Study of hypothermia on cultured neuronal networks using multi-electrode arrays

Efficient and safe use of hypothermia during various neuro-medical procedures requires sound understanding of low temperature effects on the neuronal network's activity. In this report, we introduce the use of cultivated dissociated neuronal networks on temperature controlled multi-electrode arrays (MEAs) as a simple methodology for studying the long-term effects of hypothermia. The networks exhibit spontaneous activity in the form of synchronized bursting events (SBEs), followed by long intervals of sporadic firing. Through the use of our correlation method, these SBEs can be clustered into sub-groups of similar spatio-temporal patterns. Application of hypothermia to the network resulted in a reduction in the SBE rate, the spike intensity and an increase in inter-neuronal correlations. Within 2 h following the cessation of hypothermia, the cultured network returned to its initial spatio-temporal SBE structure. These results suggest that the network survived cold exposure and demonstrate the feasibility of long-term continuous neural network recording during hypothermic conditions.     

Effective parameters for stimulation of dissociated cultures using multi-electrode arrays

Electrical stimulation through multi-electrode arrays is used to evoke activity in dissociated cultures of cortical neurons. We study the efficacies of a variety of pulse shapes under voltage control as well as current control, and determine useful parameter ranges that optimize efficacy while preventing damage through electrochemistry. For any pulse shape, stimulation is found to be mediated by negative currents. We find that positive-then-negative biphasic voltage-controlled pulses are more effective than any of the other pulse shapes tested, when compared at the same peak voltage. These results suggest that voltage-control, with its inherent control over limiting electrochemistry, may be advantageous in a wide variety of stimulation scenarios, possibly extending to in-vivo experiments.     

Far-field potentials recorded from action potentials and from a tripole in a hemicylindrical volume

There is growing evidence in support of the hypothesis that far-field potentials are recorded when action potentials encounter discontinuities in the surrounding volume. The present study found further support for this hypothesis using two methods of experimentation. The first method recorded potentials when the action potential from an isolated bullfrog sciatic nerve in a hemicylindrical volume (i) encountered a change in the shape of the surrounding volume, (ii) crossed a boundary between 2 volumes of differing resistivities, (iii) reached a bend in the nerve, or (iv) reached the functional end of the nerve. In the second method, potentials were recorded when an electrical tripole, constructed in a way to produce the electrical equivalent of an action potential, encountered the same discontinuities as well as when it was configured to simulate a curved nerve. These results are consistent with the hypothesis that dipole components of an action potential predominant in far-field recordings.     

Electrophysiological recording of re-aggregating brain cell cultures on multi-electrode arrays to detect acute neurotoxic effects

Neurotoxicity aims to understand how xenobiotics interfere with the function of the nervous system and to unravel their mechanisms of action. Neuronal activity is the primary functional output of the nervous system and deviations from its resting level may indicate toxicity. Consequently, the monitoring of electrophysiological activity in complex cell culture systems appears particularly promising for neurotoxicity assessment. To detect acute neurotoxic effects of chemicals we developed a test system based on the electrophysiological recordings from neural networks in re-aggregating brain cell cultures using multi-electrode arrays. We characterised the electrophysiological properties of the cultures and, using known neurotoxicants, evaluated their usefulness to predict neurotoxic effects. Aggregates displayed evoked field potentials and spontaneous neural activity involving glutamatergic and GABAergic synaptic transmission. Paired pulse inhibition indicated the presence of short-term synaptic plasticity via functional inhibitory networks. Cultures were treated with 0.1-100 microM of trimethyltin chloride (TMT), methyl mercury chloride (MeHgCl), parathion or paraoxon, and with 0.1-100mM of ethanol for up to 100 min. TMT (10 microM), MeHgCl (1 microM) and ethanol (100mM) all decreased the amplitude of evoked field potential. The effect of ethanol was reversible. In contrast paraoxon (10 microM) increased the amplitudes of evoked field potentials while parathion showed no significant effects. The effects of TMT and ethanol on the frequency of spontaneous activity were consistent with those obtained for evoked field potentials. All effects occurred at levels at which cytotoxic injuries were not detectable. Taken together our system expressed electrophysiological properties similar to those of established slice culture preparations. It detected known neurotoxicants at subcytotoxic levels and therefore appears suitable for the assessment of toxic insults specifically interfering with nervous system function, e.g. neuronal activity, synaptic transmission and short-term plasticity. If incorporated into testing strategies, it might represent a valuable tool for the mechanistic assessment of neurotoxic effects.     

