ULTRASOUND EXCITATION OF NEURONAL CULTURES · 2019. 3. 5. · 1.6 Group1: with PTZ and FUS, Group2: with PTZ, without FUS. Baseline EEG peaks, pre FUS, post FUS stimulation 1, post

ULTRASOUND EXCITATION OF NEURONAL

CULTURES

A Thesis

Submitted For the Degree of

Master of Technology

in the Faculty of Engineering

by

Mahek Vijaykumar Mehta

Center for Nano Science and Engineering

Indian Institute of Science

BANGALORE – 560 012

JUNE 2016

Declaration

I Mahek Mehta, hereby declare that the interdisciplinary work reported in this thesis

has been carried out in the Centre for Nano Science and Engineering(CeNSE), under the

joint guidance of Prof Bharadwaj Amrutur(EC) and Prof Sujit K Sikdar(MBU). I also

declare that this work does not form the basis for the award of any Degree, Diploma,

Fellowship, Associateship or similar title of any University or Institution.

Place: Bangalore

Date:

Mahek Vijaykumar Mehta

i

Acknowledgements

Firstly, I would like to thank my advisors Prof. Bharadwaj Amrutur (EC) for guiding

me through this interdisciplinary project and Prof. Sujit K Sikdar (MBU) for taking

regular and active interest in my progress.

I would like to thank my lab mates: Jude, Saumitra for helping me with the equipments

and Zubin and Grace for their assistance with the neuronal cultures.

I would also like to acknowledge the help and suggestions I received regarding Ultrasound

setup from Ajay, Dhananjay, and Irfan from RP’s lab, Deepak and Karthik from EC,

Debeyan from IAP and Manoj from the systems lab.

This would not have been possible without wonderful facilities provided by CeNSE, IISc,

and fundings from the Govt. of India.

ii

Abstract

Ultrasound has been shown to be able to modulate the nervous system activity. Non-

invasive brain stimulation with sub-millimeter resolution is possible using high-frequency

focused ultrasound.

This study tries to characterize the effect of ultrasound of different amplitudes and

frequencies on in-vitro neuronal cultures. 40kHz transducer has been shown to increase

the network activity with 121mW/cm2 intensity of the ultrasound in air. 450kHz and

690kHz transducers have been used, but their ultrasound intensity was not sufficient to

induce any response from the network.

iii

Contents

Declaration i

Acknowledgements ii

Abstract iii

Notation and Abbreviations ix

1 Introduction 11.1 Neurons and Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1.1 Anatomy of a Neuron . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.2 Excitable Membrane and Action Potentials . . . . . . . . . . . . . 21.1.3 Ion Channel Structure . . . . . . . . . . . . . . . . . . . . . . . . 31.1.4 Extracellular Field Potential . . . . . . . . . . . . . . . . . . . . . 41.1.5 Effect of Temperature . . . . . . . . . . . . . . . . . . . . . . . . 5

1.2 Introduction to Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . 51.2.1 Ultrasound Propagation . . . . . . . . . . . . . . . . . . . . . . . 61.2.2 Acoustic Waveforms . . . . . . . . . . . . . . . . . . . . . . . . . 71.2.3 Acoustic Impedance and Propagation through Interface . . . . . . 71.2.4 Piezoelectricity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.2.5 Ultrasound Safety Parameters . . . . . . . . . . . . . . . . . . . . 8

1.3 Past Studies showing the Ultrasound effects on the Nervous System . . . 91.3.1 Non-thermal Effects . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.4 Goal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2 Materials and Methods 132.1 Neuronal Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.2 MEA and Recording System . . . . . . . . . . . . . . . . . . . . . . . . . 13

2.2.1 Recording System . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.3 Spike Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.4 US Transducers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.5 US Stimulation Generator . . . . . . . . . . . . . . . . . . . . . . . . . . 162.6 Block Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.7 Temperature Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . 17

iv

CONTENTS v

2.8 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3 Results and Discussions 183.1 Stimulation from 400ST/R160 40kHz transducer . . . . . . . . . . . . . . 183.2 Stimulation from 450kHz transducer . . . . . . . . . . . . . . . . . . . . 253.3 Stimulation from 690kHz transducer . . . . . . . . . . . . . . . . . . . . 33

4 Conclusion 37

A Procedure for Preparing Neuronal Culture 38

B Technical Specifications 40B.1 US transducer 40k . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40B.2 STEMINC Piezoelectric disk . . . . . . . . . . . . . . . . . . . . . . . . . 41

References 43

List of Tables

1.1 Ultrasound safety guidelines for diagnostic and imaging purpose: Param-eters with maximum permissible values[11] . . . . . . . . . . . . . . . . . 8

B.1 40kHz US transducer technical specifications . . . . . . . . . . . . . . . . 40B.2 450kHz piezoelectric disk (SMD05T04R111WL) technical specifications . 41B.3 690kHz piezoelectric disk (SMD20T3R111) technical specifications . . . . 41

vi

List of Figures

1.1 Cartoon explaining structure of a neuron, Source: opensource image, Au-thor: LadyofHats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Excitable Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Ion channel structure (1) channel domains (2) outer vestibule (3) selec-

tivity filter (4) filter diameter (5) phosphorylation site (6) cell membrane.Source:public domain, Author: Outslider . . . . . . . . . . . . . . . . . . 4

1.4 Extracellular Field Potentials . . . . . . . . . . . . . . . . . . . . . . . . 51.5 Acoustic waveform: Pulsed and continuous . . . . . . . . . . . . . . . . . 71.6 Group1: with PTZ and FUS, Group2: with PTZ, without FUS. Baseline

EEG peaks, pre FUS, post FUS stimulation 1, post FUS stimulation2. [7] 91.7 (a) Visually evoked response, pre and post sonification, (b) post sonifi-

cation VEP amplitude with time (c) motor response after 1s stimulationof rabbit somatomotor region, (d) zoomed in version of the response (e)response for the case where a stimulus is given 2mm caudal to the somato-motor region. [8] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.8 one minute recording from 14 electrodes before and 10s after sonification:spiking rate, spikes/min [3] . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1.9 Response of M1 to tFUS: (a) (top) raw(black) and average(grey) USevoked MUA(Multi Unit Array) recording. TTX reduced the response.(bottom) raw and average Localized Field Potentials from M1. (b) peakEMG normalized response for different US intensities and frequencies [9] 12

2.1 Neuronal culture grown on MEA, Source: Neuroelectronics Lab, CeNSE . 132.2 120MEA200/30iR-Ti MEA from Multichannel systems, Germany . . . . 142.3 Ultrasound Transducers . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.4 Ultrasound Stimulation Generator . . . . . . . . . . . . . . . . . . . . . . 162.5 Ultrasound Stimulation System Block Diagram . . . . . . . . . . . . . . 17

3.1 Interference noise on blank (without culture) MEA from 40kHz trans-ducer: (top) total number of spikes detected from the network (bin size= 1s) (bottom) color-map showing spikes on each electrode with time . . 19

vii

LIST OF FIGURES viii

3.2 Recordings from the Culture40A: Total number of spikes detected andcolor map showing spikes on each electrode with time (bin size = 1s) (toprow) without any stimulus (spontaneous network activity) (bottom row)with 40kHz, 10Vp US stimulus . . . . . . . . . . . . . . . . . . . . . . . . 20

3.3 Recordings from the Culture40B: Total number of spikes detected (binsize = 1s)and color map showing spikes on each electrode with time . . . 21

