1

Comparative analysis of electrical stimulation versus biochemical stimulation in rd1 degenerated retina as potential candidates for retinal prosthesis

Thesis submitted in partial fulfillment of the requirements for the degree

Master of Science

Graduate School of Cellular & Molecular Neuroscience

Faculty of Science Faculty of Medicine

Eberhard-Karls-Universitt Tbingen

Presented by

Archana Jalligampala

from Cuttack, India

Tbingen, 26-05-2011

2

Thesis Advisor: PD Dr. / Prof. Dr. Elke Guenther Department of Electrophysiology, NMI, Reutlingen

Second Reader: PD Dr. Guenther Zeck Department of Microsystems and Nanotechnology, NMI, Reutlingen

I affirm that I have written the dissertation myself and have not used any sources and aids other than those indicated.

Date / Signature:

3

Acknowledgements

The journey of my master thesis has been indeed a great learning experience, challenging yet fun and would not have been possible without the following people. Firstly Prof. Dr. Elke

Guenther who gave me an opportunity to work in her lab and help me unravelling mysteries

of Electrophysiology and above all about retina electrophysiology. Irrespective of her busy

schedules she was kind enough to dedicate some precious time to listen out my progress

and failures in the project and helping me by giving her suggestions which indeed was quite a big contribution in itself. Secondly, Dr.Udo Kraushaar, more fondly as Udo, any amount

of praising would just embarrass me because I can write a whole lot of pages about his contribution to my life both personally and professionally. Without his positive

encouragements and the zeal to see science take its shape this thesis would never had

shaped out this well. His every little word of encouragements and his trust on me to make

things work made me a strong person and more determined to get this thesis into rolling.

During this course not only did I benefit scientifically from him but personally believed that if

one is determined to achieve something no barriers could pull you down. How to think like

a scientist , what could be the slightest loop holes in an experiment was what I learnt from

him and indeed would stay with me till the end of my life. Sorry for all the frustrations you

had to bear with, your contribution means a lot. Sincere thanks to Dr.Guenther Zeck, who

kindly agreed to be the second reader for my thesis. The lab members Katherine, Theresa,

Sven, Adrian, Nadine without whose co-operation my thesis would be incomplete.

I would not have been here without the support of Graduate school for Cellular and

Molecular Neuroscience. Sincere heartfelt thanks to Prof.Herbert, Katja and Tina for always being there to help and taking enormous amount of efforts on my behalf.

The zeal to work in neuroscience specially retina physiology , the love for it and a wish to

pursue further research in this field is due to the revered faculty members like Prof Euler,

4

Dr.Timm Schubert, Dr. Francois Paquet Durand. Thank you for bestowing such a vast

ocean of knowledge and making me realize my appetite for this science. My sincere thanks

to all the faculty members of the graduate school who imparted me with such an immense

amount of knowledge which would be with me for the rest of my life.

Though professionally one could achieve heights, without personal sanity one would never

be able to give ones 100%. This was possible due to few close people without whom my

life is incomplete. Firstly Varsha, she made me realize how important science is to me and

that one has to believe in oneself to understand science. The letter N of Neuroscience was

very kindly fed to me by her efforts. She has been a big driving force for me to pursue

neuroscience as my research interest. Thank you, Varsha for all the little help and being my

strength through and through the entire course of Germany. Germany would not have been

the same without you. It is often said an anchor in your life makes the journey worth while. This anchor enables you to be grounded amidst the tough tides of life. My anchor none

other than Sambit Biswal, a friend, philosopher , in a nutshell a person who kept me strong

through this entire course of time and much before. Irrespective of our long term

association the level of understanding and love bestowed by you made me a true fighter

and made me fight back at times when I thought I would break. Thank you so much for

being there, your presence in my life makes my life worthwhile. In ones life time we

encounter people who make you realize that life is just not achieving results but at times let loosing oneself. The spiritually sanity was a big role to play during this period of my life and

none other than my grandfather like Dr. S.K. Pattnaik this would not have been possible.

Finding positivity in the darkness of negativity was what he made me believe and Im

thankful to him for everything, as, if Im here today is due to his blessings and kind advice

to follow life. Last but not the least my parents and Almighty who have been my driving

force since I have stepped into this world. Without them my meaning of life would have

been pretty incomplete.

5

Table of Contents

1. Abstract 6

2. Introduction 7

3. Materials and Methods 18

4. Results 28

5. Figures and Tables 36

6. Discussions and Implications 50

7. References 57

6

Abstract

In the vertebrate eye the retina is responsible for light perception. It is a sophisticated

image processor and is the first processing step in vision. As a model system, the retina is

a highly organised structure with defined connections and circuitry. Recent attempts to

restore vision in the blind, like patients suffering from Retinitis pigmentosa (RP) or age-related macular degeneration (AMD) have met with extraordinary success. These form of blindness results in substantial loss of photoreceptors leaving the inner retinal layers

unaltered to quite some extent. Although physiological and morphological changes may

take place in the inner layers of the retina of the affected patients, there exists opportunity

for direct excitation of the residual neuron as a mean of restoring vision which forms the

basis for visual prosthesis. The main goal of the present study aimed to mimic two broad

candidates of retinal prostheses, one being the electrical stimulation and the other

biochemical stimulation (via focal glutamate application) of acutely explanted degenerated retina of murine on micro electrode array system. This study also aimed at comparative

analysis of epi-retinal stimulation versus sub-retinal stimulation on the basis of electrical

thresholds, pulse paradigms and latency. The study could help in designing an efficient

candidate of the above two prostheses models, which could encode a high spatio-temporal

resolution, thus enabling a stable retinal prosthesis for restoring vision in blind patients.

7

Introduction

The main part in our visual system is the eye. Our ability to see is the result of a process

very similar to that of a camera. A camera needs lens and film to produce an image,

similarly the eyes needs lens as well as film to process an image. The film of our eye is

represented by the Retina. It captures the image and sends it to the brain to be developed

as shown below in the figure. Retina is responsible for light perception and forms the first

processing step in vision.

