Michael Krause

Ordered networks of rat hippocampal neurons attached to silicon oxide surfaces.

The control of neuronal cell position and outgrowth is of fundamental interest in the development of applications ranging from cellular biosensors to tissue engineering. We have produced rectangular networks of functional rat hippocampal neurons on silicon oxide surfaces. Attachment and network formation of neurons was guided by a geometrical grid pattern of the adhesion peptide PA22-2 which matches in sequence a part of the A-chain of laminin. PA22-2 was applied by contact printing onto the functionalised silicon oxide surface and was immobilised by hetero-bifunctional cross-linking with sulfo-GMBS. Geometric pattern matching was achieved by microcontact printing using a polydimethylsiloxane (PDMS) stamp. In this way the produced grid pattern ranged from 3 to 20 microm in line width and from 50 to 100 microm in line distances. As shown by atomic force microscopy (AFM), line widths and line distances of the peptide pattern differ less than 0.5 microm from the used PDMS stamp. The height of the layer of immobilised PA22-2 was approximately 3.5 nm implying the layer to be monomolecular. Immobilised PA22-2 was capable of binding anti-PA22-2 antibodies indicating that the function of the peptide was not compromised by immobilisation. Rat hippocampal neurons, cultured at low density in serum-free medium, were applied to the growth matrix of PA22-2-coated substrates and, within 1-3 h of culture, formed a network-like pattern that more or less matched the printed grid. Reliability and reproducibility of neuronal network formation depended on the geometry, line width and node diameter of the grid pattern. The immobilised neurons showed resting membrane potentials comparable with controls and, already after 1 day of culture, were capable of eliciting action potentials. The suitability of the immobilised neurons for the study of man-made neural networks and for multi-site recordings from a functional neuronal network is discussed.

Cardiomyocyte-transistor-hybrids for sensor application

An extracellular recording system has been designed for the detection of electrical cell signals using p-channel or n-channel field-effect transistor (FET) arrays with non-metallized gates. Signals from rat heart muscle cell were recorded by these devices and the results described on the basis of an equivalent circuit. This technique is sensitive enough to detect minute changes of the extracellular membrane voltage and has potential applications in drug screening. We show that known cardiac stimulants (isoproterenol, norepinephrine) and relaxants (verapamil, carbamylcholine) have characteristic effects on the heart cells in terms of the changes of beat frequencies in the absence or presence of corresponding agents.

Extended gate electrode arrays for extracellular signal recordings

Abstract We have fabricated arrays of planar gold electrodes arranged in a matrix of 8×8 with active areas ranging from 6 to 30 μm in diameter. An electronic amplification circuitry based on commercial junction field-effect transistors was used where the gold sensor fields act as extended gate electrodes (EGE) of the transistors, which leads to a new approach for long-term extracellular recording systems in vitro. The high input resistance of the amplification circuitry allows the use of small planar bare gold electrodes without further modification which therefore extends the frequency range of the measuring set-up down to the DC-level. The performance of our recording system has been tested using rat cardiac myocytes cultured directly on the device surface. The recorded signals were then compared in shape and size with recordings performed with a similar extracellular measurement set-up based directly on field-effect transistors with non-metallized gate electrodes. By simulations we could show the influence of the electrode capacitance on the time-course and on the size of the measured signals.

Neuron-transistor coupling: interpretation of individual extracellular recorded signals

The electrical coupling of randomly migrating neurons from rat explant brain-stem slice cultures to the gates of non-metallized field-effect transistors (FETs) has been investigated. The objective of our work is the precise interpretation of extracellular recorded signal shapes in comparison to the usual patch-clamp protocols to evaluate the possible use of the extracellular recording technique in electrophysiology. The neurons from our explant cultures exhibited strong voltage-gated potassium currents through the plasma membrane. With an improved noise level of the FET set-up, it was possible to record individual extracellular responses without any signal averaging. Cells were attached by patch-clamp pipettes in voltage-clamp mode and stimulated by voltage step pulses. The point contact model, which is the basic model used to describe electrical contact between cell and transistor, has been implemented in the electrical simulation program PSpice. Voltage and current recordings and compensation values from the patch-clamp measurement have been used as input data for the simulation circuit. Extracellular responses were identified as composed of capacitive current and active potassium current inputs into the adhesion region between the cell and transistor gate. We evaluated the extracellular signal shapes by comparing the capacitive and the slower potassium signal amplitudes. Differences in amplitudes were found, which were interpreted in previous work as enhanced conductance of the attached membrane compared to the average value of the cellular membrane. Our results suggest rather that additional effects like electrodiffusion, ion sensitivity of the sensors or more detailed electronic models for the small cleft between the cell and transistor should be included in the coupling model.