3.4 Recording1 from the Culture40B, electrode 91 . . . . . . . . . . . . . . . 213.5 Recording2 from the Culture40B . . . . . . . . . . . . . . . . . . . . . . 223.6 Recording3 from the Culture40B, electrode 91 . . . . . . . . . . . . . . . 233.7 Recordings from the Culture40C: Total number of spikes detected (1s bin)

and color map showing spikes on each electrode with time . . . . . . . . 243.8 Measured peak-to-peak voltage of the receiver 450kHz piezoelectric disk

with frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.9 Temperature rise in the recording MEA fluid due to the heating of 450kHz

piezoelectric disk under excitation . . . . . . . . . . . . . . . . . . . . . . 263.10 Culture450A recordings: Mean spiking activity before, during and after

piezoelectric disk excitation . . . . . . . . . . . . . . . . . . . . . . . . . 273.11 Recordings from the Culture450A: Total number of spikes detected (1s

bin) and color map showing spikes on each electrode with time (top row)without any stimulus (spontaneous network activity) (bottom row) with426kHz, 9Vp US stimulus . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.12 Culture450A: normalized mean spike shapes from different recordings,with and without 426kHz, 9Vp US stimulus . . . . . . . . . . . . . . . . 29

3.13 Culture450B: (top) Total number of spikes detected (1s bin) and (middle)color map showing spikes on each electrode with time (bottom) normalizedmean spike shape with and without stimulus . . . . . . . . . . . . . . . . 30

3.14 Culture450C, 9Vp Pulsed US stimulus: (top) total number of networkspikes (bin size = 1s) (bottom) color map of the same, for each individualelectrode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.15 Culture450D, different pulsed US stimuli: (1st row) total number of net-work spikes (bin size = 1s) (2nd row) color map of the same, for eachindividual electrode (3rd row) comparison of US and non US spike on anelectrode (4th row) different spike clusters . . . . . . . . . . . . . . . . . 32

3.16 Measured peak-to-peak voltage of the receiver 690kHz piezoelectric diskwith frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3.17 Culture690A, 9Vp continuous sinusoidal US stimulus: (left) recording1(right) recording2, total number of network spikes (bin size = 1s) . . . . 34

3.18 Culture690B, 20Vpp pulsed (7% duty cycle, 30ms period) stimulus . . . . 353.19 Culture690C, 100Vpp pulsed (7% duty cycle, 30ms period) stimulus . . . 36

B.1 Measured Impedance of 40kHz US transducer with frequency . . . . . . 40B.2 Measured Impedance of Piezoelectric disks with frequency: (a) 450kHz

Piezo disk (b) 690kHz piezo disk . . . . . . . . . . . . . . . . . . . . . . . 42

Notation and Abbreviations

Abbreviations Details

AP Action Potential

FUS Focused Ultrasound

HIFU High Intensity Focused Ultrasound

LIFU Low Intensity Focused Ultrasound

MEA Micro Electrode Array

PA Pulse Average

PRF Pulse Repetition Frequency

SA Spatial Average

SP Spatial Peak

TA Temporal Average

tFUS Transcranial Focused Ultrasound

TP Temporal Peak

US Ultrasound

Notation Details

ISPPA Spatial Peak Pulse Average Intensity

ISPTA Spatial Peak Temporal Average Intensity

ITA Temporal Average Intensity

D Duty Cycle

ix

Chapter 1

Introduction

1.1 Neurons and Culture

1.1.1 Anatomy of a Neuron

Figure 1.1: Cartoon explaining structure of a neuron, Source: opensource image, Author:LadyofHats

Neurons are special kind of cells in the nervous system, which process and transmit the

signals. They are electrically excitable and communicate with the neighbors primarily

1

Chapter 1. Introduction 2

by firing an electrical impulse called Action Potential (AP). By interconnecting with

each-other, they form neural networks. A typical neuron receives signals from other

neurons through dendrites and transmits the signal through axon. Axon connects to the

dendrites of another neuron through a point connection called synapse.

1.1.2 Excitable Membrane and Action Potentials

Cell walls of mammalian cells are made of a lipid bilayer, separating intracellular matrix

from the extracellular fluid. Cell walls of neurons (cell-body (soma), axons and active

dendrites) have ion pumps and ion channels which regulate the flow of ions in and out of

the cell. Ion pumps maintain the concentration of ions inside the cell, which is different

from the extracellular fluid, making the cell electrically polarized, and the potential of

the inside relative to the outside is called resting membrane potential, which is generally

negative in human neurons. There are different kinds of ion channels in the membrane

which allow specific ions to pass through. Ion channels make the membrane excitable,

as their momentary opening and closing generate electric impulses.

(a) Equivalent electrical representation ofan excitable membrane [1]

(b) Action Potential and channel conduc-tances in a squid giant axon[1]

Figure 1.2: Excitable Membrane

Sodium, Potassium, and calcium are the main ions taking part in the electrical activ-

ities of the membrane. Their ion channel conductances are voltage dependent, and they

have equilibrium potentials because of the concentration gradients. The lipid bilayer is


non-conducting, and acts like a capacitor. The current through the membrane, as shown

by Hodgkin[1]:

I = CMdV

dt+ ḡKn

4(V − VK) + ḡNam3h(V − VNa) + ḡl(V − Vl)

wheredn

dt= αn(1− n)− βnn

dm

dt= αm(1−m)− βmm

dh

dt= αh(1− h)− βhh

CM : membrane capacitance,

VK , VNa, Vl: equilibrium potentials

ḡK , ḡNa, ḡl: conductance constants

n,m, h: gating variables

αi, βi = f(V, T ): gating kinetics variables

The ion channels conductances are negligible in the resting state. The input currents

from the stimuli depolarize the membrane, and as it reaches the threshold, the voltage-

dependent ion channels start conducting, causing the membrane to fire an AP. Fig 1.2

shows AP, and ion channel conductances in squid giant axon membrane. These APs

can propagate down the axon over large distances, stimulating other neurons through

synapses.

1.1.3 Ion Channel Structure

Ion channels are proteins that sit in the lipid bilayer, like water-filled tunnels, allowing

specific ions to pass. There is a selectivity filter to select which ions may pass through.

The pore opening or closing is often gated through a chemical, electrical or mechanical

signal. Voltage-gated ion channel conductivity is sensitive to the membrane voltage,


while the ligand-gated ion channels are opened by neurotransmitters in post-synapse,

and the leak channels are always open channels. The Fig 1.5 shows structure of a typical

voltage-gated channel.

Figure 1.3: Ion channel structure (1) channel domains (2) outer vestibule (3) selectiv-ity filter (4) filter diameter (5) phosphorylation site (6) cell membrane. Source:publicdomain, Author: Outslider

1.1.4 Extracellular Field Potential

Extracellular fluid outside the membrane is considered to be a resistor. At a given time, a

region of the membrane that is negative inside is positive outside, which is called source,

while the region that is positive inside is called sink. Extracellular currents move from

source to sink, and the polarity of the recorded voltage from outside of the cell depends

on where it is placed; near source or sink. As the current flows through a closed path,

the extracellular current is equal in magnitude to the membrane current from sink to

source.

Vext ∝ Im ∝dVmdt

hence, the time duration of the extracellular spike is similar to that of membrane AP,

and the shape is approximately proportional to the first derivative of membrane voltage.


(a) neurons near electrodes (b) extracellular spike examples

Figure 1.4: Extracellular Field Potentials

1.1.5 Effect of Temperature

The rate coefficients of the gating factors are Arrhenius in nature and depend on tem-

perature. As the temperature increases, the rate coefficients also increase. At higher

temperatures, the activation of sodium and potassium channels and the deactivation of

the sodium channels become faster, hence, the rising and falling edge of the AP become

sharper, and the amplitude reduces.

1.2 Introduction to Ultrasound

Ultrasound is an acoustic wave with a frequency above human audible range (>20kHz).