Fig 1: The above figure suggests a broad comparison of a camera with the vertebrate eye, suggesting as the film captures image and is developed to generate a processed image ,similarly in our eye the retina acts as a film and captures image and sends it the higher organisation for being developed.(Fig taken from Artificial eye, www.bestneo.com)

Being the most accessible part of the vertebrate CNS it is well separated from the rest of

the brain and connected by well-defined axonal projections branched from the ganglion call layer. It possesses a highly ordered structure and consists of different neuronal cell types,

which are organized in different layers. The retina derives its nourishment from the Retinal

Pigment Epithelium (RPE) .The photoreceptors cells are localized in the outer and inner segment (OS & IS) and its nucleus forms the outer nuclear layer (ONL). The nuclei of bipolar cells, horizontal cells and amacrine cells form the inner nuclear layer (INL).Dividing these nuclear cell layers are two synaptic layers where synaptic contacts occur i.e. inner

8

plexiform layer (IPL) & outer plexiform layer (OPL). Lastly the nuclei of ganglion cells form the ganglion cell layer (GCL). The ganglion cells then branch out to give axonal projections which form the nerve fiber layer, (as depicted in the figure below) to the lateral geniculate nucleus and further to the visual cortex for processing information.

Fig 2: The above figure suggests the highly ordered structure of retina with well characterized cellular composition. The figure in the left shows the layered structure of retina and the one in the right gives an overview of the cells which forms these layers (Fig taken from wikipedia.com)

Photoreceptor cell death (rods and cones) is the major hallmark of a group of human inherited retinal degenerations commonly referred to as Retinitis Pigmentosa (RP) pertaining to rod degeneration (Paquet-Durand et al, 2009; Christian Hamel,2006) and Age-related macular degeneration (AMD) pertaining to cone degeneration. Rods and cones form the photoreceptor layer and form the photosensitive cells in the retina. Rods

form the part of periphery vision and function in less intense light. The numbers of rod cells

are more in comparison to cone cells and they form the basis for night vision. Cones

function best in relatively bright light and are responsible for perception of light. They are

well concentrated in the macula and become sparse on moving towards periphery of the

retina. They are also responsible for visual acuity i.e. the area of highest resolution. RP

9

predominantly affects the photoreceptor layer in the periphery which is the rods and

eventually leads to a vision called as tunnel vision which eventually leads to blindness. In

AMD there is progressive loss of macula (cone) which leads to loss of central vision, precisely the visual acuity. Together; AMD & RP affect at least 30 million people in the

world (Thomas M. O Hearn et al, 2006). The figure below illustrates the appearance of image perceived by RP & AMD patients. They are the most common causes of untreatable

blindness in developed countries and, currently, there are no effective measures of

restoring vision.

(a) (b)

Fig 3: The above figure demonstrates the perception of patients suffering from retinal degeneration. (a) shows the tunnel vision as commonly seen in patients suffering from Retinitis pigmentosa due to loss in photoreceptors(rods) and (b) shows the loss of visual acuity due to degeneration of macula in Age-related macular degeneration (Fig taken from NIH National Eye Institute)

In recent times due to absence of effective therapeutic remedies for RP & AMD,

researchers and scientists are motivated to develop experimental strategies to restore

some degree of visual function to affected patients.

Much previous literatures argued that retinal degeneration such RP affect only sensory

retina. Many approaches to retinal rescue are based on this clearly incorrect assumption.

At first it may appear discouraging and even insurmountable given that the field of vision

rescue has been labouring for past 20 years to overcome the loss of photoreceptors alone,

only recently recognizing the scope of neural remodelling in the retina. Furthermore this

10

plasticity and continued neural signalling reveals possible mechanism for exploitation, and

emphasizing the need for early diagnosis and intervention to retard remodelling. The fact of

remodelling influences all rescue strategies begins before the inner retinal cells die.

Of the various retinal rescues retinal prosthesis implant strategies aim to replace

photoreceptors with electronics or artificial synapse to drive remnant retina for transmitting

visual information.

As from histological sections of the diseased retina it is evident that residual inner layers of

neural retina are spared for a long time, several approaches have been designed to

artificially stimulate this residual retina and thereby the visual system and this gave way to

a term Retinal Prosthesis. At present, two major strategies for electrical stimulation as a retinal prosthesis have been pursued.

The Epiretinal approach involves a semi-conductor based device (array) in close contact with the ganglion cell layer. In such implants information must be captured by a

camera system before transmitting data and energy to the implant (Artificial eye, 2008, www.bestneo.com). Second is the Subretinal approach which involves the electrical stimulation of inner retina from the sub-retinal space (between RPE and neural retina) by implantation of semiconductor based micro-photodiode (MPDA) array into this location or focal stimulation of the inner retina using tungsten electrode and assessing the read out at

the microelectrode array (Artificial eye, 2008, www.bestneo.com). In the former approach the electrical charge generated by MPDA in response to light

stimulus may be used to artificially alter the membrane potentials of the neurons of the

residual retinal layers in a manner to produce formed images.

11

Fig 4: The above figure gives broad overview of strategies of occular implants including the difference between epiretinal and subretinal approaches in implants (Fig taken from Bionic eye)

Significant progress has been made towards the development of long- term, implantable

retinal prostheses due to rapid advancement of micro-electrode fabrication methods (one major contribution in recent times, the group of Prof.Dr.Zrenner, in Tuebingen, Germany working on Retina Implant Project who could successfully implant a MPDA enabling a patient named Mika to read specific alphabets, Subretinal electronic chips allow blind

patients to read letters and combine them to words,Eberhart Zrenner et al 2010). However electrical stimulation has few limitations encoding important sensory features used in

normal central visual processing. Few acute and chronic human testing revealed that

patients perceived white flashes of light in response to epiretinal and subretinal stimulation.

