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Peter Fromherz

Publications and source records attributed to Peter Fromherz.

17 recordsLinked to original sources
PubMed2006

A cell-semiconductor synapse: transistor recording of vesicle release in chromaffin cells.

The release of dense-core vesicles in bovine chromaffin cells is a model for the presynaptic process in neurons. It is usually studied by microamperometry of catecholamines with carbon fibers. Here we introduce transistor recording as a tool to study vesicle release. When we stimulate a chromaffin cell placed on a field-effect transistor, the gate voltage exhibits peaks that correlate with a simultaneously performed amperometric recording. We attribute the transistor signal to a release of protons from the extruded matrix of vesicles that lowers the extracellular pH and changes the electrical surface potential of the gate oxide. The rise time of the transistor signals is similar to that of amperometric responses, whereas their duration is distinctly longer. In a model computation, the rise time is identified with the extrusion of vesicle matrix into the narrow extracellular space between cell and gate oxide, and the decay time is attributed to pH equilibration through slow diffusion in the extruded matrix. Because the transistor recording relies on protons, it can be applied to acidic vesicles with electrochemically inactive hormones or transmitters.

Animals↗
PubMed2006

The mechanism of extracellular stimulation of nerve cells on an electrolyte-oxide-semiconductor capacitor.

Extracellular excitation of neurons is applied in studies of cultured networks and brain tissue, as well as in neuroprosthetics. We elucidate its mechanism in an electrophysiological approach by comparing voltage-clamp and current-clamp recordings of individual neurons on an insulated planar electrode. Noninvasive stimulation of neurons from pedal ganglia of Lymnaea stagnalis is achieved by defined voltage ramps applied to an electrolyte/HfO2/silicon capacitor. Effects on the smaller attached cell membrane and the larger free membrane are distinguished in a two-domain-stimulation model. Under current-clamp, we study the polarization that is induced for closed ion channels. Under voltage-clamp, we determine the capacitive gating of ion channels in the attached membrane by falling voltage ramps and for comparison also the gating of all channels by conventional variation of the intracellular voltage. Neuronal excitation is elicited under current-clamp by two mechanisms: Rising voltage ramps depolarize the free membrane such that an action potential is triggered. Falling voltage ramps depolarize the attached membrane such that local ion currents are activated that depolarize the free membrane and trigger an action potential. The electrophysiological analysis of extracellular stimulation in the simple model system is a basis for its systematic optimization in neuronal networks and brain tissue.

Action Potentials↗
PubMed2006

Nyquist noise of cell adhesion detected in a neuron-silicon transistor.

Interfacing of nerve cells and field-effect transistors is determined by current flow along the electrical resistance of the cell-chip junction. We study the thermal noise of the junction by measuring the fluctuations of extracellular voltage with a low-noise transistor. We find a spectral power density of 5 x 10(-14) V2/Hz and interpret it as Nyquist noise of the cell-chip junction with a resistance of 3 MOhm. The thermal noise allows us to elucidate the properties of cell adhesion and it sets a thermodynamical limit for the signal-to-noise ratio of neuroelectronic interfacing.

Animals↗
PubMed2006

The extracellular electrical resistivity in cell adhesion.

The interaction of cells in a tissue depends on the nature of the extracellular matrix. The electrical properties of the narrow extracellular space are unknown. Here we consider cell adhesion mediated by extracellular matrix protein on a solid substrate as a model system. We culture human embryonic kidney (HEK293) cells on silica coated with fibronectin and determine the electrical resistivity in the cell-solid junction rhoJ=rJdJ by combining measurements of the sheet resistance rJ and of the distance dJ between membrane and substrate. The sheet resistance is obtained from phase fluorometry of the voltage-sensitive dye ANNINE-5 by alternating-current stimulation from the substrate. The distance is measured by fluorescence interference contrast microscopy. We change the resistivity of the bath in a range from 66 Omega cm to 750 Omega cm and find that the sheet resistance rJ is proportionally enhanced, but that the distance is invariant around dJ=75 nm. In all cases, the resulting resistivity rhoJ is indistinguishable from the resistivity of the bath. A similar result is obtained for rat neurons cultured on polylysine. On that basis, we propose a "bulk resistivity in cell adhesion" model for cell-solid junctions. The observations suggest that the electrical interaction between cells in a tissue is determined by an extracellular space with the electrical properties of bulk electrolyte.