If the particles oscillate in the direction of the energy propagation, the wave is called

longitudinal and if they oscillate in a direction perpendicular to the direction of propa-

gation, the wave is transverse. Ultrasound can propagate as longitudinal or transverse

wave in solids while it propagates only as longitudinal wave in fluids.


1.2.1 Ultrasound Propagation

Ultrasound is a pressure wave in the medium. The relation between the pressure ampli-

tude, particle displacement, and the US intensity as described in [2]:

Wave equation for one dimensional sound propagation with sound velocity c, and the

particle displacement ξ:∂2ξ

∂t2= c2

∂2ξ

∂x2

Solution for the particle displacement:

ξ(x, t) = ξ0cos(ωt− kx)

where, ξ0: particle displacement amplitude

ω: angular frequency

k: wave number.

c = dωdk

The pressure p(x, t) at a given point is related to the displacement as :

p(x, t) = −ρc2 ∂ξ∂x

The pressure, velocity and displacement amplitudes are related as:

P0 = ρ0cU0 = ρ0cωξ0

For propagating US wave, the intensity is given by:

I =ρcU0

2=P 202ρc

=ρcω2ξ20

2= 2π2ρcf 2ξ20

For wave propagation in a lossy medium with attenuation coefficient A,

ξ(x) = ξ(0)e−Ax, I(x) = I0e−2Ax


1.2.2 Acoustic Waveforms

Figure 1.5: Acoustic waveform: Pulsed and continuous

Acoustic waveforms can be pulsed or continuous waves. For pulsed waveform, the

time delay between two pulses is called pulse repetition period (T).

Pulse repetition frequency (PRF) PRF = 1/T

Duty cycle D = Ton/T

ISPTA = DISPPA

1.2.3 Acoustic Impedance and Propagation through Interface

Acoustic impedance of a medium for a planar wave : Z = ρc

For US propagating from medium 1 to 2 (normal incidence), the reflection coefficient R

at the interface is :

R =|Z2 − Z1||Z2 + Z1|

and the transmission coefficient is :

T =2|Z2||Z2 + Z1|

With incident intensity Ii, the reflected intensity is IR = IiR2 and transmitted inten-

sity is IT = IiT2Z1Z2


1.2.4 Piezoelectricity

Piezoelectric materials are dielectric crystalline materials that develop strain under ap-

plied electric field, and vice-versa. This effect is called piezoelectric effect. It can be used

to generate US, by applying a voltage of desired frequency across the piezoelectric mate-

rial. Piezoelectric coefficient (d) relates the strain to the applied voltage. Displacement

of the medium in the direction of the field is given by:

ξ = d33V, ξ0 = 0.5d33Vpp

The intensity of US generated by applying Vpp voltage:

I = 0.5π2ρcf 2d233V2pp

1.2.5 Ultrasound Safety Parameters

High-Intensity Ultrasound can cause a lesion and ablation in the tissues because of

heating and cavitation. It can also open the Blood Brain Barrier. FDA has set up some

safety guidelines on the intensity and mechanical index limits on Ultrasound stimulation

for human use, which are as follows:

Use ISPTAmW/cm2 ISPPAW/cm

2 or MIPeripheral Vessel 720 190 1.9Cardiac 430 94 1.9Fetal Imaging 94 190 1.9Opthalmic 17 28 0.23

Table 1.1: Ultrasound safety guidelines for diagnostic and imaging purpose: Parameterswith maximum permissible values[11]

Mechanical Index (MI) is a first order safety parameter for US, defined as:

MI =P nmax(MPa)√f(MHz)


where, f is the center frequency, and P nmax is peak negative pressure.

1.3 Past Studies showing the Ultrasound effects on

the Nervous System

Since the 1950s, people have started investigating the effects of Ultrasound (US) on the

nervous system. US has been shown to increase the network activity [3], change the

conduction velocity and the amplitude of an Action Potential (AP) [4], stimulate visual,

sensory and motor cortex. It can locally open Blood Brain Barrier [5], perform ablative

neurosurgery [6], and suppress epilepsy [7]. While many effects of US are thermal, it has

non-thermal effects on the nervous system also. As Ultrasound is a mechanical wave,

it can be used along with MRI system. FUS can be used as a non-invasive tool for

neuro-stimulation, and neurosurgery.

1.3.1 Non-thermal Effects

Min et al.[7] showed suppression of chemical induced epilepsy in rat brain, using FUS.

Epilepsy was induced by injecting pentylenetetrazol (PTZ), a GABA receptor antagonist,

Figure 1.6: Group1: with PTZ and FUS, Group2: with PTZ, without FUS. BaselineEEG peaks, pre FUS, post FUS stimulation 1, post FUS stimulation2. [7]


which increases the neural activity. Electrodes were inserted to measure the activity,

which reduced when irradiated with the focused US, as shown in the Fig. 1.6, 690kHz,

0.5ms pulse, with PRF 100Hz, and Ispta = 130mW/cm2. From Fig. 1.6, it can be seen

that application of FUS stimulation reduces the spiking activity, compared with the

group not irradiated with FUS stimulation.

Yoo et al.[8] from the same group were able to evoke a response in the somatomotor

region of rabbit, and reversibly suppress activity in Visual region, using FUS, with

temperature rise less than 0.7oC. Somato-motor stimulation: by application of 690kHz,

Isppa = 12.6W/cm2, 50% duty cycle trans-cranial FUS on the somato-motor region for a

duration more than 1s, a fore-paw movement was observed. The same stimulus did not

produce any response when targeted 2mm caudal to the motor cortex.

Figure 1.7: (a) Visually evoked response, pre and post sonification, (b) post sonificationVEP amplitude with time (c) motor response after 1s stimulation of rabbit somatomotorregion, (d) zoomed in version of the response (e) response for the case where a stimulusis given 2mm caudal to the somatomotor region. [8]

Visual response: After 7-8 s sonification of visual region under 5% duty cycle, Isppa =

6.6W/cm2 trans-cranial FUS, the visually evoked responses (VEP) were suppressed,

which recovered after 10-15 mins (Fig. 1.7). Note: Craniotomy was performed on the

rabbit skull to expose the brain to FUS without skull interference.

Massoud et al. [3] used a hippocampal culture on a 16 electrode, 0.75mm × 0.75mm

MEA (Multi Electrode Array), and stimulated with 7.75MHz, 1ms bursts of 50-100


W/cm2, at a rate of 2Hz, for one minute.

Figure 1.8: one minute recording from 14 electrodes before and 10s after sonification:spiking rate, spikes/min [3]

Post sonification, culture showed an increase in firing rate. In this experiment, MI

being less than 0.7, the possibility of cavitation is ruled out. Bulk temperature rise is

negligible, and the recordings were made after the sonification.

Tufail et al.[9] did a rather interesting experiment, where they non-invasively pro-

duced a motor response from rat, by using tFUS in-vivo on motor cortex. They used

US with frequency 0.25-0.5MHz, with Isppa = 75 − 225mW , and Ispta = 21-163 mW.

Attenuation by the skin and the skull at this frequency was less than 10%.

Electrode to measure LFPs and an MUA were inserted in M1 region of a live, anes-

thetized rat. On tFUS stimulus, MUA showed increased activity during the stimulus

(Fig. 1.9). Application of TTX to M1 reduced the tFUS evoked-response in M1, indi-

cating the necessity of sodium channels for a US-evoked motor response. The pressure at

the target was 0.1M Pa in these experiments. 72ms pulse duration of the pulsed US stim-

ulus produced 0.02oC temperature rise, while the used pulse durations were 100 times

shorter. From the 2D color plot of normalized peak EMG response to 20 different US

stimulus waveform with different intensities and frequencies (0.25, 0.35, 0.425, 0.5MHZ),

it can be inferred that higher intensities and higher frequencies produced lower spike


Figure 1.9: Response of M1 to tFUS: (a) (top) raw(black) and average(grey) US evokedMUA(Multi Unit Array) recording. TTX reduced the response. (bottom) raw andaverage Localized Field Potentials from M1. (b) peak EMG normalized response fordifferent US intensities and frequencies [9]

amplitude.