It was also seen that although the temporal feature of electrical stimuli was well correlated

with electro-phosphene percepts, patients demonstrated difficulty in perceiving shapes with

more complex spatial patterns of electrical stimulations. It was observed in few patients

with small diameter electrodes generated free radicals like hydrogen peroxide which

induced retinal tissue damage by causing toxicity to the lipid membranes of neuron and glia

and those with large diameter electrodes required yielded poor resolution. (Finlayson and

12

Iezzi, IOVS, July 2010). In addition to the above limitations, electrical stimulation is non-specific i.e. it stimulates both ON and OFF retinal ganglion cells, bipolar cells and very

likely amacrine cells thus reducing contrast and spatial localization within the retina.

(Finlayson and Iezzi, IOVS, July 2010). Many of the neural prostheses use electricity to depolarize the neural cells, however

stimulation in the nervous system is primarily through chemical neurotransmission. The

native use of electrical stimulation is demonstrated by the electrical gap junctions between neurons, neurotransmitters provide specificity and flexibility of stimulus parameters that gap

junction typically do not provide. (Peterman et al, 2003) To achieve useful vision, a retinal prosthesis may need to mimic the biological complexity

and resolution of the retina, including the ability to address individual cell types and to

stimulate the retina with the biological specificity achieved by using neurotransmitters, of

which glutamate acts as a primary neurotransmitter. The other common neurotransmitters

in the retina are GABA, glycine, dopamine and acetylcholine. Glutamate is the excitatory

neurotransmitter released by photoreceptor, bipolar, and retinal ganglion cells, may provide

a more natural means of stimulating RGCs (retinal ganglion cells). However in some horizontal cells and amacrine cells when labelled with glutamate antibodies showed weak

labelling. It is believed that these cells release GABA, not glutamate as their

neurotransmitter, suggesting that the weak labelling of glutamate reflects the pool of

metabolic glutamate used in the synthesis of GABA (Yang, 1996). Glutamate is incorporated into these cell types through a high affinity glutamate transporter located in the

plasma membrane. These transporters maintain the concentration of glutamate within the

synaptic cleft at low levels preventing glutamate excitotoxicity. A major share of glutamate is taken up by the glial cells of the retina, Muller cells and rapidly broken down to glutamine.

Histological techniques help in identifying the potential glutamatergic neurons by labelling

neurons containing glutamate and neurons that take up glutamate. To further determine if

13

these cell types actually release glutamate as their neurotransmitter, the receptors on the

post synaptic side have to be examined. Once released from the presynaptic terminal,

glutamate diffuses across the cleft and binds onto the receptors located on the dendrites of

the post synaptic cell(s). Two major classes of glutamate receptors have been identified. First being the Ionotropic glutamate receptors, which directly gate ion channels and Metabotropic glutamate

receptors, which may be coupled to an ion channel or other cellular functions via an

intracellular second messenger cascade. Various retinal neuronal cell types differentially

express their subtypes. These receptor types are similar in that they both bind glutamate

and glutamate binding can influence the permeability of ion channels. However, there are

several differences between the two classes. Glutamate binding on to an ionotropic

receptor directly influences ion channel activity because the receptor and the ion channel

form one complex, thus mediating fast synaptic transmission between neurons. Two

Ionotropic receptor types have been identified, NMDA receptors which bind glutamate and

the glutamate analogue N-Methyl-D-Aspartate(NMDA) and non-NMDA receptors, which are selectively agonized by kianate(GluR5/6), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA(GluR1-4), but not NMDA. The non-NMDA receptors open non-selective cation channels more permeable to sodium

and potassium ions than calcium. In retina, non-NMDA receptors have been identified on

horizontal cells, OFF-bipolar cells, amacrine cells and ganglion cells. NMDA receptors also

open non-selective cation channels on binding with glutamate, resulting in an increased

conductance, which is due to increased permeability to calcium than sodium ions. Few

retinal ganglion cells and some amacrine cells express functional NMDA receptors. The

functional organisation of Ionotropic glutamate receptors is well illustrated in Fig (5a & b).

14

(a) (b)

Fig 5: (a) Non-NMDA receptors are selectively agonized by kianate, AMPA and are permeable to Na+ and K+ ions than Ca2+ ions (Mayer and Westbrook, 1987). (b) NMDA receptors are structurally complex, with separate binding sites for glutamate, glycine, Mg2+ and Zn2+ and polyamines and are permeable to Ca2+ than Na+ ions (Fig adapted from webvision.edu)

Unlike ionotropic receptors, which are directly linked to an ion channel, metabotropic

receptors are coupled to their associated ion channels through a second messenger

pathway. Ligand binding activates a G-protien and initiates the intracellular cascade (Fig 6). These receptors are classified into three groups (I, II &III) based on structural homology, agonist selectivity and their associated second messenger cascade. Few metabotropic

receptors (like APB) are selective for the cells of the ON-pathway and some of these receptors are differentially expressed throughout the retina. Unlike NMDA and non-NMDA

receptors glutamate binding onto metabotropic receptors activates a G-protein which

further activates phospohdiesterase, that reduce the intracellular concentration of cyclic

nucleotides leading to closure of cGMP gated non-selective cation channels. This in turn

gates the entry of sodium or calcium ions into the cell thereby decreasing the conductance.

15

Fig 6: Represents the metabotropic glutamate receptors (mGluR) coupled to their associated ion channels with second messenger cascade. In the above figure when glutamate binds the mGluR receptors then the G protein is activated and the intracellular cascade of proteins are triggered thereby modulating the associated ion channel to open or close. (Figure taken from webvision.edu)

These glutamate receptors have been shown to participate in normal visual activation of

RGCs including kainate, AMPA, and NMDA receptor subtypes. To work towards the

neurotransmitter based retinal prosthesis many research groups( Noolandi et al,2003, Peterman et al,2003,Finlayson and Iezzi,2010) are developing different strategies to establish a device which could release neurotransmitter(glutamate) focally in small volumes in acute retina whole mounts. Such strategies involved developing artificial

synapse chip (Peterman et al, 2003) with biocompatible interface for localized neurotransmitter delivery on to the retina. With recent developments in microfluidic devices

there has been possibility of ejecting a pattern of neurotransmitters via an inkjet print-head adapted from a desktop printer onto the retina (Noolandi et al, 2003). In recent studies different research groups aim at developing an artificial synapse that

would be nano-scaled photovoltaic driven structure like Polypyrrol (Ppy) which would be able to bind and release glutamate as the neurotransmitter from a glutamate reservoir, as

shown below, via its tuneable charge. This would then form the basis of artificial

biochemical neural implant (ANI) which would facilitate restoration of vision.

