Electrical stimulation of nerve cell Electrical stimulation of nerve cell networks growing on microelectrode networks growing on microelectrode

arrays: stimulation efficiency and arrays: stimulation efficiency and entrainmententrainment

Honors ThesisWorking DraftVivek JainMentor: Dr. Guenter W. Gross

AcknowledgementAcknowledgementI thank the CNNS staff for their assistance with MEA fabrication and cell culture.

I also express my appreciation to Dr. Gross for his assistance with culture setup, recording, and stimulation procedures.

Finally, I express my gratitude to Dr. Eve for leading us all through many hurdles during the past three semesters.

STATEMENTSTATEMENT

StatementStatement Understanding native activity changes in

neuronal networks will lead to development of viable methods for memory storage and network ‘learning’,

Observing such changes requires repeatable and quantifiable network responses

Statement (contd.)Statement (contd.) Repeatability depends on knowledge about

the effectiveness of a stimulation pulse or a pulse train.

BACKGROUNDBACKGROUND

TerminologyTerminology MEAs: micro-electrode arrays

MMEPs (multi micro-electrode plates), for culturing, stimulating and recording neuronal activity, spikes and action potentials of individual axons neurons

MMEPs in use in CNNSMMEPs in use in CNNS

ITO Electrodes photo-etched on Glass Plates

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DefinitionsDefinitions

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1. Biphasic Monopolar Stimulation

Only one microelectrode provides the biphasic pulse relative to ground (i.e. Monopolar)

Biphasic = 2 phases

Definitions (contd.)Definitions (contd.)2. Stimulation Efficiency:

Gross et al (1993), Shahaf et al (2001) use efficiency as the Response/Stimulus ratio:

Efficiency = (# of Response Pulses) (# of Stimulation

Pulses)

Definitions (contd.)Definitions (contd.)3. Entrainment:

The “locking together of frequencies” (Winfree, 1980) so as to make the network respond in synchronization with the applied stimulus is called the entrainment. While in entrainment, the network units fire when, and only when, there is a stimulus provided

Definitions (contd.)Definitions (contd.)Entrainment: the process by which an extrinsic stimulus changes the phase of a spontaneous event cycleLeft: at 30 trains/min, the network gets entrained to the stimulus pulse train

Gross et al, 1993

Pulses TrainsPulses Trains A typical pulse train may consist of 1 (i.e. a

single pulse) to 20 pulses

A representative pulse train consisting of 20 pulses

By varying the time interval between successive trains, we change the pulse train frequency

Sample Stimulation Pulse Train (on Sample Stimulation Pulse Train (on oscilloscope)oscilloscope)

Gross et al, 1993

Repetitive PulsesRepetitive Pulses

Characteristics of StimulationCharacteristics of Stimulation Monopolar, Biphasic pulses for less cell

damage and to avoid electrode breakdown (Temel et al 2004, Gross et al 1993)

Constant Voltage stimulation because constant current may cause voltages to exceed electrolysis thresholds (~2.5 V) (Gross et al 1993)

Normal Stimulation DomainNormal Stimulation Domain

Gross et al, 1993

LIMITATION 1: Networks non-stationary dynamic (Jimbo et al,

1999); constant network ‘learning’ probably occurs

LIMITATION 2: Each culture is unique; activity differs no a priori knowledge of stimulation electrodes Replication not easy

Limitations of methodologyLimitations of methodology

Large cells near electrode cratersLarge cells near electrode craters(Morphological cell-electrode coupling)(Morphological cell-electrode coupling)

Gross et al., 1993

RESEARCH OBJECTIVERESEARCH OBJECTIVE

Research ObjectiveResearch ObjectiveTo characterize stimulation efficiency/entrainment as

a function of the following variables: Frequency (pulses/trains) Pulse durations # of pulses/trainsSignificance:

No study done yet that pins down most effective characteristics of a stimulation pulse train

May pave way for improved communication with networks and studies of information storage.

RESEARCH METHODOLOGYRESEARCH METHODOLOGY

Research MethodologyResearch Methodology Use MMEP-4

Cultures will be provided by the CNNS staff

Electrodes

Recording Matrix

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Stimulation ProtocolsStimulation Protocols Single Pulse Stimulation

Usually episodes of 20 pulses at frequencies ranging from 0.1 Hz to 10 Hz. Can be either single-site or multi-site.

Pulse Train Stimulation Episodes of 20 trains (each train around 10

pulses long) at frequencies ranging from 0.1 Hz to 10 Hz.

Cristopher Sparks, PhD Dissertation UNT, 2001

8 x 8 recording matrix (MMEP 4)8 x 8 recording matrix (MMEP 4)

Micro electrodes at different separations

Stimulation Sites

Response Sites

PRELIMINARY RESULTSPRELIMINARY RESULTS

Experiment ProtocolExperiment Protocol Used 38 day old, high density, Frontal Cortex

culture Was treated with 8 μL of 10 mM bicuculine 28 active units were found

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Pulse Train Single Pulse

Day 1

Day 2

Experiment Protocol (contd.)Experiment Protocol (contd.) Located responsive units, and then

conducted episodes of pulse train and single pulse stimulation at 5 sites that yielded the strongest auditory network response

Stimulation occurred at frequencies from 0.1 Hz to 10 Hz for pulse trains and from 0.2 Hz to 10 Hz for single pulses

Experiment Protocol (contd.)Experiment Protocol (contd.) Pulse Trains Characteristics

Train Width: 0.04s i.e. 10 pulses pulse-train @ 250 pulses per second (PPS)