1.4 Goal

Low-Intensity Ultrasound has been shown to stimulate the neurons, generate a motor

response and modify visually evoked potential. It is non-invasive and can be nonthermal.

At low intensities, it doesn’t damage the tissues as heating and cavitation effects are

absent. At higher frequencies, focused US can have a sub-millimeter spatial resolution.

Hence, it can be used for medical applications, to stimulate the brain non-invasively.

The aim of this study is to characterize the effect of US stimulation on neuronal culture

in vitro, at different frequencies and amplitudes of US stimulation; its effect on the firing

pattern and the spike shapes at the electrodes.

Chapter 2

Materials and Methods

2.1 Neuronal Culture

Neuronal cultures were grown and maintained on MEA using procedures similar to those

described in Potter et al. in [12], and are described in the appendix.

Figure 2.1: Neuronal culture grown on MEA, Source: Neuroelectronics Lab, CeNSE

2.2 MEA and Recording System

120 channel MEA : 120MEA200/30iR-Ti MEAs from Multichannel systems, Germany,

were used to culture neurons, stimulate them and record the extracellular activity.

13

Chapter 2. Materials and Methods 14

(a) top view (b) zoomed in view of the centralrecording area

Figure 2.2: 120MEA200/30iR-Ti MEA from Multichannel systems, Germany

They have 12×12 electrode grid with 120 channels and 4 reference electrodes, with

200µm spacing. The electrodes are 30µm in diameter, made of TiN, and SiN isolators

and opaque tracks of Ti. The recording area is 2.4 ×2.4 mm2.

2.2.1 Recording System

The recording system consists of a headstage, interface board, and computer. MEA2100-

HS120 headstage was used to place MEA and record from 120 channels. The MEA

culture was maintained above a preset temperature using TC02 temperature controller,

which sensed the temperature using Pt100 temperature sensor, and provided appropriate

current to the heating element. The data was acquired at 50kHz using MCS IFB 3.0

multiboot interface board, digitally filtered and transferred to PC using a high-speed USB

cable. Headstage, interface board, heating element, temperature sensor, and controller

were purchased from Multichannel systems, Germany.


2.3 Spike Detection

The extracellular field potentials appear as voltage spikes. To extract those spikes, the

signal was first filtered using a 2nd order highpass Butterworth filter with cutoff frequency

of 500Hz. Then the spikes were detected by threshold crossing at voltage levels 5x the

standard deviation of the noise. 101 samples around the peak were stored as the spike

waveform.

2.4 US Transducers

(a) 40kHz transducer fromRobokits, India

(b) 450kHz piezoelectric disk,STEMINC, USA

(c) 690kHz piezoelectric disk,STEMINC, USA

Figure 2.3: Ultrasound Transducers

1] 40kHz air type US sensor(400ST/R160, Robokits, India). It was mounted on a

plane, suspended 1-2 mm above the MEA fluid.

2] 450kHz piezoelectric disk (SMD05T04R111WL, STEMINC, USA) was stuck under

the MEA for stimulation.

3] 690kHz piezoelectric disk (SMD20T3R111, STEMINC, USA) was coated with PDMS

silicone, and kept in direct contact with the MEA fluid for US coupling.


Figure 2.4: Ultrasound Stimulation Generator

2.5 US Stimulation Generator

The excitation sine wave, generated from the function generator, drives the input to

the NMOS power stage, that generates a square wave at the output, with the same

frequency and the amplitude equals to the power supply voltage. 555 timer is used in

the astable mode to generate millisecond pulse train. These pulses gate the output of

the power stage, and hence, the output is pulsed high frequency (100kHz-1MHz), high

voltage wave (0-100Vpp). This excitation is applied to the transducer using 100Ω series

power resistor.

2.6 Block Diagram

The cultured MEA was kept on the headstage, in the incubation chamber. The transduc-

ers were either mounted on the top of the MEA or stuck under it. US excitation signal

was generated using a function generator and a pulse generator and applied to the US

transducer using BNC cable. The data was recorded by the Interface board connected

to the MEA headstage, and processed and stored in the PC. The temperature controller

maintained the MEA temperature above the preset level.


Figure 2.5: Ultrasound Stimulation System Block Diagram

2.7 Temperature Measurement

The temperature change in the MEA fluid due to US transducer was measured by the

temperature sensor of the temperature controller HW-30, Dagan, Temperature resolution

was 0.1K

2.8 Data Analysis

Post recording data analysis like total spike count of the network, spike sorting and shape

comparison, colormap of the activity has been done using custom scripts on MATLAB.

Chapter 3

Results and Discussions

This section discusses the results of US stimulation provided to the neuronal cultures at

different excitation frequencies and power.

3.1 Stimulation from 400ST/R160 40kHz transducer

The US transducer was mount on top of the MEA such that the vibrating element was

≈2 cm above the MEA solution.

Intensity at the Culture: Measured sound pressure using a hydrophone at a dis-

tance 3.5 cm from the vibrating element excited with 8Vpp, 40kHz sine wave: 296Pa

P (2cm, 10VP ) = 296(3.52

)(108

) = 647Pa

The intensity incident on the fluid surface: Ii =P 2

ρc= 647

2

346W/m2 = 121mW/cm2

Intensity transmitted: IT = IiT2Z1Z2

= 121(4)( 3461000×1500)mW/cm

2 = 111µW/cm2

Hence, for 10Vp excitation of the transducer, the intensity of US at the neuronal

culture: 111µW/cm2

Noise: The transducer excitation above 20kHz did not affect the MEA recordings, as

the interference noise was below the spike detection threshold. The fig. 3.1 shows

that continuous wave excitation of the transducer at 40kHz, 10Vp doesn’t cause

any interference with the MEA (filled with fluid) recording.

18

Chapter 3. Results and Discussions 19

0 50 100 150 200 2500

500

1000

1500

2000

2500

time (s)

tota

l spi

kes

dete

cted

15kHz, 20Vpp stimulation

(a) 20Vpp excitation at 15kHz

0 50 100 150 200 250 3000

100

200

300

400

500

600

time (s)to

tal s

pike

s de

tect

ed


(b) 20Vpp excitation at 17kHz

0 50 100 150 200 250 3000

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

time (s)

tota

l spi

kes

dete

cted


(c) 20Vpp excitation at 40kHz

Figure 3.1: Interference noise on blank (without culture) MEA from 40kHz transducer:(top) total number of spikes detected from the network (bin size = 1s) (bottom) color-map showing spikes on each electrode with time

Temperature Rise: No temperature rise was measured due to the excitation of the

US transducer.

Stimulating Culture40A

A high neuron density culture was selected, and filled with 500µl of DMEM solution. It

was recorded 5 times for 271s duration.

From the Fig 3.2, it can be seen that with high enough spontaneous activity and a large

amount of fluid (2-3 mm high column), the effects of the stimulus are not visible.

Stimulating Culture40B

A low neuron density culture with a low amount of fluid (unknown amount; regular

fluid with 2 hours of evaporation) was stimulated with US, and the correlated increased

activity can be seen in the Fig. 3.3. The third recording was taken in the absence of any

stimulus, which doesn’t show any increase in the activity.