16

Fig 7: The above figure conceptualizes the idea of an artificial biochemical neural implant which would help in restore vision in patients suffering from RP & AMD mimicking the natural way of visual processing. The figure in the left depicts the photoreceptor loss as seen in RP & AMD. The figure in the right shows the possibility of devising a glutamate reservoir which could mimic the neurotransmitter release of photoreceptors to the inner retina thereby forming a strategy to restore vision.( Fig adapted from wikipedia.org/idea conceptualized from Prof Dr.Elke Guenther poster presentation)

In the current study both electrical as well as biochemical stimulations formed suitable

candidates for retinal prostheses. The activity was seen using microelectrode array (MEA) system. The MEA system has an advantage over other systems, as one could analyse the

retinal activity at multiple sites and in turn could assess the entire retinal network activity.

Electrical stimulation for RP retina was done via epiretinal stimulation and subretinal

stimulation. The former involved stimulating the retina at a single electrode of the MEA from

the ganglion cells side and simultaneously recording electrically evoked responses from

other neighbouring multiple ganglion cells. The later involved stimulating the retina on the

bipolar cells side via bipolar tungsten electrode and recording from the ganglion cells side

placed over the MEA. In both the stimulations the read out is at the ganglion cells side in

the form of action potentials. By standardizing threshold paradigms for electrical stimulation

(for epi-retinal as well as sub-retinal) I could make a preliminary comparison between the different approaches of electrical stimulation in RP retina. Biochemical stimulation was

Glutamate Reservoir

17

done via local, small volume sub-retinal application of glutamate using patch pipette in

flattened eye cup preparations of RP retinas to investigate the feasibility of activating

ganglion cells as means of neurotransmitter-based retinal prosthesis. Aspects of the

prosthesis like duration of glutamate ejection, concentration, response latency were also standardized. Further, by different application of drugs like Picrotoxin, Strychnine and

Cyclothiazide the response pattern of glutamate receptors could be studied. This could

elucidate some preliminary approach towards an efficient neurotransmitter-based retinal

prosthesis and thus could form a platform for comparing electrical stimulation with

biochemical stimulation, to reason out a better retinal prosthesis of the two with respect to

spatio-temporal resolution.

18

Materials and Methods

Animals

Animals were housed under standard white cyclic lighting, had free access to food and

water. FVB strain (rd1) female mice were used (Charles River WIGA, GmbH, Germany).All procedures were performed in accordance with the local ethics committee for the use of

animals in ophthalmic and visual research. All efforts were made to minimize the number of

animals used and their suffering. Because most of the dynamic changes reduce

comparatively around post-natal day 60(P60), from postnatal day 28(P28) onwards, hence most of the measurements were carried out from this age and after. This inference was

made from multiple measurements made at different ages of the mice (P28, P40 & P60) to obtain a stable age for applying both electrical and biochemical stimulation.

In-vitro acute retina Preparation

Retinae from P60 and older rd1 mice were used for recording. In brief, animals were killed

by decapitation and the eyeballs were enucleated and cut open along the orra serrata. The

lens and vitreous were carefully removed. The neural retina and the pigment epithelium

were gently removed from the underlying tissue. The isolated retina was cut into retinal

patches of approximately 3X3 mm (Stett et al, 2000) (Fig.8) or at times were whole mounted depending upon the preparation .It was noted that the entire retina preparation

was done as quick as possible with less damage to preserve the retina activity. The retinal

patches/ whole mounts were placed in artificial cerebrospinal fluid (ACSF) comprised of 125mM NaCl, 25mM NaHCO3, 3.5mM KCl, 2mM CaCl2,, 25mM Glucose and 1mM MgCl2,

bubbled with 95% O2 and 5% CO2 ; Osmolarity 340mOsm, pH of 7.3 -7.4 at 30 C , a nd

then mounted onto a planar MEA. At times ACSF solution was substituted with AMES

medium (Sigma Aldrich, Germany), this substitution did not affect the retina vitality or the recordings. One of the retinal patches were placed ganglion cell layer down onto the

19

recording chamber the MEA with a continuous perfusion rate of 4-6ml/min. The inflow of

the medium into the chamber was maintained by a valve and simultaneously the outflow

was maintained by a peristaltic pump, in order to maintain the vitality of the retina. The

MEA chip was mounted to a MEA stage with integrated heating (Multichannelsystems (MCS), Reutlingen, Germany) and placed onto the table of an inverted microscope (Olympus CK2, Japan). Experiments were performed at a temperature of 37 C using a temperature controller, TC02 (MCS, Reutlingen, Germany). The remaining samples were stored at room temperature (RT) and bubbled in ACSF solution until further use

(a) (b) (c)

Fig 8: Retina Preparation. The above figure depicts the invitro acute retina preparation and mounting over planar MEA for recording. In fig a) the eyeball was enucleated and cut open along orra serrata and the retina was carefully removed clearing vitreous humour. Fig b) shows mounting of retina over a Planar MEA. It is taken care that the mounting is done carefully without any damage to the retina or the electrodes. Fig c) shows the retina mounted over a Planar MEA seen from an inverted microscope. (Figure adapted courtesy Prof.Dr.Elke Guenther, NMI, Reutlingen)

Micro-electrode array and data recording

A Planar MEA containing Titanium nitride (TiN) electrodes (circular shape, diameters: 30 m) on a glass substrate in an 8X8 square-type grid layout (MCS, Reutlingen, Germany) was used for the recording of field potential and spiking activities from RGCs (retinal ganglion cells) It was noted that the retinal patches/ whole mount covered the entire grid layout of the MEA for better recording. A single TiN MEA electrode is made of

