Appl. Phys. A 66, 459–463 (1998)
Applied Physics A Materials Science & Processing Springer-Verlag 1998
Rapid communication Transistor records of excitable neurons from rat brain S. Vassanelli, P. Fromherz∗ Department Membrane and Neurophysics, Max-Planck-Institute for Biochemistry, D-82152 Martinsried-München, Germany Received: 4 February 1998/Accepted: 7 February 1998
Abstract. Field effect transistors form electrical junctions with excitable nerve cells from rat brain which are cultured on oxidized silicon. Action potentials in the neurons give rise to voltage transients on the gate with an amplitude < 100 µV as revealed by signal averaging. The shape of the extracellular records depends on a depletion and accumulation of voltagegated sodium and potassium channels in the attached region of the cell membrane. PACS: 87.22.-q; 73.40.Mr; 8780.+s
A field effect transistor (FET) with a metal-free gate in an electrolyte detects the extracellular voltage beneath an individual neuron [1]. The voltage transient during an action potential is caused by the current through the attached cell membrane which flows along the seal resistance between the neuron and the chip. By using large neurons from segmental ganglia of the leech, it was possible to distinguish junctions dominated by capacitive [1–4], ohmic [2–4], and voltagegated ionic currents [5]. In the future, transistors may become the basis for massive parallel recording by chips with very large scale integration (VLSI). The coupling of small mammalian neurons is a central problem to be solved. Recently we observed junctions of neurons from the hippocampus of embryonal rats when stimulated by ac signals [6]. The neurons were not excitable as they were cultured for a very short time to avoid a perturbation by pro-liferating glia. In this paper we report the first records of action potentials in hippocampal neurons after prolonged culturing with sufficient suppression of glia cells. First we summarize the crucial features of the FET probe. Then the methods and the results are described. Finally the nature of the membrane currents is assigned by model computations.
∗ Corresponding
author. E-mail:
[email protected]
Transistor probe The structure and circuit of a neuron transistor are shown in Fig. 1. The cell is attached to the metal-free gate of a FET with source, drain, and bulk silicon kept at bias voltages with respect to the bath (Fig. 1a). It is stimulated by current through a pipette. During an action potential (AP), capacitive and ionic currents flow through the attached membrane and along the cleft between the cell and chip. They give rise to an extracellular voltage VJ on the gate, which modulates the current from source to drain. The current balance in the junction is described by the circuit of Fig. 1b (active point-circuit model) [3, 5]. It leads to a relation of the extracellular voltage VJ (t) (time t) and the intracellular voltage VM (t) according to (1) with the specific membrane capacitance cM , with the specific ionic conduci tances gJM of the attached membrane (ion species i, reversal voltage VJ0i ) and with the specific capacitance cOX of silicon dioxide. gJ is a specific seal conductance of the junction (total conductance of the cleft divided by the area of the contact) [5, 6]. gJ VJ = cM
d (VM − VJ ) X i dVJ + gJM VM − VJ − VJ0i − cOX . dt dt i (1)
The intracellular voltage VM is determined as usual by the current through the free membrane with area AFM (spei cific ionic conductances gM , reversal voltages V0i ) and by the injection current Iinj with some contribution of the attached membrane with an area AJM (AJM AFM in our experiments) according to (2). dVM X i + gM VM − V0i = (2) dt i " # Iinj AJM d (VM − VJ ) X i − cM + gJM VM − VJ − VJ0i . AFM AFM dt i
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Fig. 2. Neurons from the hippocampus of an embryonal rat cultivated for six days on a chip in a serum-free medium. Scale bar 100 µm. The diameter of the cells is about 20 µm. The ten gates (size 1.8 µm × 2.2 µm) are in the centers of the white circles