Validation of the use of field effect transistors for extracellular signal recording in pharmacological bioassays

The use of field effect transistors (FETs) in biomedical research has been in rapid progression in recent years. The present study aims to demonstrate a quantitative use of these devices in pharmacological bioassays. FETs were made as a 4 x 4 matrix of gates with a width of 200 microm separating each gate. The surface of the FETs (silicon oxide), covered with a layer of laminin, fibronectin, or nitro cellulose was suitable for cell adhesion. The cultured dissociated cardiac myocytes were spontaneously active within 24 to 48 h after initial plating. Simultaneous intracellular patch clamp recordings were used to verify the electrophysiological signals of cells that were coupled to the gates. All positive chronotropes (isoproterenol, norepinephrine) and negative chronotropes (verapamil, carbamylcholine, SDZ PCO400) showed their characteristic effects on heart cells in terms of changes of beat frequency. As the myocytes were in a complete syncitium on each FET, the cells need not be directly coupled to the gate in order to detect any ionic changes. This enables global cellular responses to be analyzed. The system also offers an opportunity to study the interconnections and communications between different cells. Furthermore, the changes of signal shapes in the presence of different agents could also be detected. The present study demonstrates how versatile and sensitive this recording system is in distinguishing different ionic signal shapes. The authors believe that this system has the potential to replace some currently employed in vitro methods, offering an alternative, which can substantially reduce animal use in pharmacological experiments.

64-Channel extended gate electrode arrays for extracellular signal recording

A 64-channel amplifier system for the recording of extracellular signals with planar metal microelectrodes is presented. Gold metal microelectrodes on glass wafers were fabricated using standard photolithographic techniques. The measurement system was divided into a headstage preamplifier and a main amplifier. The inherent noise of the extracellular recording system was minimized by using an independent battery supply. The metal electrodes were directly connected to the gates of low noise junction field effect transistors (JFETs) using a specially designed electronic circuit. With this set-up, it was possible to record extracellular signals with planar metal microelectrodes without any surface modification for impedance reduction. A feedback circuit in the first amplification stage compensated slow drifts of the gold microelectrodes, which made online sampling of all 64 channels with a sampling rate of 10 kHz possible. Recordings were taken from rat cardiac myocytes cultured on fibronectin coated sensor chips. The system exhibited a good signal-to-noise ratio. It was able to detect the signal propagation within the cardiac cell layer and it could be used for pharmacological investigations involving the heart.

A protein phosphatase is involved in the cholinergic suppression of the Ca2+-activated K+ current sIAHP in hippocampal pyramidal neurons

The slow calcium-activated potassium current sI(AHP) underlies spike-frequency adaptation and has a substantial impact on the excitability of hippocampal CA1 pyramidal neurons. Among other neuromodulatory substances, sI(AHP) is modulated by acetylcholine acting via muscarinic receptors. The second-messenger systems mediating the suppression of sI(AHP) by muscarinic agonists are largely unknown. Both protein kinase C and A do not seem to be involved, whereas calcium calmodulin kinase II has been shown to take part in the muscarinic action on sI(AHP). We re-examined the mechanism of action of muscarinic agonists on sI(AHP) combining whole-cell recordings with the use of specific inhibitors or activators of putative constituents of the muscarinic pathway. Our results suggest that activation of muscarinic receptors reduces sI(AHP) in a G-protein-mediated and phospholipase C-independent manner. Furthermore, we obtained evidence for the involvement of the cGMP-cGK pathway and of a protein phosphatase in the cholinergic suppression of sI(AHP), whereas release of Ca(2+) from IP(3)-sensitive stores seems to be relevant neither for maintenance nor for modulation of sI(AHP).

Serotonergic modulation of carbachol-induced rhythmic activity in hippocampal slices.

Fast rhythmic activity in a frequency range between 20 and 40 Hz occurs in vitro in hippocampal area CA3 after activation of muscarinic receptors. Here we show that carbachol-induced rhythmic activity is modulated by serotonin (5-HT). Spectral analysis reveals that 5-HT (0.3-30 microM) decreases power, but not frequency, of rhythmic activity in a concentration-dependent and reversible manner. The 5-HT(1A) agonists 8-OH-DPAT and buspirone mimic the effect of 5-HT, whereas the selective 5-HT(1A) receptor antagonist WAY-100635 (1 microM) significantly prevents the effect of 5-HT. In contrast to the effect of 5-HT(1A) agonists, the 5-HT(2) agonist DOI increases spectral power and prevents the reduction of spectral power by 5-HT. Application of WAY-100635 alone has no effect on rhythmic activity. Likewise, the 5-HT(2) antagonist ritanserin (10 microM) does not affect rhythmic activity, or its reduction by 5-HT. Finally, the 5-HT re-uptake inhibitor fluoxetine significantly decreases rhythmic activity in the presence of a low concentration of 5-HT, suggesting that 5-HT released from terminals in the slice likely reduces rhythmic activity. These results strongly implicate 5-HT(1A) and 5-HT(2) receptors in the modulation of spectral power of carbachol-induced rhythmic activity and that 5-HT(1A) receptors are responsible for the prevailing effect of 5-HT.

Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways.

The slow afterhyperpolarizing current (sIAHP) is a calcium‐dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well‐known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)‐dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP, resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons.

Model network architectures in vitro on extracellular recording systems using microcontact printing

A PDMS stamp is used to transfer a synthetic peptide in a given pattern to any suitable surface. Using this method two-dimensional neuronal model networks could be formed on glass substrates as well as on electronic devices and adjusted to the given microelectronic structure. The present work focuses on the mechanism of neurite guidance under simplified in vitro conditions, using in vitro guidance cues and outline the incorporation of these interfacial methods into microelectronic sensor devices.