200um

20

nanocolumnar 3-D structure that increases the overall surface area of the TiN electrode in

comparison to a standard 2-D aluminum or platinum electrode with the same electrode

diameter, thereby increasing the overall capacitance and reducing the noise levels,

allowing a continuous and reliable electrical stimulation over a period of several weeks,

(van Bergan et al, 2003) (Fig.9a, b & c). Four electrodes at the vertices were inactive and one electrode was used as internal reference (iR) electrode. The inter-electrode spacing from centre to centre were 200 m. Impedances of the electrodes were approximately 50

k at 1 kHz. The insulation is made of Silicon Nitride (Si3N4). Each electrode has a track to guide it to the contact pad where it contacts the amplifier. In TiN MEA electrodes the track

and contact pad is made of Titanium nitride .The MEA60 data acquisition system (MCS, Reutlingen, Germany) is a 60-channel amplifier having a compact design (165 x165x 19mm) and due to the surfacemounted technology of pre- and filter amplifiers the complete electronic circuit and amplifier hardware were built into a single housing to ensure

optimal signal to noise ratio of the recording. An input voltage range from -2048 mV to

+2048 mV; an amplifier gain of 1100 and sampling frequency of 20 kHz were used for

obtaining microelectrode recordings of retinal activity from up to 59 retinal sites (Micheal Fejtl et al, Advances in network electrophysiology using multi electrode arrays, Springer publications) The retinal activity of 59 channels could be visualized over the computer screen with the McRack software. During data acquisition a high pass filtering (second order Butterworth filter, cut off frequency of 50 Hz) was set in order to differentiate actual retinal activity from baseline drifts. Raw recorded data were stored on a hard disk. As

activities of more than one RGC can be recorded in one channel of the MEA a procedure to

sort multi-unit activities to single unit activity was also carried out using data analysis tools.

These tools could be used simultaneously and independently during the recordings.

21

(a) (b)

Fig 9: Planar MEA: Figure (a) shows a regular TiN electrode Planar MEA form Multichannel systems, Reutlingen, Germany bearing TiN electrodes in the middle and tracks guiding the electrode to their respective rectangular contact pads in. (b) shows the grid lay out of 8X8 electrodes over a planar MEA. (c) shows a single TiN MEA electrode at m and nm resolution. The nano columnar stricture of the electrode increases the overall surface of these electrodes and thereby yielding high performance of these MEAs. (Figures taken from MCS application downloads, Reutlingen, Germany)

Electrical Stimulations

Epi-retinal stimulation: This type of stimulation involved stimulating from the ganglion

cells side layer at a single electrode in the centre of the MEA and recording RGCs

responses from the other nearby electrode/channels. Charge-balanced biphasic constant

(c )

22

voltage rectangular pulse trains were generated (anodic pulse first with no temporal separation between two phases.). 50 trains of pulse was applied with a period of 4 sec. Pulse amplitude was varied from 0.5V to 2.0V along with pulse duration which varied

from 300 s-1ms in order to visualize a reliably evoked RGC response as seen from

previous work (Ryu et al,2009). The stimulation pulses were generated by STG 1004 stimulus generator (MCS, Reutlingen, Germany). McStimulus software was used to feed the stimulation paradigms and thresholds to the stimulus generator (Fig.10). It was seen that while delivering voltage pulse the recording properties of the MEA electrodes were

retained and to address this, the MEA 1060-BC amplifier in combination with the MEA

Select software was used to make it sure that the available MEA electrodes could be used

as stimulation sites (stimulation electrode) by simple mouse clicks. Moreover in order to retain the recording properties of the MEA electrodes selected for stimulation an electronic

blanking circuit (BC) has been incorporated into the amplifier to generate blanking signals which could transiently decouple all the MEA electrodes from the main amplifier input stage

during the time course of the stimulation. This is so done in order to prevent any stimulus

artifacts. The time for generating this blanking signal depends on the stimulus strength and

waveform and could be specified via Mc_Select software.

These stimulation paradigms and thresholds were common for both epi-retinal stimulation

and sub-retinal stimulation as adapted from literature survey (Ryu et al, 2009; Jenson and Rizzo 2007). However these stimulations were delivered to the tissue in two different ways depending on the type of stimulation. As stated above in epi-retinal stimulation a single

electrode of the Planar MEA was used for stimulating the ganglion cells and the

neighboring electrodes of the Planar MEA were used for recording the RGCs responses.

Sub-retinal stimulation: This type of stimulation involved stimulating the retina at the

bipolar cells side and recording RGCs responses using Planar MEA. Concentric bipolar

tungsten electrode with exposed metal shield (Length=76mm, 0.325mm x 0.126mm x

23

24

Biochemical Stimulation

Biochemical stimulation involved local and small volume sub-retinal application of

glutamate containing solutions in the retina. For local application of glutamate glass patch

pipette(Science products GmbH,Germany) with tip openings of 1-2 m was filled with solution containing 2mM glutamate.(This concentration was considered as this was the lowest concentration that was effective in eliciting RGCs response in normal retinas,

Finlayson and Iezzi,2010) dissolved in Ringer /ACSF solution. The glass patch pipettes were manipulated into the tissue using micromanipulators (Leica Microsystems, Germany). Due to sharp and fragile pipette tips it was made sure that the pipettes were positioned well

in the tissue subretinally without damaging the retina or scratching the electrodes.

Images of the electrode positions were captured to determine the focality and spread of the

drug application. It was made sure that the position of the electrode for drug applications

over period of time remained consistent as that would bring about variability in the signals.

A few m variations would not affect the data variably, as the resolution of MEA is not very

high.