Fig. 1a,b. Neuron transistor. a Schematic cross section. A neuron is attached to the silicon dioxide of a field effect transistor. It is fused to a pipette for injecting current and for recording the intracellular voltage VM . Current through the attached membrane flows along the cleft between cell and chip and changes the voltage VJ on the gate. n-type bulk silicon (B), p-type source (S), and drain (D) are kept at bias voltages with respect to the electrolyte on ground potential. b Circuit. The free cell membrane (area AFM ) and the attached membrane (area AJM ) are characterized by their specific capacitance and their voltage-dependent specific conductances for different ions (Na, K, leaks). The reversal voltages are symbolized as batteries. Iinj is the injection current. The junction itself is described by a specific seal conductance gJ . The perturbation by the probe is given by the capacitance of the oxide
Chips We used metal-free p-type field effect transistors on n-type silicon [1]. The fabrication of the chips is described in [7]. The area of a gate was 1.8 µm × 2.2 µm in a design with ten transistors (thickness of the gate oxide 12 nm) [8]. Each chip was attached to a perspex chamber (bottom diameter 3 mm). The surface was wiped with a 1-vol% solution of a liquid dish detergent, rinsed with milli-Q water (Millipore, Bedford, MA, USA), dried, and sterilized with UV light. The chips were coated with poly-L-lysine (MW 375 500, Sigma, Heidelberg, Germany) by adsorption from a 10-µM aqueous solution for 1 h and dried. Neurons Neurons were dissociated from the hippocampi of Wistar rats (Thomae, Biberach, Germany) at 18 days gestation [9].
They were preplated twice to get rid of glia cells and suspended in Dulbecco’s medium (DMEM with glutamax I, Gibco, Eggenstein, Germany, No. 61965026) supplemented with 10-vol% foetal bovine serum (Gibco, 10106078) and 1-vol% penicillin (Gibco, 15140114). The final concentration was 450 000 cells/ml. Details are described in [6]. 350 µl of the cell suspension were applied to a chamber with 100 µl of Leibovitz L-15 medium with glutamax I (Gibco, 31415029) supplemented with 5% foetal bovine serum. The chips were kept at 37 ◦ C and 10% CO2 for 2 h. Then the medium was removed and the cells were cultured in 450 µl of neurobasal medium (Gibco, 21103049) supplemented with 2-vol% B27medium (Gibco, 17504036) and 1-vol% glutamax I (Gibco, 35050038) for six days [10, 11]. The medium was replaced by 150 µl of extracellular medium (135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2 , 1 mM MgCl2 , 10 mM glucose, 5 mM Hepes, pH 7.4) before a measurement. An example of a chip is shown in Fig. 2. The density of cells was around 100 000 cm−2 . 70% of the neurons were excitable. On average we found 0.3 neurons per chamber that were excitable and coupled to a transistor. Setup Bulk silicon and sources were kept at a voltage +1 V with respect to the bath kept at ground potential with a Ag/AgCl electrode. The source–drain voltage was − 0.5 V. This working point was chosen to optimize the signal-to-noise ratio. A change of +1 mV on the gate induced a modulation of the source–drain current by − 0.02 µA. The rms noise was about 100 µV at a bandwidth of 10 kHz. A selected neuron was fused to a patch pipette with a hydrophobic shaft (sylgard) [12, 13], which was filled with intracellular medium (140 mM KCl, 2 mM MgCl2 , 1 mM EGTA, 5 mM Hepes, pH 7.3) (resistance 2–3 MΩ). The Ag/AgCl electrode in the pipette was connected to a patch-clamp amplifier (SEC10L, npi-electronics, Tamm, Germany). In the first step of an experiment we measured the access resistance RA
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of the pipette and the total capacitance CM and resistance RM of the cell membrane by voltage-clamp and currentclamp techniques. These parameters determined the voltage transfer from the head stage to the cell, together with the stray capacitance CST of the pipette and head stage. Typical values were RA = 10 MΩ, RM = 1 GΩ, CM = 10 pF, and CST = 5 pF. In a second step we determined the specific seal conductance gJ on the basis of (1) with cM = 1 µF/cm2 and cOX = 0.3 µF/cm2 under conditions where an opening of ion i channels could be neglected with gJM ≈ 0: we applied a short (200 µs) voltage pulse (without compensation) to the head stage, measured the current through the system, computed the intracellular voltage VM (t) and recorded the extracellular voltage VJ (t). During the subsequent measurements we repeated this check of the junction. In a third step we elicited APs by current injection and observed the extracellular voltage VJ (t). We did not see a defined response with a single stimulation because of the noise. 50 to 400 signals were averaged, taking the onset of the injection current as a reference. The shape of the APs in a series was invariant. Their timing was reproducible within 100 µs.
