A BIOLOGICAL NEURAL NETWORK FOR ROBOTIC

CONTROL

Towards a Human Neuroprocessor

José M. Ferrández, Victor Lorente, Javier Garrigós

Departamento de Electrónica, Tecnología de Computadores y Proyectos, Universidad Politécnica de Cartagena, Spain

Eduardo Fernández

Instituto de Bioingeniería, Universidad Miguel Hernández de Elche, CIBER-BBN, Spain

Keywords: Cultured neural network, Induced plasticity, Multielectrode recordings, Robotic control.

Abstract: The main objective of this work is to analyze the computing capabilities of human neuroblastoma cultured

cells and to define stimulation patterns able to modulate the neural activity in response to external stimuli

for controlling an autonomous robot. Multielectrode Arrays Setups have been designed for direct culturing

neural cells over silicon or glass substrates, providing the capability to stimulate and record simultaneously

populations of neural cells. This paper tries to modulate the natural physiologic responses of human neural

cells by tetanic stimulation of the culture. If we are able to modify the selective responses of some cells

with a external pattern stimuli over different time scales, the neuroblastoma-cultured structure could be

trained to process pre-programmed spatio-temporal patterns. We show that the large neuroblastoma

networks developed in cultured MEAs are capable of learning: stablishing numerous and dynamic

connections, with modifiability induced by external stimuli.

1 INTRODUCTION

Using biological nervous systems as conventional

computer elements is a fascinating problem that

permits the hybridation between Neuroscience and

Computer Science. This synergic approach can

provide a deeper understanding of natural perception

and may be used for the design of new computing

devices based on natural computational paradigms.

The brain uses millions of biological processors,

with dynamic structure, slow commutations

compared with silicon circuits, low power

consumption and unsupervised learning. This kind

of computation is more related to perceptual

recognition, due to the natural variance of the

perceptive patterns and the a priori lack of

knowledge about the perceptual domain.

There exist many research approaches based on

mimicking this bioinspired parallel processing, not

only from the algorithm perspective (Anderson and

Rosenfeld, 1998), but also from the silicon circuits

design. These bioinspired approaches are useful for

pattern recognition applications, like computer

vision or robotics, however they are implemented

over serial and artificial silicon processors with fixed

and static structure. A real biological processor with

millions of biological neurons and a huge number of

interconnections, would provide much more

computational power instead of their low transition

rates due to high number of computing elements and

the extraordinary network capability of adaptation

and reconfiguration to unknown environments. This

extraordinary capability is related with natural

unsupervised learning.

Learning is a natural process that needs the creation

and modulation of sets of associations between

stimuli and responses. For understanding the process

of learning, is necessary to define the physiological

mechanisms that support the creation and

modulation of associations and determine the

relation that modulate the configuration between

stimuli and responses associations. These

mechanisms and relation have been studied by many

508

M. Ferrández J., Lorente V., Garrigós J. and Fernández E. (2009).

A BIOLOGICAL NEURAL NETWORK FOR ROBOTIC CONTROL - Towards a Human Neuroprocessor.

In Proceedings of the International Joint Conference on Computational Intelligence, pages 508-513

DOI: 10.5220/0002335305080513

 SciTePress

neurophysiological studies at different levels mainly

in single cell experimentation.

Our learning experiments were performed in neural

cultures containing 120.000 human neuroblastoma

SY-5Y, under the assumption that this kind of cells

are able to respond electrically to external stimuli

and modulate their neural firing by changing the

stimulation parameters. Such cultured

neuroblastoma networks showed dynamical

configurations, beeing able to develop and adapt

functionally in response to external stimuli over a

broad range of configuration patterns. We are

especially interested in analizing if populations of

neuroblastoma cells are able to process and store

information, and if learning can be implemented

over this biological structure.

