Honors Thesis Working Draft Vivek Jain Mentor: Dr. Guenter W. Gross

1. Electrical stimulation of nerve cell networks growing on microelectrode arrays: stimulation efficiency and entrainment Honors Thesis Working Draft Vivek Jain Mentor: Dr. Guenter W. Gross

2. Acknowledgement I 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.

3. STATEMENT

4. Statement • 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

5. Statement (contd.) • Repeatability depends on knowledge about the effectiveness of a stimulation pulse or a pulse train.

6. BACKGROUND

7. Terminology • MEAs: micro-electrode arrays • MMEPs (multi micro-electrode plates), for culturing, stimulating and recording neuronal activity, spikes and action potentials of individual axons neurons

8. MMEPs in use in CNNS * ITO Electrodes photo-etched on Glass Plates

9. Definitions Biphasic = 2 phases • Biphasic Monopolar Stimulation Only one microelectrode provides the biphasic pulse relative to ground (i.e. Monopolar)

10. Definitions (contd.) • Stimulation Efficiency: • Gross et al (1993), Shahaf et al (2001) use efficiency as the Response/Stimulus ratio: Efficiency = (# of Response Pulses) (# of Stimulation Pulses)

11. Definitions (contd.) • 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

12. Definitions (contd.) Entrainment: the process by which an extrinsic stimulus changes the phase of a spontaneous event cycle Left: at 30 trains/min, the network gets entrained to the stimulus pulse train Gross et al, 1993

13. Pulses 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

14. Sample Stimulation Pulse Train (on oscilloscope) Gross et al, 1993

15. Repetitive Pulses

16. Characteristics 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)

17. Normal Stimulation Domain Gross et al, 1993

18. Limitations of methodology • 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

19. Large cells near electrode craters(Morphological cell-electrode coupling) Gross et al., 1993

20. RESEARCH OBJECTIVE

21. Research Objective To characterize stimulation efficiency/entrainment as a function of the following variables: • Frequency (pulses/trains) • Pulse durations • # of pulses/trains Significance: • 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.

22. RESEARCH METHODOLOGY

23. Research Methodology • Use MMEP-4 • Cultures will be provided by the CNNS staff * Recording Matrix Electrodes

24. Stimulation 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.

25. Cristopher Sparks, PhD Dissertation UNT, 2001

26. 8 x 8 recording matrix (MMEP 4) Response Sites Stimulation Sites Micro electrodes at different separations

27. PRELIMINARY RESULTS

28. Experiment Protocol • Used 38 day old, high density, Frontal Cortex culture • Was treated with 8 μL of 10 mM bicuculine • 28 active units were found

29. 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

30. 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

31. Early Results • The network response showed a steady decline as the frequency was increased for both Pulse Train and the Single Pulse stimulations

32. Early Results (contd.)

33. 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!

34. Early Results (contd.) The thick curves show the averages of individual responses in each of the two ‘colonies’ of cells

35. Early Results (contd.) 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

36. Early Results (contd.) Site 2: The black one is a 4th Order curve, while the pink is an exponential decay curve

37. Early Results (contd.) 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.

38. Early Results (contd.) Site 4: The black one is a 4th Order curve, while the pink is an exponential decay curve

39. Analysis • 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

40. 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

41. Stability of culture • Native Activity Graphs:

42. Sample Bursting (Raster Display) 4 Units on a single DSP Native Bursts Sorted Units

43. Near futurue • Culture left for 3 days Hmm… Hypothesis for the next experiment??

44. 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 

45. REFERENCES

46. References Bove, 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

47. References Manevitz, 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