Fig. 3.4 shows the mean spike shape with and without US stimulation, from the


0 50 100 150 200 250 3000

200

400

600

800

1000

1200

1400

1600

1800

2000

time (s)

tota

l spi

ke c

ount

Trial1 control

spike countspike count smoothedUS stimulation duration

(a) Recording1, control

0 50 100 150 200 250 3000

200

400

600

800

1000

1200

1400

1600

1800

2000

time (s)

tota

l spi

ke c

ount

Trial3 control


(b) Recording2, control

time

ele

ctr

ode#

0

0.5

1

time

ele

ctr

ode#

0

0.5

1

0 50 100 150 200 250 3000

200

400

600

800

1000

1200

1400

1600

1800

2000

time (s)

tota

l spi

ke c

ount

Trial2 90:260


(c) Recording3, US stimulation

0 50 100 150 200 250 3000

200

400

600

800

1000

1200

1400

1600

1800

2000

time (s)

tota

l spi

ke c

ount

Trial4 stim


(d) Recording4, US stimulation

time

ele

ctr

ode#

0

0.5

1

time

ele

ctr

ode#

0

0.5

1

Figure 3.2: Recordings from the Culture40A: Total number of spikes detected and colormap showing spikes on each electrode with time (bin size = 1s) (top row) without anystimulus (spontaneous network activity) (bottom row) with 40kHz, 10Vp US stimulus


0 50 100 150 200 250 3000

10

20

30

40

50

60

time (s)

tota

l spi

ke c

ount

Trial9 50:150


(a) Recording1, US stimulus

0 50 100 150 200 250 3000

10

20

30

40

50

60

70

time (s)

tota

l spi

ke c

ount

Trial10 50:110


(b) Recording 2, US stimulus

0 50 100 150 200 250 3000

10

20

30

40

50

60

time (s)

tota

l spi

ke c

ount

Trial11 control


(c) Recording 3, control

time

ele

ctr

ode#

0

0.5

1

time

ele

ctr

ode#

0

0.5

1

timeele

ctr

ode#

0

0.5

1

Figure 3.3: Recordings from the Culture40B: Total number of spikes detected (bin size= 1s)and color map showing spikes on each electrode with time

0 20 40 60 80 100 120-80

-60

-40

-20

0

20

40

60USt: 50:150, number of US spikes: 1991, and non US spikes: 581

time (0.02ms)

volta

ge ( µ

V)

non USUS

(a) average spike shape with and withoutUS stimulation

(b) spike clustering along two principaleigenvectors (basis vectors)

Figure 3.4: Recording1 from the Culture40B, electrode 91


record 1, electrode 91. The shapes were similar, and both of them (with and without

US) fell under similar clusters. Similar observation was made for electrode 92 also.

0 20 40 60 80 100 120-80

-60

-40

-20

0

20

40


time (0.02ms)

volta

ge ( µ

V)

non USUS

(a) electrode 91, average spike shape withand without US stimulation

(b) electrode 91, spike clustering alongtwo principal eigenvectors (basis vectors)

0 20 40 60 80 100 120-80

-60

-40

-20

0

20

40


time (0.02ms)

volta

ge ( µ

V)

non USUS

(c) electrode 99, average spike shape withand without US stimulation

-350 -300 -250 -200 -150 -100 -50 0 50-100

-80

-60

-40

-20

0

20

40

60spike clustering: o: control, *: US stimulation spike

(d) electrode 99, spike clustering alongtwo principal eigenvectors (basis vectors)

Figure 3.5: Recording2 from the Culture40B

Recording 2 from the CultureB was interesting. Electrode 91 showed similar spike

clusters and spike shapes for both; US stimulation and control activity, while electrode

99 was active for a short time during US stimulus, with one type of spikes similar to the

control spike shape, while one more cluster was present that was different (Fig. 3.5).

Recording 3 from the CultureB was the control activity. Electrode 91 (and 92, not

shown) showed spike shape similar to those in recordings 1 and 2. Notably, all the spikes

of the electrodes were very similar, falling under one cluster (Fig. 3.6).


0 20 40 60 80 100 120-60

-50

-40

-30

-20

-10

0

10


time (0.02ms)

volta

ge ( µ

V)

non USUS

(a) average spike shape with and withoutUS stimulation

-10000 -8000 -6000 -4000 -2000 0 2000-3000

-2500

-2000

-1500

-1000

-500

0

500

1000

1500


(b) spike clustering along two principaleigenvectors

Figure 3.6: Recording3 from the Culture40B, electrode 91

Stimulating Culture40C

Culture40C was also a low-density culture, with only 200µl(


0 50 100 150 200 250 3000

10

20

30

40

50

60

70Trial4 stim 70:170

spike countsmoothed spike countUS stimulus duration


0 50 100 150 200 250 3000

50

100

150

200

250Trial5 90:190


(b) Recording2, US stimulus

time

ele

ctr

ode#

0

0.2

0.4

0.6

0.8

1

time

ele

ctr

ode#

0

0.5

1

0 20 40 60 80 100 120-80

-60

-40

-20

0

20


time (0.02ms)

volta

ge ( µ

V)

non USUS

(c) Recording1, electrode 2: aver-age spike shape with and without USstimulation

-100 -80 -60 -40 -20 0 20 40 60 80-60

-40

-20

0

20

40

60


(d) Recording1, electrode 2:spike clustering along two principaleigenvectors

0 20 40 60 80 100 120-40

-30

-20

-10

0

10


time (0.02ms)

volta

ge ( µ

V)

non USUS

(e) Recording2, electrode 90: aver-age spike shape with and without USstimulation

-100 -80 -60 -40 -20 0 20 40 60 80 100-100

-80

-60

-40

-20

0

20

40


(f) Recording2,electrode 90: spike clustering alongtwo principal eigenvectors

Figure 3.7: Recordings from the Culture40C: Total number of spikes detected (1s bin)and color map showing spikes on each electrode with time


3.2 Stimulation from 450kHz transducer

This US transducer was stuck under MEA with Fevicol adhesive.

350 400 450 500 550 600 650 700 750 8000

0.5

1

1.5

2

2.5

3

3.5

frequency (kHz)

Vol

tage

pea

k-to

-pea

k (V

)

450kHz piezoelectric disk frequency response peaks

450kHz voltage peaksglass transmission

(a) 20Vpp continuous sine wave excitation

100 150 200 250 300 350 400 450 500 550 6000

1

2

3

4

5

6

7X= 442Y= 7

frequency (kHz)

Vol

tage

pea

k-to

-pea

k (V

)

450kHz disk frequency response peaks (Pulsed Signal

(b) 45Vpp pulsed sine wave excitation

Figure 3.8: Measured peak-to-peak voltage of the receiver 450kHz piezoelectric disk withfrequency

Intensity at the Culture: The transducer was stuck under the MEA (1mm thick

glass), and a similar piezoelectric disk was stuck on the top of the glass (in the well

of a test MEA), to measure the received US intensity. US Intensity at the culture

can be assumed to be the same as that is measured by the piezoelectric transducer

at its place, for back of the envelope calculation. The expression for converting the

received peak-to-peak voltage to the intensity is:

I =2π2f 2ρc(0.5d31Vpp)

2

kp

I =2π2(4.5× 105)2 × 7900× 2500× (0.5× 140Vpp × 10−12)2

0.58W/m2

I = 0.669(Vpp1V

2

)W/m2 = 0.067(Vpp1V

2

)mW/cm2

For 3.3Vpp received signal, I = 0.66mW/cm2. For the peak input voltage (80Vpp),

the received signal was 13Vpp, and the intensity I = 11.3mW/cm2. Hence, this


piezoelectric disk could provide 0.66mW/cm2 intensity US with continuous wave,

or upto ISPPA =11.3mW/cm2, with pulsed US stimulation.