All the drug applications were applied with a pressure ejection module, the two channel Toohey Spritzer pressure system IIe (Science products GmbH, Germany). The glass patch pipette was placed in the pipette holder in the micromanipulator and connected to the

pressure outlet controlled by a valve, of the Toohey Spritzer. The pressure, pulse duration

were digitalized during recordings. For generating puff applications as trigger pulses during

data acquisition / recording as analog data, TTL positive pulse from the Toohey Spritzer

was connected to one of the three analog channels of the MEA set up. The trigger to

dispense/ puff drug could be controlled with a remote attached to the Toohey Spritzer or

could be done manually. The drug application was done every 1min interval (this marked the number of sweeps in McRack software) for different ejection duration (starting from 10ms -100ms). However reliable RGCs response to puff application was visible for ejection

25

duration of 20ms. A slight electronic shift delay of 20-30ms from the command pulse/

trigger was observed as this was the time delay introduced by the capacitance of the

pressure system ( valve outlet) . The drug application (glutamate) was done in area of the retina displaying spontaneous activity .The RGCs responses and spatial

distribution/focality of the responses was visualized using the data acquisition software,

McRack. The acquired data were further analyzed using data analysis tools like Igor Pro

software.

Data Analysis

Electrical stimulation: Epi-retinal and Sub-retinal stimulation

The acquired evoked responses were analyzed using McRack software (MCS, Reutlingen, Germany) and Neuroexplorer v 4.0 by generating Per-event raster plots from averaged data of the total number of trails. For generating a peri-event raster from acquired evoked

responses a Trigger detector was set in McRack software that marked the stimulus pulse.

This was so done to align a common point for the stimulus pulse over the number of trials.

Further Spike sorter from the McRack software was used to sort actual retinal activity from

the rest of the data by the use of a threshold detection limit over the total number of trials. It

was made sure that the detection limit values were set keeping in consideration the raw

data as well as filtered data to prevent any biasing. The sorted data files generated were

exported to the Neuorexplorer v 4.0 software for generating peri-event rasters and peri-

stimulus time histograms using the option Peri-event rasters from the data analysis tools

from Neuroexplorer software. It was made sure that all the scales, time bins were set

properly in the analysis window to generate relevant rasters for the data.

Peri-event rasters analyzing tool helps in visualizing the retinal activity before and after the

stimulus thereby giving a clear representation of the data and helps in differentiating

background or spontaneous activity from stimulus-correlated activity.

26

Biochemical stimulation: The data was acquired and analyzed using McRack software

and Neuroexplorer v 4.0. The averaged data was represented in form of perievent rasters

over the total number of trials. During data acquisition to mark the puff application of

glutamate a Trigger detector was set. The threshold for triggering on biological signals was

defined from Analog Raw Data streams. The trigger event was considered at time t=0 but

one could set the time scale to negative values to display pre-trigger time (in this case it was set at -100ms) i.e. how long before the trigger event the data is considered and how long after the trigger event the data recording is stopped i.e. post-trigger time (in this case it was 800ms). This total time gives the window extent (in this case being 900ms) as shown below in the figure.

Fig 11: Above figure illustrates how trigger detection works. Time t=0 specifies the trigger event ,pre and post trigger time together give the window extent displaying the time the data is being recorded. (Fig from MCS, website) Further the data stream collected is spike sorted to differentiate actual retinal activity from

rest of the data using Spike sorter from McRack software. It was very essential to define

the spike cut out as the data acquired was in triggered mode. To detect the spike activity in

the given window frame spike cut out ranges. The spike cutout ranges were defined

depending upon the detection event, which is the time when the threshold (of the spikes) has been crossed (in this case pre-trigger time was set at 1ms and post trigger time was

27

set at 2ms), additionally a Dead time was set (in this case 2ms) which determines for how long after a detection event no new detection event is accepted and to save computer

performance it was recommended that the dead time was at least nearing the post trigger

time. The sorted data files generated were exported to the Neuorexplorer v 4.0 software for

generating peri-event rasters and peri-stimulus time histograms using the option Peri-event

rasters from the data analysis tools from Neuroexplorer software. It was made sure that all

the scales, time bins were set properly in the analysis window to generate relevant rasters

for the data. The generated per-event rasters represent the data for the time window

considered for the trigger event for total number of trials.

Offline data analysis: The data generated from the peri-event rasters were exported to

excel files and were analyzed offline. To determine the effect of glutamate on RGCs, all

counts/ spikes above Mean+2 S.D (threshold) was considered. In order to graphically represent the response latencies with respect to spatial distribution and spike activity with

respect to duration of glutamate ejection Igor Pro software was used. To generate statistical significance in spike activity with respect to duration of glutamate ejection, Kruskal-Wallis test was performed using Igor Pro software. Bar graphs representing the

success ratio of different modes of stimulation were generated using Matlab.

28

Results

1. Optimization of signal to noise ratio of Planar MEA

The rate limiting step involved at achieving a good signal to noise ratio over a Planar MEA.

It was observed that due to fragile vitreous humour and dead cells resulting from retina

preparation the RGCs signals were masked thereby yielding a poor signal to noise ratio

(Fig 12a). For overcoming this issue it was necessary that a tight contact was made between the electrodes and the retina to prevent any leak of current yielding a poor signal

to noise ratio. Hence a platinum grid with a closely space nylon mesh was placed over the

whole mounted retina to adhere tightly over the electrode. This resulted in good signal to

noise ratio thereby making feasible to record RGCs activity over MEA. (Fig 12b)

2. Electrical Stimulations

2.1 Stimulus dependent activity of rd1 degenerated retina (P60) via epi-retinal and sub-retinal stimulation:

It was seen that due to loss of photoreceptors in rd1 retina and decreased retinal thickness

upto 50 %( Zrenner et al, 1999; Sekirnijak et al, 2009) which leads to increased hyperexcitability thereby increasing the level of spontaneous activity. Previous work

suggests that ganglion cells of the degenerated retina require high voltage to reach the

threshold for stimulation of the remaining neural retina (Chen et al 2006, Jensen and Rizzo 2007). In degenerated retina, for both epi-retinal and sub-retinal stimulation, the pulse amplitude (biphasic rectangular pulse, anodic followed by cathodic pulse without any interphase delay) was varied from +/- 0.25 to 1V with a fixed duration of 900s for a period of 4s. For epi-retinal stimulation, reliable ganglion cell response was found at +/- 0.75V (+/- 750mV, n=10/50) and for sub-retinal stimulation it was seen at +/- 0.5V (+/- 500mV, n=5/40). By varying the pulse duration from 300s to 1ms with fixed amplitude of +/- 0.75V for epi-retinal and +/- 0.5V for sub-retinal, reliable time duration was found at +/- 450s

