Pizzi quantum mind 2007

Neurons react to ultraweak Neurons react to ultraweak electromagnetic fieldselectromagnetic fields

Rita Pizzi

Department of Information Technologies University of Milan

LiVinG NeTWorkS LaBLiVinG NeTWorkS LaB Since 2002 the Living Since 2002 the Living

Networks Lab (Department of Networks Lab (Department of Information Sciences, Information Sciences, University of Milan ) works University of Milan ) works with cultures of neurons on with cultures of neurons on MEAs (MicroElectrode MEAs (MicroElectrode Arrays)Arrays)

The group is composed by The group is composed by physicists, electronic physicists, electronic designers, computer designers, computer scientists and scientists and biotechnologists, with the biotechnologists, with the support of external biological support of external biological labs.labs.

LiVinG NeTWorkS LaBLiVinG NeTWorkS LaB

Aim of the group is the development of research Aim of the group is the development of research in the field of computational biology, bionics and in the field of computational biology, bionics and Artificial IntelligenceArtificial Intelligence

Many experiments have been performed by Many experiments have been performed by developing and analyzing organized structures developing and analyzing organized structures of biological neural networksof biological neural networks

It has been possible to train these networks It has been possible to train these networks using digital patterns as simulated perceptions.using digital patterns as simulated perceptions.

The neuronsThe neurons In the last experiments we In the last experiments we

stimulated the network by stimulated the network by means of directional patternsmeans of directional patterns

The patterns are 8x8 bitmapsThe patterns are 8x8 bitmaps The bit duration is 300 ms Each stimulation is followed

by a 1 s pause: during that, the Artificial Neural Network elaborates the signals

The stimulation pulse is an alternated low-voltage signal (+/- 30 mV, 733 Hz).

The bionic creatureThe bionic creature

The analysis confirms the quite satisfactory responses of the hybrid system.

The statistical evaluation after the learning phase and the delivery of 25 random patterns presents an accuracy of 80.11% and a precision of 90.50%.

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“Cremino” is the first hybrid creature endowed with a small human brain

Aim of this research is to reacha better understaning of the

neurophysiological mechanism of memory and learning

an efficient interface between neurons and electronics

A step forward to the development of neuroelectronic prostheses

The neuronsThe neurons

•Up to now we used human

neural stem cells •Cells are plated at a density of

3500 cells/cm2 in a chemical

medium containing EGF and

FGF-2 growth factors •The cells are cultured for 15 days

in order to get mature neurons •We cultured them directly on

MEAs previously coated with a

matrigel substrate.

The Hardware/Software systemThe Hardware/Software system We developed a system that

interfaces the cells by means of their direct adhesion to MEAs (Multielectrode Arrays)

A MEA is a glass Petri dish where small electrodes are inserted. Each electrode is connected by means of an isolated track to a pad suitable for the external connection.

Our Panasonic MEAs have 64 ITO (Indium Tin Oxide)- Platinum microelectrodes.

The microelectrode size is 50 µ, the interpolaristance 150 µ.

Very low impedance (10kΩ) critical to achieve a good signal-to-noise ratio

The Hardware/Software systemThe Hardware/Software system It records the activity of

cells simultaneously on different channels

It can record the cellular activity for a long time without damaging the cultures

It is suitable for our experiments that study the dynamical behavior of a whole neural network

The figure shows one of our cultures on MEA

The Hardware/Software systemThe Hardware/Software system

The system was The system was changed and improved changed and improved many times , adopting many times , adopting more and more more and more powerful acquisition powerful acquisition cards and dedicated cards and dedicated controllerscontrollers

The Hardware/Software systemThe Hardware/Software system At the moment we use At the moment we use

a state-of-the-art National a state-of-the-art National Instruments equipment: Instruments equipment: external rack PXI 1031 with external rack PXI 1031 with a high-speed optimized a high-speed optimized DAQ board PXI 6251 (16 DAQ board PXI 6251 (16 Analog Inputs, 24 Digital Analog Inputs, 24 Digital I/O, 2 Analog Outputs ) , I/O, 2 Analog Outputs ) , sample rate 1.25 MS/ssample rate 1.25 MS/s

Labview 8.0 for the Labview 8.0 for the management of themanagement of theacquisition card and signal acquisition card and signal recordingrecording

Quantum processes in brainQuantum processes in brain

In this frame we work on a second line of In this frame we work on a second line of research, devoted to investigate possible research, devoted to investigate possible quantum processes inside neuronsquantum processes inside neurons

We use the same experimental set-up and We use the same experimental set-up and perform experiments on the neurons by perform experiments on the neurons by stimulating them with electrical and laser stimulating them with electrical and laser pulses and by analysing their signals.pulses and by analysing their signals.