The main objective of this work is to analyze the

computing capabilities of human neuroblastoma

cultured cells for controlling a robot. Multielectrode

Arrays Setups have been designed for direct

culturing neural cells over silicon or glass substrates,

providing the capability to stimulate and record

simultaneously populations of neural cells . This

paper describes the process of growing human

neuroblastoma cells over MEA substrates and tries

to change the natural physiologic responses of these

cells by external stimulation of the culture provided

by the robot sensors. Modifying the global responses

of some cells with a external pattern stimuli means

adjusting the biological network behaviour due to

changes in synaptic efficiency or long-term

potentiation (LTP). Therefore, the neuroblastoma-

cultured structure could be trained to process pre-

programmed spatio-temporal patterns. In what

follows, we show that the large neuroblastoma

networks developed in cultured MEAs are capable

of learning: stablishing numerous and dynamic

connections, with modifiability induced by external

stimuli.

2 HUMAN NEUROBLASTOMA

CULTURES

The physiological function of neural cells is

modulated by the underlying mechanisms of

adaptation and reconfiguration in response to neural

activity. Hebbian learning describes a basic

mechanism for synaptic plasticity wherein an

increase in synaptic efficacy arises from the

presynaptic cell's repeated and persistent stimulation

of the postsynaptic cell. The theory is commonly

evoked to explain some types of associative learning

in which simultaneous activation of cells leads to

pronounced increases in synaptic strength. The N-

methyl-D-aspartate (NMDA) receptor, a subtype of

the glutamate receptor, has been implicated as

playing a key role in synaptic plasticity in the CNS

(Bading and Greenberg, 1991), where as dopamine

receptors are involved in the regulation of motor and

cognitive behaviors. For most synaptic ion channels,

activation (opening) requires only the binding of

neurotransmitters. However, activation of the

NMDA channel requires two events: binding of

glutamate (a neurotransmitter) and relief of Mg2+

block. NMDA channels are located at the

postsynaptic membrane. When the membrane

potential is at rest, the NMDA channels are blocked

by the Mg2+ ions. If the membrane potential is

depolarized due to excitation of the postsynaptic

neuron, the outward depolarizing field may repel

Mg2+ out of the channel pore. On the other hand,

binding of glutamate may open the gate of NMDA

channels (the gating mechanisms of most ion

channels are not known). In the normal

physiological process, glutamate is released from the

presynaptic terminal when the presynaptic neuron is

excited. Relief of Mg2+ block is due to excitation of

the postsynaptic neuron. Therefore, excitation of

both presynaptic and postsynaptic neurons may open

the NMDA channels, this is closely related with

Hebbian learning.

Another important feature of the NMDA channel is

that it conducts mainly the Ca2+ ion which may

activate various enzymes for synaptic modification,

even nictric oxide has been identified as a relevant

element in synaptic regulation. The enhancement of

synaptic transmission is called the long-term

potentiation (LTP), which involves two parts: the

induction and the maintenance. The induction refers

to the process, which opens NMDA channels for the

entry of Ca2+ ions into the postsynaptic neuron. The

subsequent synaptic modification by Ca2+ ions is

referred to as the maintenance of LTP.

A human neuroblastoma SY5Y cell line, that

express clonal specific human dopamine receptors,

and also NMDA receptors, will be the biological

platform for studying learning in cultured cells.

Neuroblastoma SH-SY5Y cells are known to be

dopaminergc, acetylcholinergic, glutamatergic and

adenosinergic, so in this line they respond to

different neurotransmitters. The cells have very

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different growth phases, as it can be seen in Figure

1. The cells both propagate via mitosis and

differentiate by extending neurites to the

surrounding area. The dividing cells can form

clusters of cells which are reminders of their

cancerous nature, but chemicals can force the cells

to dendrify and differentiate, in some kind of

neuritic growth.

Figure 1: Human neuroblastoma cells.

As conclusion, neuroblastoma culture cells show

electrophysiological responses similar to standard

neurons, as potential actions generation sensible to

tetrodotoxin (TTX) and acetylcholyn. They have

neurotransmitters synthesis process and are able to

neuritic growth in culture medium.