29

(t=900s) for both the stimulation. (Fig 13a and 14a). The short latency spikes (around 2ms) could not be observed probably due to the contamination with stimulus artifacts,observed were long latency spikes (around 20ms-50ms) post electrical stimulation. For both epi-retinal and sub-retinal stimulation the read out was ganglion cell response, as

a result of which the stimulus and stimulus correlated signals from RGCs were similar .This

long latencies, was possibly due indirect activation of retinal ganglion cell network (Jensen and Rizzo, 2007) or could be due to different type of ganglion cells responsible for the different latencies (Jensen and Rizzo, 2008). Therefore it was seen that the threshold parameters (pulse amplitude and pulse duration) and pulse paradigms (adapted from literature) were comparable for both epi-retinal and sub-retinal stimulation.

2.2 Peri-event raster plot for determining stimulus correlated activity for epi-retinal

and sub-retinal stimulations:

Peri-event rasters represented the spontaneous activity of the rd1 retina followed by epi-

retinal stimulus locked activity of the retinal ganglion cells. It was observed that in epi-

retinal stimulation the activity lasted around 100ms post electrical stimulation, before it

returned to its baseline (Fig 13b). Interestingly, it was observed that in sub-retinal stimulation the retinal ganglion cells followed a stimulus correlated oscillatory

pattern(n=2/4), with a frequency of around 10Hz , which was prominent for few milliseconds and then subsequently fades away (as shown in fig 14b, around 350ms). To figure out the reason for such an oscillatory pattern a bath application of strychnine and picrotoxin

cocktail was done during sub-retinal stimulation (figure not shown). It was observed that the oscillatory pattern was disrupted, which suggests that since sub-retinal stimulation explores

the remnant retinal circuit, these oscillatory pattern could be notably due to the inhibition of

lateral pathway onto the excitatory vertical pathway. In both the type of stimulations, the

latencies were measured upon the spontaneous activity. The threshold for measuring

30

activity responsible due to electrical stimulation was calculated as (mean + 2xs.d) of spontaneous activity (counts/bin) before the command pulse. Such a calculation considers the marginal errors and fluctuations in the data set and additionally could give unbiased

estimation of the data set. The activity that crossed the threshold post onset of command

pulse was the activity responsible due to electrical stimulations. The respective latencies

were determined as the time from the command pulse to the first time bin in the peri-

stimulus histograms that exceeded the threshold activity. Mean latency for epi-retinal

stimulation was around 24.3 +/- 0.94ms and for sub-retinal stimulation was 27 +/- 3.2ms

(Latency in terms of Mean+/- SEM, Table 1).

3. Biochemical (Glutamate) stimulations The lack of well defined spatial resolution due to electrical stimulation and lack of cellular

specificity electrical stimulation had few limitations in encoding important sensory features

used in normal central visual processing. To circumvent these limitations a more

naturalistic means of stimulation of RGCs was hypothesized which could encode a natural

vision. The natural vision is encoded as neuro-transmitter signals with glutamate as the

primary transmitter. Thus to determine the feasibility to develop such a hypothesis and

standardize different parameters of such a neuro-transmitter based prosthesis local and

small volume application of glutamate was performed in rd1 retina invitro.

3.1 Suppressive effect of local glutamate application on rd1 retinal ganglion cells:

Local exogenous glutamate application at the site of spontaneous activity induced an initial

suppression of firing activity followed by increased activity /excitation (n=5/10). (Fig 15). On application of 2mM glutamate (the minimum concentration of glutamate which could elucidate retinal ganglion cell response in normal retina, Finlayson & Iezzi, 2010). The peri-event rasters show an activity window of 900ms of which 100ms is prior to the application

31

of the glutamate puff (at time, t=20ms) followed by suppression of activity. The suppression was followed by an initial increase in activity which persisted over the entire time window or

at times returned to baseline activity. This suppression could be possibly due to two major reasons. Firstly, glutamate application leads to rapid desensitization of AMPA receptors

present on the retinal ganglion cells (Massey and Miller, 1988). Another possible reason could be the excitation of glutamate receptors on amacrine cells by glutamate applications

which in turn released GABA or glycine to induce an inhibitory effect to the ganglion cell

activity (Bloomfield and Dowling, 1985).

3.2 Oscillatory behaviour of RGCs activity of rd1 retina on local glutamate

application( n=2/5): In few experiments it was seen that local exogenous application of glutamate not only did

induce an initial suppression of retinal ganglion cell activity followed by an increase in

activity but also could show oscillatory behaviour which was persistent over the entire time

window (Fig 16, t=800ms). The oscillations were at a frequency of approx. 10 Hz. The oscillations could probably be due to the activation of glutamate receptors present on the

amacrine cells which could in turn release GABA or glycine over a period of time causing

inhibition. This inhibitory effect shapes the activity from the retinal ganglion cells thereby

resulting in an oscillatory pattern. (Bloomfield and Dowling, 1985; Yang XL, 2004) .

3.3 Spatio-temporal effectiveness of local glutamate stimulation in rd1 degenerated

retina:

For elucidating the effectiveness of spatio-temporal resolution of local glutamate

stimulation, the response latencies were measured with respect to the distance from the

site of glutamate application. The latencies were measured upon the spontaneous activity.

The threshold for measuring activity responsible due to glutamate stimulation was

32

calculated as (mean +/- 2xs.d) of spontaneous activity (counts/bin) before the command pulse. Such a calculation considers the marginal errors and fluctuations in the data set and

additionally could give unbiased estimation of the data set. The activity that crossed the

threshold post onset of command pulse was the activity responsible due to glutamate

stimulation. The respective latency was determined as the time from the command pulse to

the first time bin in the peri-stimulus histograms that exceeded the threshold activity. It was

seen that mean latency at the site of ejection was 16+/-3 ms. The mean latency of the onset of activity at an inter electrode distance of 200m was 20+/- 4.2ms. Additionally at a

diagonal inter electrode distance of 282m the mean latency was 25+/- 2.25ms. No

prominent effect of glutamate was visible at >=400m distance from the site of ejection (all values Mean+/-SEM; for n=5; Table 2). There was no significant difference between the latency responses with respect to the spatial distribution from the site of glutamate ejection (Fig 17) which suggested that the spatio-temporal resolution was not well achieved which could be due to the resolution of MEA or probably that the effectiveness does not depend

upon the distance from the site of ejection of glutamate rather on the cell type of retinal ganglion cells. (Finlayson et al, 2010).