Noise: No interference noise was registered in the recording during continuous wave

stimulation of the disk at 442kHz, 20Vpp, while pulsed stimulation above 45Vpp

interfered with the recording and registered false spikes (not the extracellular field

potentials).

Temperature Rise: Pulsed (7% duty cycle) sine wave (80Vpp) excitation to the 450kHz

piezoelectric disk produced ∆T = 1.2oC (24.6oC-25.8oC) at the fluid. The tem-

perature rise in the fluid because of 20Vpp continuous sine wave excitation to the

piezoelectric disk was significant (∆T = 4oC). The temperature rise with time has

been shown in the Fig. 3.9.

0 5 10 15 20 25 30 35 40 45 5026

26.5

27

27.5

28

28.5

29

29.5

30

30.5

time (s)

Tem

pera

ture

T(o C

)

450kHz piezoelectric disk induced temperature rise

9Vpp15Vpp18Vpp

Figure 3.9: Temperature rise in the recording MEA fluid due to the heating of 450kHzpiezoelectric disk under excitation


Mean activity recorded from the CultureA before, during and after the US stimulation is

shown in the Fig.3.10. Recordings 1,2 and 9 were made in the absence of any stimulation.

Rest of them were made with 426kHz, 20Vpp US stimulation.


1 2 3 4 5 6 7 8 9 10 11 120

50

100

150

200

250

300

350

400

450

#recording

mea

n nu

mbe

r of n

/w s

pike

s

Culture450A recording

before USUSpost US

Figure 3.10: Culture450A recordings: Mean spiking activity before, during and afterpiezoelectric disk excitation

Recordings 1,9,3 and 4 are shown in the Fig.3.11. During these recordings, the spikes

on the electrodes were similar in shape and fell in similar clusters (data not shown). It

should be noted from the Fig. 3.12, which shows the normalized mean spike shape with

and without the stimulus for different electrodes, that the spikes during the stimulus

were sharper.


Culture450B compares the response of MEA to the US stimulation, with thermal stim-

ulation (increasing the temperature by similar amount as it increases during the US

stimulation.

Fig. 3.13 shows three recordings from the Culture450B: (left) only US stimulus,

(center) control, (right) only thermal stimulation. Normalized mean spike shapes were

similar in all the three recordings. Even though the stimuli for the recording 1 and 3

were different, they showed similar rise time (time to reach peak activity, after stimulus

onset) and fall time (time to reach the baseline activity, after the stimulus turn-off). The


0 50 100 150 200 250 3000

50

100

150

200

250

300

350

400

450

time (s)

tota

l spi

ke c

ount

Trial1


(a) Recording1, control

0 50 100 150 200 250 3000

100

200

300

400

500

600

time (s)

tota

l spi

ke c

ount

Trial9 control



time

ele

ctr

ode#

0

0.5

1

time

ele

ctr

ode#

0

0.5

1

0 50 100 150 200 250 3000

100

200

300

400

500

600

700

800

900

1000

time (s)

tota

l spi

ke c

ount

Trial3 (60:170)


(c) Recording3, US stimulus

0 50 100 150 200 250 3000

100

200

300

400

500

600

700

800

900

time (s)

tota

l spi

ke c

ount

Trial4 (50:170)


(d) Recording4, US stimulus

time

ele

ctr

ode#

0

0.5

1

time

ele

ctr

ode#

0

0.5

1

Figure 3.11: Recordings from the Culture450A: Total number of spikes detected (1s bin)and color map showing spikes on each electrode with time (top row) without any stimulus(spontaneous network activity) (bottom row) with 426kHz, 9Vp US stimulus


0 20 40 60 80 100 120-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial3 (60:170)

ControlUS

0 20 40 60 80 100 120-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial4 (50:170)

ControlUS

0 20 40 60 80 100 120-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial5 (70:260)

ControlUS

0 20 40 60 80 100 120-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial6 (100:210)

ControlUS

0 20 40 60 80 100 120-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial8 (100:160)

ControlUS

0 20 40 60 80 100 120-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial11 (100:200)

ControlUS

Figure 3.12: Culture450A: normalized mean spike shapes from different recordings, withand without 426kHz, 9Vp US stimulus

temperature rise was similar in both the cases.


Culture450C was stimulated with pulsed US excitation of 20Vpp, 7% duty cycle. The low

voltage was one third of the high voltage. It did not produce any temperature change in

the MEA.

Pulsed US stimulus (Ton =2 ms, T = 30 ms) showed no change in the network activity.

While the temperature rise was negligible, US intensity was comparable to the previous

culture stimuli, though for a shorter time.

Stimulating Culture450D

This culture was stimulated with different frequencies and amplitudes of pulsed US

stimulus. The temperature rise was negligible. Three recordings are shown in Fig. 3.15.

Recording1 was made during pulsed US stimulus of 45Vpp, which showed no change in


0 50 100 150 200 250 3000

50

100

150

200

250

300

350

400

time (s)

tota

l spi

ke c

ount

Trial2 100:170, 32.9oC, pulsing US


(a) Recording1, 418kHz, 20VppUS stimulus

0 50 100 150 200 250 3000

50

100

150

200

250

time (s)

tota

l spi

ke c

ount

Trial3 control



0 50 100 150 200 250 3000

50

100

150

200

250

300

350

400

450

time (s)

tota

l spi

ke c

ount

Trial4 (100:170,...) 38oC thermal


(c) Recording1, thermal step,∆T = 6oC, from 32oC − 38oC

time

ele

ctr

ode#

0

0.5

1

time

ele

ctr

ode#

0

0.5

1

time

ele

ctr

ode#

0

0.5

1

0 20 40 60 80 100 120-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial2 100:170

ControlUS

0 20 40 60 80 100 120-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial3 control

ControlUS

0 20 40 60 80 100 120-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1Trial4 (100:170),... thermal

ControlUS

Figure 3.13: Culture450B: (top) Total number of spikes detected (1s bin) and (middle)color map showing spikes on each electrode with time (bottom) normalized mean spikeshape with and without stimulus


0 50 100 150 200 250 3000

20

40

60

80

100

120

140

160

180

200Trial4 (50:100 150:200,...) 33.9oC


0 50 100 150 200 250 3000

20

40

60

80

100

120

140

160

180

200Trial5 440kHz (50:100 20Vpp/7Vpp 150:200 10Vpp/4Vpp) 34.3oC

(b) Recording2, US stimulus

time

elec

trode

#

0

0.5

1

timeel

ectro

de#

0

0.5

1

Figure 3.14: Culture450C, 9Vp Pulsed US stimulus: (top) total number of network spikes(bin size = 1s) (bottom) color map of the same, for each individual electrode

the network activity. The spike shapes with and without stimulus were similar. Record-

ings 2 and 3 showed an increase in the activity with the stimulus (60,50,80 Vpp), while

their spike shapes changed from that of the control activity. As shown in the Fig.3.15,

there were spikes only during US stimulus on that particular electrode (110). Recording

3 spike shape with and without US stimulus were different. The stimulus spike shapes

during the recordings 2 and 3 were noise artifacts, which was confirmed by recording on

a blank MEA, with a similar stimulus.