Animals↗
PubMed2006

Genetic targeting of individual cells with a voltage-sensitive dye through enzymatic activation of membrane binding.

Optical recording of the electrical activity of individual neurons in culture or in a tissue requires cell-selective staining with a fluorescent voltage-sensitive dye. In a proof-of-principle experiment, we implement a novel approach to genetically targeted staining. The method relies on a water-soluble precursor dye and an overexpressed cell-surface enzyme that transforms the precursor into a hydrophobic dye that binds to the targeted cell. We fused an alkaline phosphatase to a specifically designed general-purpose membrane anchor, and the fusion protein was expressed on the surface of HEK293 cells, as was corroborated by immuno- and histochemical staining. We next synthesised an amphiphilic hemicyanine dye containing two enzymatically cleavable phosphate groups at its hydrocarbon tails. When the phosphate groups were removed, the binding to membranes was enhanced by a factor of a thousand, as shown by titration with lipid vesicles. We observed selective staining of enzymatically active cells by fluorescence microscopy in a mixed population of phosphatase-transfected and untransfected HEK293 cells. The critical parameters of enzyme-induced cell-selective staining were elucidated by a simple kinetic model to guide further developments of the method.

Base Sequence↗
PubMed2005

Functional Na+ channels in cell adhesion probed by transistor recording.

Cell membranes in a tissue are in close contact to each other, embedded in the extracellular matrix. Standard electrophysiological methods are not able to characterize ion channels under these conditions. Here we consider the area of cell adhesion on a solid substrate as a model system. We used HEK 293 cells cultured on fibronectin and studied the activation of Na(V)1.4 sodium channels in the adherent membrane with field-effect transistors in a silicon substrate. Under voltage clamp, we compared the transistor response with the whole-cell current. We observed that the extracellular voltage in the cell-chip contact was proportional to the total membrane current. The relation was calibrated by alternating-current stimulation. We found that Na(+) channels are present in the area of cell adhesion on fibronectin with a functionality and a density that is indistinguishable from the free membrane. The experiment provides a basis for studying selective accumulation and depletion of ion channels in cell adhesion and also for a development of cell-based biosensoric devices and neuroelectronic systems.

Action Potentials↗
PubMed2004

Giant lipid vesicles impaled with glass microelectrodes: GigaOhm seal by membrane spreading.

Giant unilamellar lipid vesicles could be perfect systems to study ion channels in the environment of lipid membranes with defined chemical and physical properties. Prerequisite for electrical measurements is an intravesicular electrical contact. We describe the impalement of giant lipid vesicles by glass micropipet electrodes with a tight seal. To avoid displacement or burst during impalement, the vesicles are immobilized in relaxed conditions by microscopic picket fences of polyimide. The outer surface of the pipets is selectively coated with silanes or polylysine. Structurally, the impalement is verified by ejecting a fluorescent solution out of the pipet. For electrical characterization, current pulses are applied to the pipet and voltage transients are recorded. The data are evaluated in terms of the capacitance and effective resistance of the membrane. Directly after impalement, we observe a seal resistance up to 1.2 GOmega that continuously decays within a period of up to 20 min until it suddenly disappears without burst of the vesicle. During impalement, a spreading of the vesicle membrane along the outer surface of the pipets is observed using a fluorescent membrane-bound dye. We assign the tight pipet-vesicle contact to spreading of the lipid bilayer by a rolling mechanism and the loss of resistance to micro- and macropores that are induced by the resulting membrane tension. Limitation of spreading is attempted with barriers on the pipet.

Electric Impedance↗
PubMed2004

Neuron-semiconductor chip with chemical synapse between identified neurons.

Noninvasive electrical stimulation and recording of neuronal networks from semiconductor chips is a prerequisite for the development of neuroelectronic devices. In a proof-of-principle experiment, we implemented the fundamental element of such future hybrids by joining a silicon chip with an excitatory chemical synapse between a pair of identified neurons from the pond snail. We stimulated the presynaptic cell (VD4) with a chip capacitor and recorded the activity of the postsynaptic cell (LPeD1) with a transistor. We enhanced the strength of the soma-soma synapse by repetitive capacitor stimulation, establishing a neuronal memory on the silicon chip.

