase in the reliability of monosynaptic transmission.

Segmentation of the response of a neuronal network into clusters with similar activity.

An important question in the analysis of the electrical activity of a large population of neurons is the detection of families of neurons having a similar pattern of electrical activity, so that the original neuronal network can be decomposed into distinct clusters. This paper describes how it is possible to segment the activity of a neuronal network into clusters of sites with similar patterns of activity. Such a segmentation gives insight on how the network is organized, on how it functions and on its behavior as a dynamical system. Simulation and experiments on real data suggest that the correct approach to solve these problems must use multiresolution analysis. The method has been applied to both synthetic data and real data coming from a network of dissociated cortical neurons from neonatal rat brain.

Spontaneous calcaneal fracture after deep heel burns with diabetes.

Two patients with diabetes mellitus sustained spontaneous calcaneus fracture after deep heel burns. Both cases had avulsion type fracture of the os calcis. The authors discuss the incidence and possible etiology with literature review.

Haemodynamic changes in early phase reflex sympathetic dystrophy.

We studied six patients with early phase reflex sympathetic dystrophy (RSD). Osteoporotic changes were noted in the bones of the affected limb. Despite higher temperatures indicated by thermography, laser speckle image sensing showed no increase in blood flow on the skin surface. Digital subtraction angiography showed arteriovenous shunting or increased density of perfused vessels. Based on these results, we speculate that in RSD persistent vascular contraction caused by pain leads to the formation of arteriovenous shunts in the affected limb with an ischaemic state in the peripheral subcutaneous tissue which is indicated by pain and swelling.

Development of low magnesium-induced spontaneous synchronized bursting and GABAergic modulation in cultured rat neocortical neurons.

Development of spontaneous synchronized bursting in the early stages of rat neocortical neuronal cultures was studied by whole-cell and extracellular recordings. Neocortical neurons from rat embryos were cultured on planar electrode arrays, and low Mg(2+)-induced spontaneous activity was recorded from 5 to 16 days in vitro (DIV). At 5-6 DIV the current synchronized to the bursting had only a slow component lasting 3-5 s, whereas in older cultures a fast transient component was dominant. A gamma-aminobutyric acid (GABA)A receptor antagonist, bicuculline methiodide, had little effect on the spontaneous activity at 5-6 DIV, whereas in older cultures it had a marked effect on the slow current component. These results suggest a role of GABAergic transmission in the development of synchronized activities.

Spontaneous periodic synchronized bursting during formation of mature patterns of connections in cortical cultures.

Long-term recording of spontaneous activity in cultured cortical neuronal networks was carried out using substrates containing multi-electrode arrays. Spontaneous uncorrelated firing appeared within the first 3 days and transformed progressively into synchronized bursting within a week. By 30 days from the establishment of the culture, the network exhibited a complicated non-periodic, synchronized activity pattern which showed no changes for more than 2 months and thus represented the mature state of the network. Pharmacological inhibition of activity only during the period when regular synchronized bursting was observed was capable of producing a different mature activity pattern from the control. These results suggest that periodic synchronized bursting plays a critical role in the development of synaptic connections.

Periodic synchronized bursting and intracellular calcium transients elicited by low magnesium in cultured cortical neurons.

1. In Mg(2+)-free external solution, rat cortical neurons in cultured networks entered a stable firing mode, consisting of regular bursts of action potentials superimposed on long-lasting depolarizations. The average separation between bursts varied from culture to culture, but was usually between 5 and 20 s. The distribution of burst intervals followed a Gaussian or normal distribution, with a standard deviation of typically 10% of the average burst period. 2. A gradually depolarizing pacemaker potential was never observed between bursts, but the threshold for action potentials during the quiescent phase was > or = 10 mV above the resting potential. No progressive change in conductance or excitability was observed during the quiescent period. Intracellular stimulation of action potentials did not reproduce the long-lasting depolarization. 3. Switching from current clamp to voltage clamp at the resting potential revealed large postsynaptic currents, mainly excitatory but with a small inhibitory component, at the same phase and frequency as the spike bursts, showing that periodic synaptic input is responsible for the burst-depolarizations. The current could be eliminated by local application of 2-amino-5-phosphonovaleric acid (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the postsynaptic cell. In the presence of tetrodotoxin, irregular miniature excitatory postsynaptic currents were observed. 4. A fluorescent calcium indicator (fluo-3, 100 microM) was included in the whole-cell pipette solution, to allow simultaneous electrical and calcium measurements in the same cell. In current clamp, transient intracellular calcium increases were found, which were synchronized to the spike bursts. The Ca2+ rise lasted as long as the action potential burst, and was followed by an exponential decay considerably slower than that of the membrane potential. Calcium transients disappeared during voltage clamp at the resting potential, suggesting that calcium influx through voltage-dependent calcium channels greatly exceeds that through synaptic channels. 5. Multisite Ca2+ recording, after loading with fluo-3 acetoxymethyl (AM) ester, revealed that the onsets of burst-related calcium transients were synchronized in all active cells of each view-field, to within approximately 20 ms. Occasionally, secondary rhythms were observed in which only a subset of cells participated. The times to peak and the decay times of calcium transients varied among synchronized cells. 6. The pharmacology of the burst-related calcium transients was investigated by bath application of a variety of compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous measurement of intracellular calcium and electrical activity from patterned neural networks in culture.

Multisite extracellular electrical activity and intracellular calcium were recorded simultaneously. Electrical signals were measured using microelectrode array substrates. A novel cell positioning technique was combined with a method for controlling neurite outgrowth, which allowed cell-electrode contacts to be established easily, thus facilitating the electrical recording. Intracellular calcium was measured optically using the indicator fluo 3. Under low-magnesium conditions, cultured rat cortical neurons showed periodic transients of fluo-3 fluorescence, which were synchronized with the periodic bursting observed electrically. The intervals between bursts could be determined by electrical stimulation through the substrate electrodes. The results suggest that functional synaptic connections are formed in the culture system.