To summarize, 450kHz, 20Vpp continuous sine wave produced temperature rise and

increased activity (Culture450A). Similar increase in the activity was produced by tem-

perature stimulus, without US (Culture450B). US stimulus with similar intensity but

lower duty cycle could not increase the temperature of the MEA, but also didn’t induce

any increase in the activity (Culture450C). The Pulsed US with higher voltages (up

to 45Vpp) didn’t increase the activity, but higher voltages started to interfere with the

recording (Culture450D). The spike sharpening observed in Culture450B can also be ex-

plained by increasing temperature. Higher temperature increases the kinetic coefficients


0 50 100 150 200 250 3000

20

40

60

80

100

120

140

160

180

200

time (s)

tota

l spi

ke c

ount

Trial4 450kHz 45Vpp 2ms pulses of 30ms period 53:70, 150:170 37oC


(a) Recording1, 45Vpp stimulus

0 50 100 150 200 250 3000

50

100

150

200

250

time (s)

tota

l spi

ke c

ount

Trial5 441kHz, 60Vpp,2ms pulses of 30ms period,37oC, 50:100, 150:200


(b) Recording2, 60Vpp stimulus

0 50 100 150 200 250 3000

100

200

300

400

500

600

time (s)

tota

l spi

ke c

ount

Trial7 34oC, 2ms pulses of 30ms period, 50:100, 418kHz, 50Vpp, 150:200, 80Vpp, 441kHz


(c) Recording3, 50Vpp, 80Vppstimulus

time

elec

trode

#

0

0.5

1

time

elec

trode

#

0

0.5

1

time

elec

trode

#

0

0.5

1

0 20 40 60 80 100 120-80

-60

-40

-20

0

20

40


time (0.02ms)

volta

ge ( µ

V)

non USUS

(d) Recording1, electrode 103

0 20 40 60 80 100 120-10

-5

0

5

10

15

20

25


time (0.02ms)

volta

ge ( µ

V)

non USUS

(e) Recording2, electrode 117

0 20 40 60 80 100 120-40

-30

-20

-10

0

10

20


time (0.02ms)

volta

ge ( µ

V)

non USUS

(f) Recording3, electrode 107

-500 -400 -300 -200 -100 0 100-150

-100

-50

0

50

100

150


-60 -40 -20 0 20 40 60-30

-20

-10

0

10

20

30

40


-100 -80 -60 -40 -20 0 20 40 60 80-50

-40

-30

-20

-10

0

10

20

30


Figure 3.15: Culture450D, different pulsed US stimuli: (1st row) total number of networkspikes (bin size = 1s) (2nd row) color map of the same, for each individual electrode (3rdrow) comparison of US and non US spike on an electrode (4th row) different spike clusters


of the gating variable dynamics. Hence, the channels open and close faster, making the

action potential sharper.

3.3 Stimulation from 690kHz transducer

690kHz piezoelectric disk coated with PDMS bio-compatible silicone was placed in direct

contact with the MEA fluid for US stimulation.

100 200 300 400 500 600 700 8000

1

2

3

4

5

6

7

8

9

10

frequency (kHz)

Vol

tage

pea

k-to

-pea

k (V

)

690kHz piezoelectric disk frequency response peaks

X= 758Y= 10

(a) 20Vpp continuous sine wave

100 200 300 400 500 600 700 8000

10

20

30

40

50

60

70

frequency (kHz)

Vol

tage

pea

k-to

-pea

k (V

)

690kHz disk frequency response peaks (Pulsed Signal)

690kHzwires directly

(b) 100Vpp pulsed sine wave

Figure 3.16: Measured peak-to-peak voltage of the receiver 690kHz piezoelectric diskwith frequency

Intensity at the Culture: The intensity of the US reaching the culture was measured

using a 690kHz piezoelectric disk similar to the transmitter, placed 5mm away, in

water. Converting the received voltage on the disk to the US intensity, as done for

450kHz disk:

I =2π2f 2ρc(0.5d33Vpp)

2

kt

I =2π2(6.9× 105)2 × 7900× 2500× (0.5× 320Vpp × 10−12)2

0.45W/m2

I = 10.56(Vpp1V

2

)W/m2 = 1.056(Vpp1V

2

)mW/cm2


For received signal of 10Vpp, I = 1.06× 100mW/cm2 = 106mW/cm2

For received signal of 67Vpp, I = 1.06 × 642 = 4341mW/cm2 = 4.3W/cm2 (the

baseline coupling was 3V). Hence, it can provide US stimulations with intensities

106mW/cm2 for continuous wave and up to 4W/cm2 for pulsed wave, during the

’on’ pulse.

Noise: Continuous wave stimulation of 20Vpp, 758kHz did not affect the MEA record-

ing, but higher pulsed voltages induced noise on the electrodes being recorded.

Temperature Rise: Temperature change in the recording medium due to 690kHz

piezoelectric disk excitation at continuous sine wave of 18Vppwas less than 1oC.

For low duty cycle (7%) pulsed US, the temperature change in the medium was

negligible. ∆T = 0.1oC, at 24oC for 758kHz, 80Vpp pulsed US excitation.


0 50 100 150 200 250 3000

50

100

150

200

250

300

350



0 50 100 150 200 250 3000

50

100

150

200

250



Figure 3.17: Culture690A, 9Vp continuous sinusoidal US stimulus: (left) recording1(right) recording2, total number of network spikes (bin size = 1s)

This culture was stimulated using 690kHz piezoelectric disk excited by 20Vpp contin-

uous 745kHz sine wave, which didn’t increase the activity of the culture (3.17), nor have

any effect on the spike shapes (not shown). Temperature rise was negligible.


0 50 100 150 200 250 3000

100

200

300

400

500

600

700

800

time (s)

tota

l spi

ke c

ount

Trial9 50:150 755k, 735k 32oC 4th June


(a) Recording1, total number of networkspikes with and without stimulus (bin size= 1s)

0 50 100 150 200 250 3000

100

200

300

400

500

600

700

800

900

1000

time (s)

tota

l spi

ke c

ount

Trial10 50:132 735k, 132:150 755k 33oC 4th June


(b) Recording2, total number of networkspikes with and without stimulus (bin size= 1s)

0 20 40 60 80 100 120-30

-25

-20

-15

-10

-5

0

5


time (0.02ms)

volta

ge ( µ

V)

non USUS

(c) Recording1, electrode 42: averagespike shape with and without stimulus

(d) Recording 1 electrode 42: spike clus-ters with and without stimulus

Figure 3.18: Culture690B, 20Vpp pulsed (7% duty cycle, 30ms period) stimulus



This culture was stimulated with low voltage (20Vpp) pulsed US. It didn’t show any

change in the network activity during the stimulus, nor the spike shapes of the individual

electrode showed any change. (Fig.3.18)


This culture was recorded with 100Vpp pulsed (30ms period, 77% duty cycle) US stimulus.

Stimulus voltage interfered with the recordings and induced a large number of false spikes

on all the electrodes (Fig. 3.19). Both noise and the action potential spikes have distinct

spike shapes. Hence, high pulsed voltage stimulation of 690kHz piezoelectric dish induced

noise.