3 EXPERIMENTAL SETUP

The neuro-physiology setup provides a complete

solution for stimulation, heating, recording, and data

acquisition from 64 channels. The MEA

(microelectrode array) system is intended for

extracellular electrophysiological recordings in vitro

of different applications that include acute brain,

heart, and retina slices; cultured slices; and

dissociated neuronal cell cultures.

The basic components of the proposed system are

shown in Figure 2. These components are:

• A microelectrode array is an arrangement of 60

electrodes that allows the simultaneous

targeting of several sites for extracellular

stimulation and recording. Cell lines or tissue

slices are placed directly on the MEA and can

be cultivated for up to several months. Almost

all excitable or spontaneously active cells and

tissues can be used.

• Raw data from the MEA electrodes are

amplified by MCS filter amplifiers with custom

bandwidth and gain, which are built very small

and compact using SMD (Surface Mounted

Devices) technology. The small-sized amplifier

combines the interface to the MEA probe with

the signal filtering and the amplification of the

signal. The compact design reduces line pick up

and keeps the noise level down. The amplifiers

are mounted over an inverted microscopes.

• The analog input signals are then acquired and

digitized by the MC-Card that is preinstalled on

the data acquisition computer, that supplies the

power for the amplifiers, and the pattern stimuli

to the stimulators.

• The robot sends information about the

environment to the computer using a bluetooth

link. The sensor consists in infrared sensors for

detecting obstacles.

Figure 2: Experimental Setup.

4 METHODS

Human neuroblastoma cultures were produced using

the commercial line SH/SY5Y . Neural cells were

then plated on Micro-Electrode Arrays -MEAs

(MultiChannel Systems, Reutlingen, Germany).

Initially the nitrogen frozen cells, was immersed in a

37 degree bath, and centrifuged at 1000 rpm during

5 minutes. When cells have grown in a uniform

mono-layer process, they are washed three time with

buffer Phosphate-buffered saline (PBS) for keeping

the pH approximately constant. 0,5 per cent trypsin

was added to the solution in order to re-suspend cells

adherent to the cell culture dish wall during the

process of harvesting cells. The cells were kept in

the incubator for 5 minutes and passed through a 40

microm cell strainer (Falcon, Bedford, MA) to

remove large debris. Finally the cells are transferred

to a specific medium in order to inactivate trypsin,

and centrifugated again during 5 minutes at 1000

rpm.

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For seeding the plate cells are stained with trypan

blue, (because cells that loose their permeability get

colorred with this solution) and counted with a

Neubauer chamber. Finally, 80.000 or 120.000 total

neuroblastoma cells have been placed over the MEA

substrate.

Maintaining cells in culture is essential for studying

their physiological properties. Cell culturing is

dependent on the growth surfaces and cells must

adhere to the electrode substrate in order to establish

the best connection with the electrodes material. For

most cultures coated tissue culture plates are

prerequisite for seeding. The most commonly used

coatings are positively charged polymers. In this

work, the insulation layer (silicon nitride) of some of

the plates was pre-treated with polyethyleneimine

(PEI), showing no advantages compared with no

covered plates.

The neuroblastoma cultures are maintained in a 37

degree humidified incubator with 5 per cent CO2

and 95 per cent O2 with serum-free Neurobasal

medium. Under the aforementioned conditions we

were able to record stable electrophysiological

signals over different days in vitro (Div). The

medium was replaced one-half of the medium every

5 days.

5 RESULTS

The cultured neuroblastoma cells establish synaptic

connections. In Figure 3 it can be seen differentiated

and non-differentiated neuroblastoma cell bodies

growing around the whole electrode population. The

dendritic arborescence is more evident in the

magnification Figure 3 where differentiated neural

cells surround the four electrodes while the rest of

the cells are in their growing process. This Figure

corresponds to 80.000 neuroblastoma cells seeded in

a no-PEI MEA at 2nd day in vitro (div).