Estimating the duration of intracellular action potentials in muscle fibres from single-fibre extracellular potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet possible, and consequently, knowledge concerning certain IAP characteristics is still limited. According to the core-conductor theory, close to a fibre, a single fibre action potential (SFAP) can be assumed to be proportional to the IAP second derivative. Thus, we might expect to be able to derive some characteristics of the IAP, such as the duration of its spike, from the SFAP waveform. However, SFAP properties not only depend on the IAP shape but also on the fibre-to-electrode (radial) distance and other physiological properties of the fibre. In this paper we, first, propose an SFAP parameter (the negative phase duration, NPD) appropriate for estimating the IAP spike duration and, second, show that this parameter is largely independent of changes in radial distance and muscle fibre propagation velocity. Estimation of the IAP spike duration from a direct measurement taken from the SFAP waveform provides a possible way to enhance the accuracy of SFAP models. Because IAP spike duration is known to be sensitive to the effects of fatigue and calcium accumulation, the proposed SFAP parameter, the NPD, has potential value in electrodiagnosis and as an indicator of IAP profile changes due to peripheral fatigue.     

Estimating the correlation between bursty spike trains and local field potentials

To further understand rhythmic neuronal synchronization, an increasingly useful method is to determine the relationship between the spiking activity of individual neurons and the local field potentials (LFPs) of neural ensembles. Spike field coherence (SFC) is a widely used method for measuring the synchronization between spike trains and LFPs. However, due to the strong dependency of SFC on the burst index, it is not suitable for analyzing the relationship between bursty spike trains and LFPs, particularly in high frequency bands. To address this issue, we developed a method called weighted spike field correlation (WSFC), which uses the first spike in each burst multiple times to estimate the relationship. In the calculation, the number of times that the first spike is used is equal to the spike count per burst. The performance of this method was demonstrated using simulated bursty spike trains and LFPs, which comprised sinusoids with different frequencies, amplitudes, and phases. This method was also used to estimate the correlation between pyramidal cells in the hippocampus and gamma oscillations in rats performing behaviors. Analyses using simulated and real data demonstrated that the WSFC method is a promising measure for estimating the correlation between bursty spike trains and high frequency LFPs.     

Intracranially recorded compound action potentials from the human trigeminal nerve

Potentials from the sensory root of the trigeminal nerve in the cerebello-pontine angle, evoked by electrical stimulation of the different trigeminal divisions, were recorded during neurosurgical operations. Two components of the compound action potential could be distinguished: one early with large amplitude and one late with small amplitude. It is concluded that the two components represent activity in A alpha and A delta fibers respectively.     

Recording action potentials from cultured neurons with extracellular microcircuit electrodes

Dissociated cell cultures of neurons from neonatal rat superior cervical ganglia have been grown in specially prepared dishes, the bottoms of which consist of glass coverslips on which thin-film microcircuits have been deposited. The microcircuit provides 32 microelectrodes per dish, each approximately 8 × 10 μm in area. Extracellular recordings of action potentials from individual neurons have been made, with good signal-to-noise ratios, for cells within 40 μm of the electrode centers. The microelectrodes are also suitable for passing the current required for extracellular stimulation and action potentials have been evoked by stimulating cell bodies and processes through the electrodes.     

A simple circuit for producing vertical arrays of analogue signals and dot raster displays of nerve and muscle action potentials

It is often helpful to display analogue signals such as electromyograms, electroencephalograms, nerve action potentials, etc., in a regular vertical array. A special case of this display is the widely-used dot raster. This paper describes a simple circuit which enables up to 31 analogue signals to be thus displayed on an analogue storage oscilloscope screen or X-Y plotter. A modification permits up to 255 trials to be displayed. The same circuit produces a standard, dot raster display from standard pulses representing nerve or muscle cell action potentials.     

Compound action potentials recorded intracranially from the auditory nerve in man

We describe the characteristics of the compound action potentials recorded from the intracranial part of the auditory nerve in man and the ralation between this potential and the various peaks in the auditory brain stem evoked potential. Recorded using a monopolar electrode, the shape of this potential varies from subject to subject. It often has a triphasic shape as can be expected of the field potentials of a long nerve. The main negative peak occurs 3.0 to 3.8 ms after the onset of a 2000-Hz stimulus tone at 90 dB. Recordings from two sites on the nerve have a similar wave shape. The difference in the latencies of these two recordings is associated with the estimated conduction velocity of the nerve. The latency of the main negative peak in the compound action potential of the auditory nerve was found to match the latency of the second mastoid-positive (vertex-negative) peak of the brain stem evoked potentials recorded from the scalp.     

Telemetry system for reliable recording of action potentials from freely moving rats

Recording single cells from alert rats currently requires a cable to connect brain electrodes to the acquisition system. If no cable were necessary, a variety of interesting experiments would become possible, and the design of other experiments would be simplified. To eliminate the need for a cable we have developed a one-channel radiotelemetry system that is easily carried by a rat. This system transmits a signal that is reliable, highly accurate and can be detected over distances of > or = 20 m. The mobile part of the system has three components: (1) a headstage with built-in amplifiers that plugs into the connector for the electrode array on the rat's head; the headstage also incorporates a light-emitting diode (LED) used to track the rat's position; (2) a backpack that contains the transmitter and batteries (2 N cells); the backpack also provides additional amplification of the single cell signals; and (3) a short cable that connects the headstage to the backpack; the cable supplies power to the headstage amplifiers and the LED, and carries the physiological signals from the headstage to the backpack. By using a differential amplifier and recording between two brain microelectrodes the system can transmit action potential activity from two nearly independent sources. In a future improvement, two transmitters with different frequencies would be used telemeter signals from four microelectrodes simultaneously.     

