how everything started…

1Neuro-Transistors– JASS 2005

How everything started…

Direct link between brain and computer.

1985:

Peter Fromherz, Max Planck Institute of Biochemistry

How to design a neuron-silicon junction?


Content

1. Neuronal signalingi. Neuron architectureii. Membrane potentialiii. Action potential

2. Neuro-Transistorsi. Point-Contact Modelii. Transistor recordingiii. Capacitive stimulationiv. Two neurons and a chip


Architecture of a neuron

Basic functional units:Input component

Trigger component

Long-range conducting component

Output component

Dendrites

Cell body (soma)

Axon

Presynaptic terminals


Neurons communicate by electrical signaling.

Action potential:

Brief, invariant and large electrical pulse

All-or-none signal

Frequency-coded

Signaling


The cell membrane

Double layer of hydrophobic lipid molecules

Membrane proteins:

1. voltage-gated ion channels

2. ligand-gated (chemically- controlled) ion channels

3. energy consuming ion pumps

Control transport of ions throughthe cell membrane.


Separation of charges across membrane

Membrane potential: outinM VVV

Reduction in charge separation: Depolarization

Increase in charge separation: Hyperpolarization

Potential determined by:Ionic conductances of the cell membrane

Distribution of ions across the membrane(mainly potassium and sodium )K Na

Membrane Potential


Charge separationacross the membrane.

Potential difference acrossthe membrane driving -ions back into the cell.

-ions concentrated inside the cell.

K

Chemical force drivingthem outside downconcentration gradient.

chemF elF

K

Membrane Potential


Equilibrium potential:

i

oBK K

K

ez

TkV ln Nernst Equation

Giant squid axon: mVmV

VK 75400

20ln

1

25

Passive process, consumes no energy.

Energy is needed to set up initial concentration gradients.

Ion pumps: Proteins in cell membrane. Ion transport through hydrolysis of ATP to ADP.

Equilibrium:

Chemical force = Electrical force

Membrane Potential


Equilibrium with several ion species:

Ion flux = (electrical force + chemical force) x membrane conductance

Influence of each ion species by concentration gradient and permeability of membrane

Membrane potential described by Goldman-Hodgkin-Katz equation:

oCliNaiK

iCloNaoKBm ClPNaPKP

ClPNaPKP

e

TkV ln GHK Equation

usually around -60mV. mV


Generation of an action potential

Membrane potential and ionic conductancescomputed from the Hodgkin-Huxley model.


Recording electrical signals from neurons:

Small glass micropipettes (d < 1µm) filled with concentrated salt solutionare inserted into the cell.

Connection via an amplifier to an oscilloscope.


Voltage-clamp technique:

Difficult to examine ion conductances and because of their strong voltage dependence.

Holding (clamping) the potential in the cell at a certain value.

Opening of voltage-gated ion channels does not affect membrane potential.

Kg Nag


Patch-Clamp Technique

Allows measurement of currentsthrough single ion channels.

Seal between electrode and membrane.

Reduction of electronic noise.

Suction


Signal Propagation along the Axon

Axon can be described as one-dimensional cable.

Cell membrane: insulating coat Intracellular fluid: conductive core

Conduction velocity depends on:

Diameter of axon (Giant squid axon d=1mm)

Insulation of axonal membrane


Myelination

Myelin: electrically insulating layer around axons.

Nodes of Ranvier

Conduction velocity:

Unmyelinated: v = 5 to 10

Myelinated: up to v = 150

smsm


Synapses

Chemical signal transmission across the synaptic cleft.

Action potential

Neurotransmitter release

Neural plasticity:Regulation of synaptic strength.

Learning and memory


Today: Trying to understand fundamental principles of neuron-silicon junctions.

Physical rationalization of junction to optimize neuron-silicon interfacing

Hybrid systems with neuronal networksand microelectronic devices.

………..?

Today Science-Fiction

Where to go?


Principles of coupling

(a) Electrical field across the membrane polarizes the silicon dioxide on the chip.

(b) Electrical field across the silicon dioxide polarizes the membrane affecting voltage- gated ion-channels.Unfortunately:

(c) Neuronal activity leads to ionic and displacement currents through the membrane.

Proteins from cell membrane keepmembrane at certain distance fromthe chip.

(d) Voltage transient applied to silicon causes displacement current through oxide.

Transductive Extracellular Potential (TEP)

Transductive Extracellular Potential (TEP)

Current spreads along the cleft.


MC

JM

JJ A

Gg

JM

MM A

Cc

SCJM

SS A

Cc JG

JM

iJMi

JM A

Gg

Capacitance of the chip: ,

Ionic conductances:iJMG

Conductance of the cleft: ,

Capacitance of the Membrane:

Point-Contact Model

,

,

Transductive extracellular potential mediates coupling of neuron and silicon.

TEP is determined by current balance in junction.