Animals↗
PubMed2004

Silicon chip with capacitors and transistors for interfacing organotypic brain slice of rat hippocampus.

Probing projections between brain areas and their modulation by synaptic potentiation requires dense arrays of contacts for noninvasive electrical stimulation and recording. Semiconductor technology is able to provide planar arrays with high spatial resolution to be used with planar neuronal structures such as organotypic brain slices. To address basic methodical issues we developed a silicon chip with simple arrays of insulated capacitors and field-effect transistors for stimulation of neuronal activity and recording of evoked field potentials. Brain slices from rat hippocampus were cultured on that substrate. We achieved local stimulation of the CA3 region by applying defined voltage pulses to the chip capacitors. Recording of resulting local field potentials in the CA1 region was accomplished with transistors. The relationship between stimulation and recording was rationalized by a sheet conductor model. By combining a row of capacitors with a row of transistors we determined a simple stimulus-response matrix from CA3 to CA1. Possible contributions of inhomogeneities of synaptic projection, of tissue structure and of neuroelectronic interfacing were considered. The study provides the basis for a development of semiconductor chips with high spatial resolution that are required for long-term studies of topographic mapping.

Animals↗
PubMed2004

Imaging neuronal seal resistance on silicon chip using fluorescent voltage-sensitive dye.

The electrical sheet resistance between living cells grown on planar electronic contacts of semiconductors or metals is a crucial parameter for bioelectronic devices. It determines the strength of electrical signal transduction from cells to chips and from chips to cells. We measured the sheet resistance by applying AC voltage to oxidized silicon chips and by imaging the voltage change across the attached cell membrane with a fluorescent voltage-sensitive dye. The phase map of voltage change was fitted with a planar core-coat conductor model using the sheet resistance as a free parameter. For nerve cells from rat brain on polylysine as well as for HEK293 cells and MDCK cells on fibronectin we find a similar sheet resistance of 10 MOmega. Taking into account the independently measured distance of 50 nm between chip and membrane for these cells, we obtain a specific resistance of 50 Omegacm that is indistinguishable from bulk electrolyte. On the other hand, the sheet resistance for erythrocytes on polylysine is far higher, at approximately 1.5 GOmega. Considering the distance of 10 nm, the specific resistance in the narrow cleft is enhanced to 1500 Omegacm. We find this novel optical method to be a convenient tool to optimize the interface between cells and chips for bioelectronic devices.

Cell Culture Techniques↗
PubMed2004

High sensitivity of Stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge.

Sensitivity spectra of Stark-shift voltage sensitive dyes, such as ANNINE-6, suggest the use of the extreme red edges of the excitation spectrum to achieve large fractional fluorescence changes with membrane voltage. This was tested in cultured HEK293 cells. Cells were illuminated with light at the very red edge of the dye's excitation spectrum, where the absorption cross section is as much as 100 times smaller than at its peak. The small-signal fractional fluorescence changes were -0.17%/mV, -0.28%/mV, and -0.35%/mV for one-photon excitation at 458 nm, 488 nm, and 514 nm, respectively, and -0.29%/mV, -0.43%/mV, and -0.52%/mV for two-photon excitation at 960 nm, 1000 nm, and 1040 nm, respectively. For large voltage swings the fluorescence changes became nonlinear, reaching 50% and -28% for 100 mV hyper- and depolarization, respectively, at 514 nm and 70% and -40% at 1040 nm. Such fractional sensitivities are approximately 5 times larger than what is commonly found with other voltage-sensing dyes and approach the theoretical limit given by the spectral Boltzmann tail.

Algorithms↗
PubMed2002

Effect of neuritic cables on conductance estimates for remote electrical synapses.