in the rising phase of the AP and a broad maximum just after the peak of the AP. There was a jump downwards at the end of the injection current. A similar record of a weaker junction with gJ = 1200 mS/cm2 is shown in Fig. 3b. 373 sweeps were averaged. We observed ten junctions with similar positive extracellular transients. In three junctions we observed a weak negative extracellular transient during the rising phase of the AP. An example is shown in Fig. 3c (specific seal conductance gJ = 1800 mS/cm2 ). Model We assigned the extracellular voltage to the currents through the attached membrane on the basis of (1). The Hodgkin– Huxley model [14] was used to describe the ionic conductances, as there is no parametrization available for the neurons used in our experiments. We integrated (1) and (2) numerically together with the voltage-dependent rate equations for the ion channels [5]. The maximum conductances in the free membrane were lowered to 15% of the original values in the Hodgkin–Huxley model to match the experimental time constant in the resting state of 10 ms, Na K using g¯ M = 18 mS/cm2 and g¯ M = 5.4 mS/cm2 at a leak conL 2 ductance g = 0.045 mS/cm . The reversal voltages were V0Na = 65 mV, V0K = −62 mV, and V0L = − 39.4 mV (resting voltage of VM = −50 mV). They were assumed to be identical in the free and attached membrane with VJ0i = V0i . Areas AFM = 1250 µm2 and AJM = 78.5 µm2 for the free and attached membrane were estimated from the shape of the cells. The capacitances of membrane and oxide were cM = 1 µF/cm2 and cOX = 0, 3 µF/cm2 [5, 8], the specific seal conductance was gJ = 700 mS/cm2. We applied an injection currrent of 0.5 nA for 6 ms starting from a holding po-
FET records
VJ [mV]
VM [mV]
Iinj [nA]
A selected neuron was kept at a voltage VM ≈ −80 mV. Then current pulses of 0.5 nA with a duration of 5–10 ms were applied, separated by intervals of 1 s. The record of a junction with a specific seal conductance gJ = 600–700 mS/cm2 is shown in Fig. 3a. The intracellular transient with an amplitude of 80 mV induced an extracellular transient with an amplitude of about 150 µV and a characteristic structure was revealed by averaging 63 sweeps. There was a jump of 50 µV at the onset of the injection current. A narrow peak appeared
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Fig. 3a–c. FET records of action potentials. Injection current Iinj at the top, intracellular voltage VM in the middle row and FET record VJ at the bottom (bandwidth 10 kHz). a Averaged record VJ of 63 sweeps. Specific seal conductance gJ = 600–700 mS/cm2 . b Averaged record VJ of 373 sweeps with gJ = 1200 mS/cm2 . c Averaged record VJ of 150 sweeps with gJ = 1800 mS/cm2 . Traces a and b were sampled at 10 kHz, trace c at 5.4 kHz
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Iinj VM
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a height of 50 µV – matching the duration of the injection current – and of a positive peak in the rising phase of the AP followed by a trough in the falling phase. VJ (t) rei flected the first derivative of the signal VM (t) for gJM ≈0 according to (1) for small changes of VJ . dVM/ dt was determined by the injection current and the ionic currents through the free membrane according to (2) for AJM AFM . The rectangular pulse and the narrow positive peak correlated with features of the experiment in Fig. 3a, though the peak is too high. The model of capacitive coupling did not account, however, for the dominant broad peak of the experiment.