Point-Contact Model


i

iJM

iJM

ionicM VVVgI 0

dt

dV

dt

dVcI JMM

capM

dt

dV

dt

dVcI JSS

capS

i

iJM

iJM

JMM

JSSEJJ VVVg

dt

dV

dt

dVc

dt

dV

dt

dVcVVg 0

Kirchhoff‘s law: ionicM

capM

capS

ohmiccleft IIII

EJJohmiccleft VVgI

Point-Contact Model


Current balance in the cell:

i

iJM

iJM

JMMJM VVVg

dt

dV

dt

dVcA 0

i

iEM

iFM

EMMFM VVVg

dt

dV

dt

dVcA 0

:Area of the free membrane.

:Area of the attached membrane.JMA

FMA

Point-Contact Model


Remarks:ionic conductances depend on voltage difference across the membrane (Hodgkin-Huxley-Model).point-contact model assumes that all currents flow through one point in the membrane. Parameters , , represent average values.point-contact model is a simplification of an area-contact modelwhere depends on the position in the junction.

iJMg

Jg

Mc Sc

yxVJ , yx,

Point-Contact Model

Efficient recording and stimulation:

small distance high specific resistancelarge radius

high ionic conductanceshigh capacitance of the chip

JdJ

Jasmall

Jg


ESV

DSV

Stimulation voltage

Source-drain-voltage

Electrolyte-source-voltage

Source-drain current is controlled by gate-source voltage

Resulting current is changed to a voltage, amplified and watched on an osciloscope.

Calibration measurement without cell to determine voltage-current characteristic GSD VI

DI SJGS VVV

Transistor recording


Leech neuron on FET contacted with patch-pipette in whole cell configuration.

Ac-voltage is amplified. Response recorded with transistor.

M

J

V

V

tVJ

Plot of transfer spectrum :

tVM

Two different types of spectra observed:

A-type: - small amplitude at low frequencies.- increase of phase around 10Hz.- increase of amplitude above 1000Hz.

B-type: - high amplitude at low frequencies.- just minor change in phase.- further increase of amplitude above 1000Hz.

Ac-stimulation with transistor recording:


Interpretation using the point-contact-model:

Insert intracellular stimulation and extracellular response tiMM eVtV

tiJJ eVtV

i

iJM

iJM

JMM

JSSEJJ VVVg

dt

dV

dt

dVc

dt

dV

dt

dVcVVg 0

with 0EV 0dt

dVS and iJMg JMg

No ion channels, just leak conductance.

in

MSJMJ

MJM

M

J

ccigg

cig

V

V

JMJ

JM

M

J

gg

g

V

V

0

Low frequency limit:

A-type: Small amplitude at low frequencies low membrane conductanceB-type: Enhanced amplitude at low frequencies larger membrane conductance

Further increase at high frequencies large conductance

JMg

Jg

dt

dVcgVcc

dt

dVggV M

MJMMMSJ

JMJJ


With the values and which are known, data fitting gives us:

236.0 cmmSg JM 2217 cmmSg J

25.38 cmmSg JM 28.40 cmmSg J

25 cmFcM 23.0 cmFcS

A-type:

B-type:

Crucial difference: leak conductance of the attached membrane differs by two orders of magnitude.

A-type = Capacitive junction

B-type = Ohmic junction

Ac-stimulation with transistor recording:


Transistor recording of neuronal activity:

Small signal approximation:

Small extracellular potential

No capacitive current to the chip

MJ dVdV

0

dt

dV

dt

dVc JSS

i

iM

iJM

MMJJ VVg

dt

dVcVg 0

INJMi

iM

iJMM

iFM

MMM jVVgg

dt

dVc 11 0

JM

FMM A

A

iMJ VVV 0 ,


A-, B- and C-type response:

No voltage-gated ion channels in the membrane:

dt

dVcVVgVg MM

i

iM

iJMJJ 0

Negligible leak conductance TEP proportional to first derivative

A-type junction

Dominating ohmic leak conductance TEP reflects intracellular waveform

B-type junction

INJi

iM

iJMM

iFM

M

MM jVVgg

dt

dVc

01

1

Insert

in


dt

dVcVVgVg MMMJMJJ 0


i

INJi

MiJMM

iFM

Mi

iM

iJMJJ jVVggVVgVg 00 1

1

i

INJi

MiFM

iJM

MJJ jVVggVg 01

1

TEP of an action potential relies on inhomogeneity of the membrane.Wide spectrum of waveforms depending on distribution ofvoltage-gated ion channels.Details must be treated by numerical simulation.

tVJ



Transistor records of a leech neuron

Two positions of the neuron:

(a)Cell body right on the transistor.(b) Axon stump on transistor array.

Action potential elicited by currentinjection with a micropipette.

Intracellular potential measured with pipette, extracellular potential with transistor.


Transistor records of a leech neuron

Three types of records:

A: first derivative of waveformcapacitive junction

B: waveform itselfohmic junction

C: numerical simulation: accumulation of and -channels in attached membrane.

Depletion of ion-channels in cell body.High density of ion-channels in axon

K Na


Transistor records of neurons from rat hippocampus

Action potentials are elicited by current injection.Records with signal averaging of transistor signals.