The conductance of electrical synapses is usually estimated from voltage recordings at the neuronal somata under the assumption that each cell is isopotential. This approach neglects effects of intervening neurites. For a cell pair with unbranched neurites and an electrical synapse at their ends, we used cable theory to derive an analytical expression that relates the synaptic conductance to voltage recordings at the cell bodies and to the neurite properties. The equation implies that the conventional method significantly underestimates the actual synapse conductance if the neurite length is comparable to the electrotonic length constant and if the synaptic conductance is similar to the serial neurite conductance. For an experimental test, we cultured pairs of snail neurons on protein patterns, resulting in a geometry that matched the theoretical model. Using the isopotential theory, we estimated the synapse conductances and found them to be rather weak. To obtain the cable properties, we recorded spatiotemporal maps of signal propagation in the neurites using a voltage-sensitive dye. Fits of these maps to a passive cable model showed that the snail neurons are electrotonically rather compact. Given these features of our experimental system, the synaptic conductances derived with the nonisopotential model deviated from the estimates of the isopotential theory by about 13%. This discrepancy, although small, shows that even in electrotonically compact neurons coupled by weak synapses the impact of the neuritic cables on conductance estimates cannot be neglected. When applied to less compact and more strongly coupled cell pairs in vivo, our approach can supply the realistic estimates of synaptic conductances that are necessary for a better understanding of the role of electrical coupling in neural systems.

Animals↗
PubMed2002

Sheet conductor model of brain slices for stimulation and recording with planar electronic contacts.

Current and voltage in a brain slice are considered, taking into account the boundary conditions at the surface to an electrolyte bath and at the substrate of an electron conductor. A sheet conductor model is introduced with ohmic leak conductance to the bath and capacitive coupling to the substrate. It assigns a current-source density of neuronal activity to extracellular field potentials recorded by planar contacts, and it relates the current of planar capacitive contacts to the field potential that elicits neuronal activity. Two examples are analytically solved: the recording across a layered brain slice and the stimulation by a circular electrode. The study forms the basis for neurophysical experiments with brain slices or retinae on microelectronic chips.

Action Potentials↗
PubMed2002

Electrical interfacing of nerve cells and semiconductor chips.

The electrical interfacing of individual nerve cells and silicon microstructures is considered, as well as the assembly of elementary hybrid systems made of neuronal networks and semiconductor microelectronics. Without electrochemical processes, coupling of the electron-conducting semiconductor and the ion-conducting neurons relies on a close contact of cell membrane and oxidised silicon with a high resistance of the junction and a high conductance of the attached membrane. Neuronal excitation can be elicited and recorded from the chip by capacitive contacts and by field-effect transistors with an open gate. Integrated iono-electronic system are obtained by the outgrowth of neuronal networks on the surface of the silicon chip, by implementing electrical circuits in the chip and by two-way interfacing of the neuronal and the electronic components.

Animals↗
PubMed2002

Transistor array with an organotypic brain slice: field potential records and synaptic currents.

Linear transistor arrays on a silicon chip were used to map evoked extracellular field potentials in organotypic brain slices of rat hippocampus. The shape and amplitude of the transients were similar to those from records with micropipette electrodes. The spatial resolution was 21 and 4.6 microm. The sampled profile of the field potential showed a wide and shallow trough of transients in the stratum radiatum and a narrow but higher ridge in the stratum pyramidale. Due to the high resolution, the profile could be interpreted quantitatively in terms of synaptic currents. Transistor chips may become a novel tool for neurophysiological and pharmacological studies in brain slices.

Animals↗
PubMed2001

Recombinant Kv1.3 potassium channels stabilize tonic firing of cultured rat hippocampal neurons.

We transfected cultured hippocampal neurons with the cDNA of the voltage-gated K+ channel Kv1.3 to investigate the mechanisms by which a specific ion channel influences excitability. In transfected neurons under voltage clamp we observed an additional outward current that was blocked selectively by margatoxin. Under current-clamp conditions, Kv1.3-expressing neurons fired tonically over a wide range of stimulation intensity. In non-transfected neurons, or in Kv1.3-expressing cells blocked with margatoxin, only a few action potentials were elicited before a stationary depolarized state was reached. We attribute the specific effect of Kv1.3 to its particularly slow deactivation near the resting potential. A computational model showed that a continuous outwards current arises in Kv1.3-expressing neurons during the interspike intervals. It expands the dynamic range so that these neurons still fire tonically at stimulus current intensities at which non-transfected cells have already been driven into a stationary depolarized state.

Action Potentials↗