Since October 2002 we started our “quantum” Since October 2002 we started our “quantum” experiments.experiments.

We tried to verify if an electrical/optical We tried to verify if an electrical/optical stimulation on one neural network could in some stimulation on one neural network could in some way reach another completely separated neural way reach another completely separated neural network under electromagnetic shieldingnetwork under electromagnetic shielding

If verified, this experiment could confirm many If verified, this experiment could confirm many “quantum brain” theories (Penrose/Hameroff, “quantum brain” theories (Penrose/Hameroff, Tuszynski, Thaheld).Tuszynski, Thaheld).


Experimental set-up:Experimental set-up: Two MEAs with neuronsTwo MEAs with neurons Same acquisition card and controller as described beforeSame acquisition card and controller as described before Faraday cage around one of the MEAs.Faraday cage around one of the MEAs.

Quantum processes in brainQuantum processes in brain Experimental settings:Experimental settings:

Two MEAs with neurons, one electromagnetically Two MEAs with neurons, one electromagnetically shieldedshielded

The non-shielded MEA was stimulated in turn with:The non-shielded MEA was stimulated in turn with: Electrical bursts (e.g. 40 Hz, 30 mV, 1 ms pulses for Electrical bursts (e.g. 40 Hz, 30 mV, 1 ms pulses for

300 ms)300 ms) LED and laser stimulations (e.g. 1 ms pulses for 100 LED and laser stimulations (e.g. 1 ms pulses for 100

ms or for 3 seconds)ms or for 3 seconds)

The signals of both MEAs were recordedThe signals of both MEAs were recorded


In order to avoid biases:In order to avoid biases: blank acquisitions before stimulationsblank acquisitions before stimulations the same experiments were performed with control the same experiments were performed with control

MEAs filled with culture liquid , matrigel, fibroblastsMEAs filled with culture liquid , matrigel, fibroblasts different distances from the LED/laser sources were different distances from the LED/laser sources were

tested (5 cm, 10cm, 1. 5 m)tested (5 cm, 10cm, 1. 5 m) TTX tests to ascertain the neural origin of the TTX tests to ascertain the neural origin of the

recorded electrical signalsrecorded electrical signals Tests with different wave frequencies, Tests with different wave frequencies,

amplitudes, duration and frequencies of the amplitudes, duration and frequencies of the electrical and optical pulseselectrical and optical pulses


Bench tests results:

The channel-to-channel cross talk was < -110 dB (from DC to 100 Hz; up to 10 k source resistance).

We also generated spikes to check possible propagation to other channels. All the test completely excluded possible cross talks.

The electrode-to- electrode cross talk is< - 80 dB (from DC to 1000 Hz; up to 10 k source resistance).

The MEAs have a glass support that ensures perfect isolation.


Voltages and currents are so low that they cannot generate interference (they should be several orders of magnitude higher).

In order to avoid spikes when the laser is turned on, the supply cables of the laser were put far from the MEAs

We also minimized the power supply ripple using a capacitor with low ESR, to avoid a possible ripple in the generated signals.

The second basin was shielded by a thick aluminium Faraday cage.


The connection cable between electrodes and the

acquisition/stimulation circuit was shielded against EMI

emissions with a suitable copper jacket.

The electronic circuit was included in a plastic box whose

walls have been treated with special varnishes that

efficiently shield EMI noise.

All the cables used for the connection between culture

basins, stimulation circuit and acquisition card were

carefully and separately shielded.

First resultsFirst results

In a typical experiment with two neural MEAs we In a typical experiment with two neural MEAs we stimulated them with electrical and laser pulses stimulated them with electrical and laser pulses (laser diode 658 nm)(laser diode 658 nm)


The first finding was an increase of The first finding was an increase of crosscorrelation and coherence during the both crosscorrelation and coherence during the both electrical and laser stimulations:electrical and laser stimulations:

Coherence graphCoherence graph


Crosscorrelation graph


The autocorrelation functions suggest The autocorrelation functions suggest similar behaviours of the two MEAssimilar behaviours of the two MEAs

First resultsFirst results As crosscorrelation could be due to the As crosscorrelation could be due to the

high autocorrelation values, we also high autocorrelation values, we also performed an autoregressive ARIMA performed an autoregressive ARIMA model to eliminate the factors that in model to eliminate the factors that in the first time series could affect the the first time series could affect the second series.second series.