The electrophysiological properties of the

neuroblastoma cultures were analized by recording

the spontaneous activity of the network. Time course

of experiments was over 15 days; recordings were

done using two MCS-Meas with two neuroblastoma

cell cultures (but only in one the cells survived till

day 15). In vitro neuroblastoma networks show

spontaneously firing. This firing rates change during

the culture development with marked day

differences and the global rate is closely related to

the age of the network.

Figure 3: Biological neural network over multielectrode

array.

The physiological recordings correspond to

neuroblastoma cultures in the range of 1-7 div. They

show bursting and spiking activity, with usually

negative depolarisations. Figure 4 show the spiking

activity of the neural population with an automatic

detection level for each electrode. This is very

convenient if you have multiple channels for

extracting spikes.

Figure 4: Spontaneus neural activity detected by the

multielectrode array.

The standard deviation of each data trace is used to

estimate its spike threshold. A time interval of 500

ms is used to calculate the standard deviation. By

fixing the factor, by which the standard deviation is

multiplied, the sign of the factor determines whether

the spike detection level is positive or negative, only

values above this will be extracted as spiking

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activity. A value between -1 and -4 is appropriate for

most applications the threshold was fixed at standard

deviation equal to -3 with respect to the electrode

activity in order to identify spikes embedded in the

noisy signals.

During the neuroblastoma development, a wide

range of population bursting or synchronized

activity has been observed, according to some

studies in neural cultures preparations (Wagenaar,

Pine, and Potter, 2006). The burst usually contains a

large number of spikes at many channels, with

variable duration, from milliseconds to seconds.

5.1 Tetanic Stimulation

Spontaneous activity was recorded for intervals of 3

minutes before stimulation (PRE-data), and the total

number of spikes extracted was counted. The

biphasic stimulus consists in a 10 trains of a 100

anodic-first waveform with 1 Volt amplitude

delivered to all 60 electrodes in order to propagate a

tetanization stimulus to the neuroblastoma culture.

In neurobiology, a tetanic stimulation consists of a

high-frequency sequence of individual stimulations

of a neuron. It is associated with long-term

potentiation, the objective of this work. High-

frequency stimulation causes an increase in

transmitter release called post-tetanic potentiation

(Antonov, Antonova, Kandel, 2003). This

presynaptic event is caused by calcium influx.

Calcium-protein interactions then produce a change

in vesicle exocytosis. Some studies (Jimbo,

Robinson, and Kawana, 1998) use repetitive

stimulation for training neural cultures, achieving

activity potentiatiation or depresion

Once the tetanization stimulus was applied to the

whole population 5 minutes after the stimulation a 3

minutes interval was recorded (POST-data). Only

neuronal signals which had at least a 2:1 signal:noise

ration were valued as "spikes". Again, the total

number of spikes extracted was counted. This

process was made for cultures at 1 day in vitro (div),

5 div and 16 div. Figure 5 represents the counted

spikes with bar charts for the different recordings.

The conclusion from this Figure is:

1) While the neuroblastoma culture is growing

new connections are created, and the number

of spikes increases as the culture expands

over the MEA.

2) After a tetanic stimulation the cells continue

with their increased spiking rate, providing a

persistent change in the culture behaviour.

When this change in the network response

lasts, these changes can be called learning.

In all the experimentation performed, tetanic

stimulation was applied as training method, and the

electrophysiological properties of the neuroblastoma

culture change, getting a potentiation effect on the

spontaneous firing, modulating in this way the

culture neural activity.

Figure 5: Induced neural activity by tetanization stimuli.

5.2 Robotic Control

For controling the direction of the robot we propose

to compute the vector resulting from neural activity

recorded in the human neuroblastoma culture. This

vector will be provided to the robot in order to guide

his movement. The sensors will detect the obstacles,

and the information will be passed to the computer

in order to induce a selective tetanization of the

biological neural network for changing the resulting

direction vector. In Figure 6, the selected electrodes

for the tetanization are shown in order to selective

induce a persistent change in the biological neural

network behaviour.