Pulse Duration: 300 μs Inter-pulse Period: 4 ms Amplitude: 0.8 V

Single Pulse Characteristics Pulse Duration: 300 µs Amplitude: 0.8 V

Early ResultsEarly Results The network response showed a steady decline as the

frequency was increased for both Pulse Train and the Single Pulse stimulations

Netw ork Response to Single Pulse Stimulation

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0.2 0.25 0.5 0.75 1 1.25 1.55 1.75 2 3 5 10

Frequency (Hz)

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Early Results (contd.)Early Results (contd.)Netw ork Response to Pulse Train Stimulation

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0.1 0.2 0.25 0.5 0.75 1 1.25 1.55 1.75 2 3 5 10

Frequency (Hz)

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Early Results (contd.)Early Results (contd.) Pulse Train Stimulation

Stimulated 4 different sites Recorded the response for each of the 28 units that

responded to the stimulation Some responses were very minimal, and therefore

any unit that responded less than 50% at 0.1 Hz was eliminated when response curves were sorted

Distinct “colonies” of cells seem to appear, whose response curves show similar characteristics!

Early Results (contd.)Early Results (contd.)

-20%

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0.1 0.2 0.25 0.5 0.75 1 1.25 1.55 1.75 2 3 5 10

Frequency

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AVG1

AVG2

Poly. (AVG1)

Poly. (AVG2)

Poly. (AVG2)

The thick curves show the averages of individual responses in each of the two ‘colonies’ of cells

Early Results (contd.)Early Results (contd.)Site 1

0%

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0.1 0.2 0.25 0.5 0.75 1 1.25 1.55 1.75 2 3 5 10

Frequency (Hz)

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Poly. (AVG1)

Expon. (AVG2)

Site 1: This is the cleaned out graph from the previous slide. The black one is a 4th Order curve, while the pink is an exponential decay curve

Early Results (contd.)Early Results (contd.)Site 2

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Poly. (AVG1)

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Site 2: The black one is a 4th Order curve, while the pink is an exponential decay curve

Early Results (contd.)Early Results (contd.)Site 3

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0.1 0.2 0.25 0.5 0.75 1 1.25 1.55 1.75 2 3 5 10

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Poly. (AVG1)

Expon. (AVG2)

Site 3. The black one is a 4th Order curve, while the pink is an exponential decay curve. There seems to be another colony active in this site.

Early Results (contd.)Early Results (contd.)Site 4

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Frequency (Hz)

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Expon. (AVG2)

Poly. (4a)

Site 4: The black one is a 4th Order curve, while the pink is an exponential decay curve

AnalysisAnalysis Further studies pending, it seems that there is no

‘ideal’ frequency to get the network respond at high efficiency.

In the 5 previous slides, the ‘black colony’ of cells stayed at almost 100% response efficiency levels till a ‘cutoff’ frequency

The ‘pink colony’ however started at just over 50% efficiency levels, and their response decreased exponentially with increase in frequency

Analysis (contd.)Analysis (contd.) It also seems that as the frequency is lowered (0.1

Hz really means a pulse every 10 seconds!), the network simply responds to the pulse / pulse train, because it is much below the network’s native natural frequency

The network remained ‘resilient’ through my stimulation experiment – i.e., after every stimulation episode, it ‘recovered’ to its native bursting rate, showing that the results are valid and therefore avoid Limitation 1 mentioned earlier

Stability of cultureStability of culture Native Activity Graphs:

Sample Bursting (Raster Display)Sample Bursting (Raster Display)

4 Units on a single DSP

Native Bursts

Sorted Units

Near futurueNear futurue Culture left for 3 days

Hmm…

Hypothesis for the next experiment??

Future (contd.)Future (contd.) What Next?

Repeat, Repeat, Repeat Conduct more experiments before

concluding firmly whether we have found what we think we have

…actually did an experiment last week– data analysis still pending

REFERENCESREFERENCES

ReferencesReferencesBove, M., M. Grattarola, M. Tedesco and G. Verreschi. Characterization

of growth and electrical activity of nerve cells cultured on microelectronic substrates: towards hybrid neuro-electronic devices, Journal of Materials Science: Materials in Medicine, 5: 684-687.

Babalian, A. L., D. K. Ryugo and E. M. Rouiller. 2003. Discharge properties of identified cochlear nucleus neurons and auditory nerve fibers in response to repetitive electrical stimulation of the auditory nerve, Exp Brain Res, 153: 452-460

Gross, G. W., B. Rhoades, D. Reust and F. Schwalm. 1993. Stimulation of monolayer networks in culture through thin-film indium-tin oxide recording electrodes, Journal of Neuroscience Methods, 50: 131-143

Jimbo, Y., T. Tateno, H. P. C. Robinson. 1999. Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons, Biophysics Journal, 76: 670-678

ReferencesReferencesManevitz, L. M. and S. Marom. 2002. Modeling the Process of

Rate Selection in Neuronal Activity, Journal of Theoretical Biology, 216: 337-343

Shahaf, G. and S. Marom. 2001. Learning in Networks of Cortical Neurons, Journal of Neuroscience, 21 (22): 8782-8788

Temel, Y., V. V. Vandewalle, M. van der Wolf, G. H Spincemaille, L. Desbonnet, G. Hoogland & H. W. M. Steinbusch. 2004. Monopolar versus bipolar high frequency stimulation in the rat subthalamic nucleus: differences in histological damage, Neuroscience Letters, 367: 92-96

Winfree, A. T. 1980. Ring Population, The Geometry of Biological Time: 114