Two positive transients in ,one in rising phase of AP andone in falling phase.

tVJ

Interpretation:

Positive peak in falling phase is related with outward potassium current through attached membrane.

With 00 NaM VV 0 Na

FMNaJM gg

Sodium inward current through free membrane gives riseto capacitive outward current through attached membrane.

00 KM VV 0 K

FMKJM gg

Positive peak in rising phase is related with sodium current.

Observation:


Capacitive stimulation of neuronal activity

Changing voltage applied to stimulation spot

Capacitive current through insulating oxide

Current along the cleft Transductive extracellular potential TEP

Voltage-gated ion channels in the membrane may open

Action potential may arise.

tVtV SS 0

tVM

A-type stimulation:

Voltage step

Exponential response of membrane potential.

J

t

MM eVtV 0

with very short time constant sJ 3for mammalian neurons.

tVS


B-type stimulation: tVtV SS 0Voltage step

Exponential response due to capacitive effects.

stat

t

MrestM VeVVV J 0

Also stationary change of intracellular potential.

C-type stimulation: depends on channel sorting. Must be treated with numerical simulation.

tVM

Step stimulation of a leech neuron:

Voltage step with = 4.8, 4.9, 5.0V0SV

Stimulation below threshold cannot elicit an action potential (4.8V).

Capacitive stimulation of neuronal activity


Burst stimulation of snail neuron:

Excitation is achieved only when aburst of voltage pulses is applied.

After each pulse responds with short capacitive transients at rising and falling edge of

tVM

tVS

After the third pulse the intracellular potential rises so that an action potential is elicited.


Circuits with two neurons on a chip

Probing with FET Signal processing Capacitive stimulation tVJ

Neuronal activity tVM Action potential

(i) Transformation of and amplification.

(ii) Identification of an action potential with a threshold device.

(iii) Delay line(iv) Generation of a train of voltage

pulses.(v) Suppression of crosstalk from

stimulator to transistor by refractory unit.


Connection between a spontaneously firing neuron A along the chip to aseparate neuron B.After each action potential in neuron A a burst of voltage pulses is generatedand applied to neuron B.

Neuron B fires in correlation to neuron A.

Circuits with two neurons on a chip


Signaling chip-neuron-neuron-chip

Action potential Synaptic connection Neuronal activity tVM

Probing with FET tVJ

Problem:

Growing neurites exert strong forces on thecell bodies of neurons.

They pull them of their contacts.

Capacitive stimulation


Mechanical fixation of the cell bodies

Picket fences made out of polyimid (plastic) around each contact.

Two neurons from animmobilized network ofsnail neurons.

Stimulation with burst ofseven voltage pulses.

Third action potential in neuron 1leads to a postsynaptic excitationin neuron 2.

Perturbations in transistor signaldue to capacitive coupling with stimulator.


Towards defined neuronal nets

Systematic experiments on network dynamics require:

(i) Noninvasive, long term supervision and stimulation of neurons(ii) Fabrication of neuronal nets with defined topology of synaptic

connections.

Control of neuronal outgrowth:

1.) Chemical guidance:

Motion of neuronal outgrowth isguided by chemical patterns.

Linear patterns of extracellularmatrix proteins are able to guideneuronal outgrowth and let themform synapses.


2.) Topographical guidance

Grown neurites are immobilized bymicroscopic grooves.

Cell bodies are placed in into the pits ofa polymer structure.Neurites grow along the grooves and split at bifurcations.

Problem: Neuritic tree is not uniquelydefined by the guiding pattern.

Alternative: Disordered growth of neuronal nets on closely packed transistor arrays.

Towards defined neuronal nets


Transistor arrays

12 neurons cultured on an array of 128x128 transistors ( ).

Neurons I, II and III are connected by synapses.

Burst of action potentials elicited at Neuron I with a micropipette.

21mm


Alternative materials

Drawbacks of silicon:(i) Electrochemical instability of silicon dioxideLong-term shift of electrical properties of the FETs.

(ii) High noise-level of Si-based devices.Difficult to observe small signals from neurons.

Realization of EOFETs with AlGaN/GaNheterostructure FETs.

Much higher signal-to-noise ratio thanSi-based devices.

These materials are stable under physiological conditions.


m50

Cardiac myocyte cells cultivated on surface of a AlGaN/GaN array

Alternative materials


Summary and Outlook

Basic principles of neuron-silicon junctions are fairly understood.

Properties of the cleftTransistor recordingCapacitive stimulation

Optimization of neuron-silicon contact:Larger capacity of stimulation contactsLower noise of transistorsDeeper understanding of electrical properties of the cell membrane

Neuronal networks:Small defined networks of neurons with learning synapsesLarge networks of neurons on closely packed transistor arrays


Thank You for your Attention!

The End


FLIC Microscopy

Fluorescence interference contrast microscopy

Membrane labelled with fluorescent dye molecules.Excitation and fluorescence of the dye depend onthe distance between membrane and silicon.

Membrane and silicondioxide are not in closecontact.

how everything started…

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