Homogeneous membrane
b
VJ
c
VJ
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In a second simulation, the cell membrane was chosen to be chemically homogeneous with identical maximum conductances for sodium and potassium in the attached and free Na Na K K region as g¯ JM = g¯ M and g¯ JM = g¯ M . The extracellular response VJ (t) is shown in Fig. 4b. It had the shape of a rectangular pulse of 50 µV matching the injection current. There was no visible effect of the AP. The capacitive and ionic currents compensate each other, as seen directly from (1) and (2) in the case of weak coupling with VJ VM . The homogeneous model of an active junction did not accout for the experimental record of neural activity.
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Fig. 4a–e. Simulation of FET records (extracellular voltage VJ ) with the Hodkgin–Huxley model at a specific seal conductance gJ = 700 mS/cm2 . Current injection Iinj and intracellular voltage VM as a function of time are shown at the top. a Capacitive coupling with complete depletion of sodium and potassium conductance in the attached membrane. b Homogeneous cell membrane with compensation of capacitive current by local ionic currents. c Depletion of sodium conductance in the attached membrane and homogeneous potassium conductance. d Partial depletion of sodium conductance (65%) and accumulation of potassium conductance (120%) in the attached membrane. e Accumulation of sodium conductance (230%) and of potassium conductance (160%) in the attached membrane. (Identical scale for all traces a–e)
tential of −50 mV. The width of a computed AP was smaller than in the experiments and its amplitude was larger (Fig. 4, top). Capacitive junction In a first simulation, the attached membrane was depleted completely of sodium and potassium conductances with Na K g¯ JM = g¯ JM = 0. The extracellular response VJ (t) is shown in Fig. 4a. It was a superposition of a rectangular pulse with
In a third simulation the attached membrane was depleted Na for sodium conductance with g¯ JM = 0 whereas the potassium K K conductance was homogeneous with g¯ JM = g¯ M . The result is shown in Fig. 4c. The extracellular transient was a superposition of a rectangular pulse, a peak in the rising phase of the AP, and a peak in the falling phase. Here the early positive peak of capacitive current remained uncompensated due to the missing sodium current, which dominates the rising phase of the AP. On the other hand the negative trough of capacitive coupling was transformed by the potassium current to a late peak. The shape was similar to the experimental response in Fig. 3a, though the early peak was too high. Introducing sodium conductance into the junction lowers both the early and the late peak of Fig. 4c. In order to match the experimental transient, we depleted the sodium conductance partially and accumulated the potassium conNa ductance. The result with 65% sodium conductance (¯gJM = 2 K 12 mS/cm ) and 120% potassium conductance (¯gJM = 6.5 mS/cm2 ) is shown in Fig. 4d. The remaining difference between Fig. 4d and Figs. 3a and 3b was due to the different relative duration of injection current and AP. A negative peak in the rising phase of the AP (Fig. 3c) was simulated by accumulating both sodium and potassium conductance in the junction. A result with 230% sodium conNa ductance (g¯ JM = 41.4 mS/cm2 ) and 160% potassium conducK tance (¯gJM = 8.7 mS/cm2) is shown in Fig. 4e. The experimental response (Fig. 3c) was smaller due to the higher seal conductance there (gJ = 1800 mS/cm2 ).
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Conclusions
Firing neurons from mammalian brain in cell culture could be observed with a transistor by using signal averaging. A comparison with simulations showed that the extracellular response was the result of a failure of local compensation of capacitive and ionic currrent through the attached membrane. The records required and indicated different electrophysiological properties of the membrane in the region of adhesion as compared to the free membrane. An inhomogenous distribution of ion channels and an interaction with the coated surface may play a role. Detailed studies under voltage-clamp conditions – with different substrates using specific toxins – will lead to a further characterization of the effect. The extracellular record of an AP had an amplitude < 100 µV as expected for a specific seal conductance > 500 mS/cm2 . Recordings in a single sweep will require a suppression of the noise, an improvement that is crucial in order to take advantage of VLSI technology for massive parallel recording in neuronal nets.
Acknowledgements. We thank Doris Eckerlein for the most skillful culturing of neurons, Richard Schätzthauer for the simulation program, and Martin Jenkner for careful reading of the manuscript. The project is supported by the Bundesministerium für Forschung und Technologie.
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