First resultsFirst results The analysis shows that The analysis shows that the two series

are so strongly correlated that, even after

correcting for a substantial amount of

self-correlation, the values of one series

impact the prediction of the other in a

highly significant way.

This does not happen using the only

culture liquid (though highly conductive)

Evolution of the systemEvolution of the system We changed the experimental set-up in We changed the experimental set-up in

order to improve further the isolation order to improve further the isolation conditions and performancesconditions and performances

Evolution of the systemEvolution of the system Higher acquisition frequency (333 MHz, Higher acquisition frequency (333 MHz, DAQ

PCI-6036E National Instruments) We built a plexiglass incubator where the MEAs We built a plexiglass incubator where the MEAs

are placedare placed The incubator is wrapped by a Faraday cage The incubator is wrapped by a Faraday cage

made by a 1 mm brass net, connected to the made by a 1 mm brass net, connected to the groundground

Faraday cage /incubator

Acquisition card

Computer

Controller:Receives the neural signals and amplifies them before sending them to the PC

The controllerThe controller•The device is designed to ensure the maximum shielding between channels

•The controller interfaces the MEAs with the acquisition card and includes a high impedance amplifier (d)

•After the amplifier the signals pass through a 50 Hz Notch filter to eliminate power supply disturbances (e)

•Then the signals are transferred to the card after a complete isolation by means of special Texas Instruments (ISO124) electronic circuits that avoid coupling between circuits (g).

The experimental set-upThe experimental set-up

•To avoid possible spurious signals all the circuits are closed into a thick metallic box connected to ground

•The acquisition card is installed on a shielded PC

•The controller power supply is furnished by Lithium inside batteries.

•The stimulation is supplied by a red 2mW laser diode (630 nm)•The laser circuit is completely separated by the described circuits and supplied by a 8 V negative stabilizer

New experimentsNew experiments

•We used just laser pulses to avoid doubts on the origin of possible electrical correlations

•Two neural basins, one optically and electromagnetically shielded ( thick plastic box wrapped by double aluminium foil)

•Both MEAs in the Faraday cage

•The laser sends a random set of 1-2.5 sec bursts of 1 ms pulses


•Both MEAs show spikes simultaneous to the laser activation

The sharply higher frequency acquisition reveals new interesting features in the signals


•A suitable software procedure reveals that the spikes in the two MEAs are coincident


•As in the past experiments, both MEAs present high and nearly coincident autocorrelation functions


•The FFTs, although different, present some common peaks, in particular a common peak around 930 Hz


•Periodograms (intensities of frequencies) are nearly coincident

New experimentsNew experiments•Our last experiment was designed with a different criterion

•Just one neural basin, to avoid the bias that one basin transfers information to the other, whereas they just could receive the pulses simultaneously

•Three MEAs:•Neurons•Matrigel + culture liquid•Culture liquid

•All the MEAs in the Faraday cage

New experimentsNew experiments•One MEA is covered by a thick plastic box wrapped by a double aluminium foil, acting both as e-m and optical shielding•A further opaque box covers the two MEAs not hit by the laser

•The laser diode hits the non-covered MEA

•During the experiment all the MEAs exchange in turn their position and shielding conditions

New experimentsNew experiments•The laser sends a random set of 1-2.5 sec bursts of 1 ms pulses

•On a second phase the laser source is covered by a double aluminium foil

•On a third phase the laser is put 1.5 meters apart

The “Q” effectThe “Q” effect•We collected and analysed more than 1 Gb of data•During all the described phases the laser pulse arouses a simultaneous spike in the only neural basin•This graph shows (gray and lilac channels) the peaks of the non-shielded neural MEA (Laser: red channel)

The “Q” effectThe “Q” effect

•These graph show the peaks of the neural MEA when shielded (left) and when 1.5 meters apart (right)•All the other channels are related to the control basins

The “Q” effectThe “Q” effect•Using a LED (430 nm) instead of the laser, with the same set of random bursts, the signals never show any peaks.

•No peaks were shown at the end of each burst of pulses.