0 50 100 150 200 250 3000

500

1000

1500

2000

2500

3000

3500

4000

time (s)

tota

l spi

ke c

ount

Trial8 34oC 758kHz, very noisy


(a) Recording: total number of net-work spikes with and without stimu-lus (bin size = 1s)

time

elec

trode

#

0

0.5

1

(b) Recording: colormap of the ac-tivity on all the electrodes

0 20 40 60 80 100 120-60

-40

-20

0

20

40

60

80

100


time (0.02ms)

volta

ge ( µ

V)

non USUS

(c) electrode 108 average spike shapewith and without stimulus

-150 -100 -50 0 50 100 150 200 250-120

-100

-80

-60

-40

-20

0

20

40

60


(d) electrode 108 spike clusters withand without stimulus

Figure 3.19: Culture690C, 100Vpp pulsed (7% duty cycle, 30ms period) stimulus

Chapter 4

Conclusion

40kHz ultrasonic transducer was able to increase the network activity with US Intensity

of 121mW/cm2 in air (111µW/cm2 < I < 121mW/cm2 at the culture) when the fluid

film covering the culture was thin enough. 450kHz piezoelectric dish with US intensity

0.66mW/cm2 heated the MEA and increased the activity, but the increase in the activity

was thermally induced because a temperature stimulation could cause the same effect

while US stimulus of the same intensity, but lower duty cycle couldn’t. Also, the spikes

were narrower than the control, which can be caused be increased temperature. 650kHz

piezoelectric dish with US intensity 100mW/cm2 (continuous or pulsed) could not change

the network activity, as the intensity may be insufficient to stimulate the culture, while

121 mW/cm2 was sufficient at 40kHz, As I ∝ P 2 ∝ ξ2ω2, higher frequency US may

require more intensity to cause the same amount of particle displacement as the lower

frequency US.

The attempts to increase the intensity of the US by increasing the pulse voltage amplitude

were met with increased noise and/or increased temperature. To get significant and

reliable US effects on the culture activity, the intensity should be increased without

increasing the temperature. This could be achieved by keeping the US source at a

distance, and using water coupling or waveguide coupling. Air interface should be avoided

as it significantly reflects the US. If the stimulation has to be provided through air

medium, then high power, focused US transducer should be used.

37

Appendix A

Procedure for Preparing Neuronal

Culture

Whole hippocampus of 0-2 day old rat pup was digested in Papain, and the dissoci-

ated cells were seeded on MEA. For sterilization and reuse, 120 channel MEAs were

soaked overnight with Tergazyme detergent (Sigma-Aldrich, USA), thoroughly rinsed

with MilliQ water and allowed to dry under a laminar hood, sterilized with 70% ethanol

and UV light exposure. Sterilized MEA were coated with 0.05% (w/v) polyethyleneimine

solution in borate buffer, rinsed thoroughly with MilliQ water and allowed to dry and

kept under a laminar hood until cell seeding.

Wistar rats were decapitated according to approved protocols by the Animal Ethics

and Welfare Committee of Indian Institute of Science, Bangalore, India. The brain

was removed, chilled with ice PBS (Phosphate Buffer Saline) and the hippocampus was

micro-dissected under sterile conditions. Papain solution was prepared according to Se-

gal et al. [14], and divided in 1.5 ml and stored at -20oC, and thawed at 37oC before

use. Hippocampus was digested in 2 ml papain solution for 20 min at 37oC stirring

manually. The papain solution was aspirated and the pieces were pulverized three times,

three passes each with 1 ml of medium, using a P-1000 Pipetman. Cells were plated on

the electrode region (2.4 × 2.4 mm2) of MEA in a 20µL containing 50,000-2,00,000 cells,

forming a dense monolayer. The MEAs were coated with laminin and incubated for 30

38

Appendix A. Procedure for Preparing Neuronal Culture 39

min just before seeding. The dishes were flooded with 1 ml of medium (adapted from

[15], Dulbecco’s modified Eagles medium with 10% FBS serum, stored in the incubator

to equilibrate the pH and temperature before feeding) after the cells had adhered to

the substrate (45 min), and stored with ethylene-propylene membrane lids (MEA-MEM

membranes, ALA Scientific Instruments Inc., USA) in a 65% RH incubator (37oC, 5%

CO2 ambient). Antibiotic drugs were used to control contamination. For feeding the

culture, 50% of the medium was replaced twice per week.

The cultured MEA were placed in a separate incubator (5% CO2 and 37oC ambient)

while recording and stimulating. Cultures started showing significant activity after 10-

15 days.

Appendix B

Technical Specifications

B.1 US transducer 40k

Impedance with different excitation frequency

0 10 20 30 40 50 60 70 800

2000

4000

6000

8000

10000

12000

14000

X: 40.24Y: 324.1

frequency (kHz)

impe

danc

e ( Ω

)

impedance vs Frequency for 40kHz transducer

Figure B.1: Measured Impedance of 40kHz US transducer with frequency

SpecificationsResonant Frequency 40kHzImpedance @ resonance 329ΩContinuous power dissipation 200mWTransmitting sound pressure 120dB, 0dB = 0.0002µbar /10Vrms, at 30cmReceiver Sensitivity -65dB, 0dB = 1V/µbar

Table B.1: 40kHz US transducer technical specifications

40

Appendix B. Technical Specifications 41

B.2 STEMINC Piezoelectric disk

SpecificationsResonant Frequency 450 ± 10 kHzDimensions 5 mm × 0.4 mmStatic Capacitance 1.05nFElectromechanical coupling coefficient (kp) 0.58Vmax 5V/mill×16mill = 80VPower Dissipation 3W/cm2 × 0.2cm2 = 0.6WPiezoelectric coefficient (d31) -140 pm/VRelative Density 7.9

Table B.2: 450kHz piezoelectric disk (SMD05T04R111WL) technical specifications

SpecificationsResonant Frequency 690 ± 21 kHzResonant Impedance (Z) < 3.6ΩDimensions 20 mm × 3 mmStatic Capacitance 1.265nFElectromechanical coupling coefficient (kt) 0.45Vmax 5V/mill×80mill = 400VPower Dissipation 3W/cm2 × 3.14cm2 = 10WPiezoelectric coefficient (d33) 320 pm/VRelative Density 7.9

Table B.3: 690kHz piezoelectric disk (SMD20T3R111) technical specifications

Appendix B. Technical Specifications 42

100 200 300 400 500 600 700 800 900 10000

1000

2000

3000

4000

X: 442Y: 343.8

450kHz transducer Impedance vs frequency

frequency (kHz)

Impe

danc

e M

agni

tude

( Ω)

100 200 300 400 500 600 700 800 900 1000-200

-100

0

100

200

Impe

danc

e P

hase

100 200 300 400 500 600 700 800 900 10000

1

2x 10

4

X: 109Y: 169

690kHz transducer Impedance vs frequency

frequency (kHz)

Impe

danc

e M

agni

tude

( Ω)

X: 757Y: 89.31

100 200 300 400 500 600 700 800 900 1000-200

0

200

Impe

danc

e P

hase

Figure B.2: Measured Impedance of Piezoelectric disks with frequency: (a) 450kHz Piezodisk (b) 690kHz piezo disk

References

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DeclarationAcknowledgementsAbstractNotation and AbbreviationsIntroductionNeurons and CultureAnatomy of a NeuronExcitable Membrane and Action PotentialsIon Channel StructureExtracellular Field PotentialEffect of Temperature

Introduction to UltrasoundUltrasound PropagationAcoustic WaveformsAcoustic Impedance and Propagation through InterfacePiezoelectricityUltrasound Safety Parameters

Past Studies showing the Ultrasound effects on the Nervous SystemNon-thermal Effects

Goal

Materials and MethodsNeuronal CultureMEA and Recording SystemRecording System

Spike DetectionUS TransducersUS Stimulation GeneratorBlock DiagramTemperature MeasurementData Analysis

Results and DiscussionsStimulation from 400ST/R160 40kHz transducerStimulation from 450kHz transducerStimulation from 690kHz transducer

ConclusionProcedure for Preparing Neuronal CultureTechnical SpecificationsUS transducer 40kSTEMINC Piezoelectric disk

References

Documents

ULTRASOUND EXCITATION OF NEURONAL CULTURES · 2019. 3. 5. · 1.6 Group1: with PTZ and FUS, Group2: with PTZ, without FUS. Baseline EEG peaks, pre FUS, post FUS stimulation 1, post