A computerized discriminator for action potentials

A system for recognizing extracellular action potentials in the presence of noise and other pulses was developed for a PDP11/23 computer. Data can be processed either on-line or off-line from a tape recorder. There are 4 parts: (1) An RC bandpass filter attenuates noise. (2) The data input program digitizes the signal every 55 mu sec. If the signal exceeds a threshold, 12 samples of the signal and the time are written onto the disk. Up to 300 pulses/sec can be processed. (3) The pulse discriminating program recognizes an action potential by fitting the 12 points with this function: v(t) = (a + bt + ct2) exp(-t/tau). It is biphasic, has two zeroes, and decays to zero at long times. There are 4 parameters, a, b, c and tau. The operator chooses a value of tau giving good fits for all pulses of this neuron. For each pulse the computer determines values of a, b and c by the least squares technique. The zeroes of v(t) are computed. For acceptance, the total pulse height (difference between extreme voltages), the position of the first zero, and the time between zeroes must be within limits. The 12 points and v(t) can be displayed. Summary statistics and histograms are printed. It takes 80 msec/pulse for these off-line computations. (4) Occasionally a pulse may be missed or an extra one recorded. The pulse insertion-deletion program edits the disk files. The instantaneous-interspike-frequency plot is displayed and the operator moves cursors to insert or delete pulses.     

Somatosensory evoked potentials and action myoclonus

The authors studied the somatosensory evoked potentials (SEPs) in 16 cases of myoclonic encephalopathies: 8 cases of dyssynergia cerebellaris myoclonica (DCM); 2 cases of dyssynergia cerebellaris progressiva (DCP); 2 cases of Lafora's disease; 1 case of ceroid lipofuscinosis; 3 unclassifiable myoclonic syndromes. The mean age of the patients was 18 years and the mean duration of pre-study evolution was 10 years. All the patients had been treated by anticonvulsant drugs (phenobarbital, valproic acid, benzodiazepines). The amplitude of the complex P1N2 at the level of the contralateral parietal cortex, with stimulation of the median nerve at the wrist, was found to be enlarged in only 6 cases and giant responses (over 40 microV) were obtained in 2 cases. Only half of the patients with DCM presented a high amplitude response. There was no correlation either with clinical parameters (and in particular, certain patients with marked action myoclonic jerks have a normal SEP), or with the EEG data: on the contrary, the amplitude variations of the SEPs are most often similar to variations of the visual evoked potentials.     

Multi-component synaptic potentials of rubrospinal neurons of the cat induced by corticofugal spike trains

Compound nature of EPSPs in rubrospinal neurons evoked by stimulation of the sensorimotor and associative parietal region of the cerebral cortex was shown in acute experiments on nembutalized cats by means of intracellular technique. Monosynaptic nature of the first two components of EPSPs evoked by corticofugal impulses propagating with an average velocity of 18.5 m/s and 7.5 m/s was revealed. These components are supposed to arise as a result of activation of slow conducting pyramidal and corticorubral neurons. In some rubrospinal neurons the first EPSP component evoked by corticofugal impulsation had a fast rising phase and reflected activation of axo-somatic synapses. The results are discussed in the light of the mechanisms of reorganization of cortical synaptic inputs to the red nucleus neurons.     

Habituation of auditory event-related potentials: a comparison of self-initiated and automated stimulus trains

Habituation of auditory event-related potentials, recorded from the vertex, was investigated with two methods of initiating trains of stimuli (self-initiated and automated) and two interstimulus intervals (ISIs) within trains. In all conditions, habituation was asymptotic by the second stimulus in a train of 15 stimuli. The first EP in a train was unaffected by ISI, but the procedure for starting the train directly affected the amplitude and latency of N1, the latency of P2 and the occurrence of P3: all measures were greater in the automated condition than in the self-initiated. For the second through the fifteenth EPs, the procedure for starting the train had no effect, but the amplitude of P2 was directly affected by ISI.     

Simulation of myopathic motor unit action potentials

Normal motor units (MUs) were simulated and their architecture altered to simulate the changes produced by myopathy. The concentric needle electromyographic recordings of motor unit action potentials (MUAPs) from the MUs were then also simulated. These simulated MUAPs showed features that are seen in myopathy: normal amplitude and slightly reduced area, MUAPs with simple waveform and reduced duration, and complex MUAPs with normal or increased duration. The MUAP waveforms were complex because of increased variability of fiber diameter and not because of loss of muscle fibers. The MUAP duration increased when the variability of fiber diameter increased. Finally, MUAPs similar to those seen in neurogenic diseases were produced from MUs in which the only abnormality was increased variability of fiber diameter.