The “Q” effectThe “Q” effect• Same analysis as in the past experiment:comparing the FFTs of the neural MEA and the liquid control MEA, the FFTs are completely different

The “Q” effectThe “Q” effect• Same analysis as in the past experiment:The periodograms of the neural MEA and the control liquid MEA are completely different

The “Q” effectThe “Q” effect

• Same analysis as in the past experiment:The autocorrelation functions are different, and the autocorrelation of the control liquid is low

Equipment analysisEquipment analysis

•Benchmark test on the acquisition card , injecting up to 1V signals on more channels: crosstalk < -110 dB

•We simulated spikes in one channel and verified that they do not propagate through the other channels

•Crosstalk preamplifier tests, injecting up to 80 mV ( from 100 to 500 Hz) into each channel: noise < 2 mV

•High current sparks were injected in the preamplifier circuit: no interferences were shown.


•Electromagnetic shielding tests:• We introduced an antenna in the brass Faraday cage.•The antenna was connected to a spectrum analyzer, detecting frequencies from 100 Khz to 3.5 GHz. The instrument did not detect any activity during the laser pulses•The antenna was connected to an oscilloscope to verify presence of frequencies in the range 0-100 KHz. The instrument did not detect any activity during the laser pulses.


•Optical shielding test:

•We verified it using a WATEC super-high sensitivity camera ( 3 x 10-4 lux). •We did not perceive any luminescence during the laser emission.

On the other hand, the phenomenon was present also in the third phase, where the laser was shielded further on, put 1.5 meters apart and directed to an opposite direction.

First conclusionsFirst conclusions

•The “Q” effect is visible only in neural MEAs irradiated by a laser source•The matrigel MEAs don’t show the effect•The culture liquid MEAs don’t show the effect•The optical shielding does not inhibit the effect•The electromagnetic shielding does not inhibit the effect•The distance from the laser source (up to 1.5 meters) does not inhibit the effect•The LED light does not induce the effect


•Looking at the whole series of experiments, we note that:•We changed completely the experimental set-up:

•biological lab•PC•Acquisition card• MEAs model•Hardware controller•Faraday cage


•Nonetheless, we always verified the same reactions of the neural MEAs to the laser pulses•Separated neural MEAs show similar spectra•After substituting the acquisition card with a more powerful one, a series of spikes appeared simultaneous to the laser pulses•No reactions ever appeared with control MEAs or with LED pulses

•The “Q” effect appeared hundreds of times•It is perfectly repeatable and happens every time the laser circuit is turned on.

New considerationsNew considerations•In order to identify the physical source of the effect, we substituted the laser with a dummy load simulating an equivalent current absorption•The same peaks were present•No peaks were present using a dummy load equivalent to the LED absorption•We concluded that the phenomenon should not be due to the laser itself but to an electromagnetic field coming from the laser supply circuit•The field should be too weak to be detectible by our measure instruments

New considerationsNew considerations•Neurons appear to receive and amplify a signal whose value through the air , measured with a filar antenna (suitable to detect electromagnetic frequencies), and before reaching the Faraday cage, is under 2 mV (sensitivity threshold of our oscilloscope)

•It must be stressed that in order to cause an action potential (spike), a neuron needs to be stimulated inside the cell with a 30 mV pulse

New considerationsNew considerations

•In order to evaluate the intensity of the field we used a high-sensitive Gaussmeter, whose sensitivity threshold is around 70 µG

•The laser supply circuit, when turned on, generates in the near of the Gaussmeter around 0.002 G

•When moving away the Gaussmeter beyond 30 cm, the field intensity gets under the Gaussmeter sensitivity.

New considerationsNew considerations•During the experiments the laser circuit was at least 50 cm far

•We could not assess the intensity of the magnetic field (if any) received by the neurons during the experiments because it is so weak that it gets under both the oscilloscope and the Gaussmeter sensitivity

•It should be reminded that•The MEA structure is not suitable to act as antenna•MEA control circuit and laser activation circuit are completely separated•The spikes are always present just in the neural MEAs

ConclusionsConclusions

•We believe that neurons are the active receiving elements of the whole system

•Neurons act as antennas for extremely weak electromagnetic fields

•The origin of the effect is unknown

•The effect is hard to be explained in the frame of classical physics

Future projectsFuture projects

•We will continue our measures of the reactivity of neurons to ultraweak electromagnetic fields

•We acquired a mu-metal box, that contains the Faraday cage and ensures a perfect electric and magnetic shielding

Future projectsFuture projects

•It will be possible to evaluate the reactivity of neurons to fields of variable intensity

•We aim to identify general laws that could rule the phenomenon under investigation: in particular, its dependance from the distance , intensity of frequency of the pulses.

Technology

Pizzi quantum mind 2007