Figure 6: Selective electrode tetanisation.

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512

When the robot detect an obstacle in his left path, an

stimilation signal will be sent to the system for

tetanizing the right tissue. By tetaniztion the

electrodes of the right part of the array, an increase

in the firing rate of the neural cells that lie in the part

of the culture will be achieved, and the direction

vector will point to the right in this particular case.

We expect to apply some basic Braitenberg

principles to the system in order to study the

biological neural network behaviour induced by a

tetanization learning scheme.

6 DISCUSSION

Learning in cultured neuroblastoma networks by a

stimulation process, without the involvement of a

natural adaptation process to the environment

requires identifying the correct stimuli to provide to

the neurons maintained ex vivo. These

neuroblastoma networks form a large culture

covering the whole electrode array and generating a

rich dendritic configuration. The connectivity can be

modulated by external stimulation as has been

described in many studies, but also the activity of

the network can be modulated with the appropriate

stimulation scheme.

Tetanization consists in high-frequency stimulation

to the culture, in order to cause an increase in

transmitter release called post-tetanic potentiation.

The results illustrate the existence of qualitatively

different responses to stimulation. Our results

indicate the existence of a clear facilitation

mechanism in response to the tetanization stimuli at

different stages of cell development. Since this kind

of stimulation has been used in attempts to induce

plasticity in neuroblastoma, refining some crucial

aspects of the stimulation is still indispensable.

It is very important to adjust the frequency of the

train pulses of the stimulation for suppressing

bursting in the culture. While in vivo networks

suppress bursting naturally with the tissue

development and sensory inputs, ex-vivo cultures

need to reduce this synchronized activity by

adjusting the stimulation parameters. Also, for

superimposing a desired behaviour on the biological

networks it is necessary to stimulate locally some

part of the culture in order to facilitate some parts of

the networks, or achieve some kind of electrical

stimulation that depress the local activity of a

restricted location. With this local potentiation-

inhibition scheme the culture global behaviour could

be controlled.

Future work consists in determine the optimal

stimulation to apply for inducing permanent firing

changes in the culture, and the strategies for

connecting the robot sensors to the stimulation

patterns. These aspects will then constitute the basis

for inducing stable goal-directed plasticity, and

hence for designing new biological neuroprocessors

applied to robotics.

ACKNOWLEDGEMENTS

This work was supported by the Spanish

Government through grants TIN2008-06893-C03,

TEC2006-14186-C02-02 and SAF2008-03694,

Cátedra Bidons Egara, Fundación Séneca

08788/PI/08, CIBER-BBN and by the European

Comission through the project "`NEUROPROBES"'

IST-027017.

REFERENCES

Anderson J.A., and Rosenfeld E., 1998. “Neurocomputing:

Foundations of research'', MIT Press Cambridge, MA,

USA.

Bading H. and Greenberg M.E., 1991. “Stimulation of

protein tyrosine phosphorylation by NMDA receptor

activation '', Science, 253, Issue 5022, pp. 912-914.

Wagenaar, D. A., Pine, J. and Potter, S. M., 2006. “An

extremely rich repertoire of bursting patterns during

the development of cortical cultures.'', BMC Neuro-

science, 7:11.

Antonov I., Antonova I., Kandel E.R., 2003. “Activity-

Dependent Presynaptic Facilitation and Hebbian LTP

Are Both Required and Interact during Classical

Conditioning in Aplysia '', Neuron, Volume 37(1), pp.

135-147.

Jimbo Y., Robinson H.P., Kawana A., 1998. “Strength-

ening of synchronized activity by tetanic stimulation

in cortical cultures: application of planar electrode

arrays'', IEEE transactions on Biomedical

Engineering, 45(11), pp. 1297-1304.

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