3.4 RGC response dependence on glutamate ejection duration in rd1 degenerated retina:

RGC responses were directly controlled by varying the duration of glutamate ejection. The number of spikes increased with increased duration of glutamate ejection. The latency of responses, as may expected, did not vary with the duration of ejection. The number of spikes evoked by glutamate application increased with the duration of ejection to a maximum which was 100ms and then decreased with further increase of duration to

200ms. A significant difference of RGC activity from 20ms to 100ms was seen. (Kruskal Wallis test, n=3, P< 0.05, Fig 18).

33

3.5 Local glutamate application on rd1 retina in presence of GABA and glycine

blockers:

To reason out the suppression effect post glutamate application due to inhibitory effects

from amacrine cells, local exogenous application of glutamate on the retina perfused with a

bath solution of strychnine (2M) and picrotoxin (50M) in ACSF was performed. It was observed that due to blockade of GABA and glycine, the overall activity of the retinal

ganglion cells increased( 39 +/- 13%,mean+/-s.d,n=5/10). However it was seen that although the suppression effect, post glutamate stimulation had reduced but was not

completely removed (Fig 19 a & b). This suggested that apart from inhibitory effects from the amacrine cells and horizontal cells, there exists another possibility accounting for this

suppression.

3.6 Local glutamate application on rd1 retina in presence of only cyclothiazide (CTZ): The second probable reason accounting for suppression in RGCs activity was addressed

by local application of glutamate+CTZ (50M) on to the retina perfused in a bath solution of cyclothiazide in ACSF was performed. It was seen that the suppression of RGCs

decreased to a considerable amount with an overall increase in activity as shown in the

figure (Fig 20 a & b). CTZ is said to destabilize the AMPA receptors present on the retinal ganglion cells and also inhibit some GABAA receptors thereby preventing rapid

desensitization of the AMPA receptors and increasing the overall activity (5+/- 4%, mean +/- s.d, n=4/10). Therefore the results demonstrate that a substantial portion of this suppressive actions account from the desensitization of AMPA receptors.

34

3.7 Local glutamate application on rd1 retina in presence of cyclothiazide,

strychnine and picrotoxin:

To further confirm the suppression effect accounted due to the above two probable

reasons, i.e., the inhibitory effects due to GABA and glycine and desensitization effect of

AMPA receptors due to puff application of glutamate, thereby ruling out any other possibility

contributing to this suppression effect; local application of glutamate+CTZ (2mM +50M) was done in presence of a bath of ACSF containing CTZ (50M), strychnine (2M) and pictrotoxin (50M). It was seen that the suppression effect due to glutamate application completely disappeared with an overall increase in activity (30+/- 10%, mean +/- s.d, (n=3/7) (Fig 21a & b). This suggested that glutamate application is likely to have both direct and indirect effect on the retinal ganglion cells.

4. Success ratio between electrical stimulation and biochemical stimulation To sum up the entire set of successful stimulus correlated responses a normalized success

ratio plot was generated with respect to different modes of stimulation. It was seen that

glutamate stimulation successfully yielded stimulus correlated RGCs activity for 50%

(n=5/10) of the experiments. However the success ratio was low for electrical stimulation in both epi-retinal and sub-retinal stimulation with 20 %( n=10/50) and 12.5 %( n=5/40) respectively (Fig 22a). This could suggest that glutamate application, being a more naturalistic phenomenon, has a higher probability of eliciting response in comparison to

electrical stimulation. Furthermore sub-retinal stimulation was not well achieved (matter of number of successful stimulations) in comparison to epi-retinal stimulation. This suggests that further standardization with regards to positioning of the tungsten electrode in the

retina is required, in order to get comparable set of recordings as epi-retinal stimulation.

Further more a summary graph (Fig 22b) depicting the mean latencies of electrical (epi-retinal and sub-retinal) stimulation and biochemical stimulations showing the comparative

35

temporal effectiveness in rd1 retina. The overall spatial resolution in Table 3 showed that

the biochemical stimulation although did not have a well defined spatial resolution (around 282m) but had a restrictive focality in comparison to electrical stimulation, which could again suggest that the glutamate stimulation has a better spatio-temporal effectiveness in

comparison to electrical stimulation. However to draw a conclusion further investigations

needs to be done.

36

Figures & Tables

(a)

(b)

Fig 12: Above figure (a) shows a poor signal to noise ratio obtained retinal ganglion cells placed on a planar MEA (30m electrode diameter, 200m inter-electrode distance). Fig (b) shows a good signal to noise ratio obtained from the RGCs placed on the same planar MEA. The signals in (a) were poor due to the masking of signals because of fragile vitreous humour or preparation yielding dead cells or loose contact with the MEA electrodes. In (b) post optimization with the platinum grid enabled a good contact with the MEA electrodes which yielded a good signal to noise ratio.The red circles encircles retinal ganglion cell activity.

80ms

8V

80ms

8V

37

(a)

(b)

Fig 13: Above figure (a) shows high pass (50Hz) filtered spike waveform, evoked electrically from RGCs of rd1 retina via epi-retinal stimulation. The arrow in black indicates the biphasic pulse stimulus and the red circle encircles the RGCs activity post stimulus application. Fig (b) is the generated peri-event raster of the spike response in (a), the green arrow indicates the spontaneous activity prior to stimulation, the red arrow shows clear stimulus correlated activity of the RGCs post stimulus. The stimulus correlated activity lasts for over 100ms and then returns to baseline activity. The mean latency of the first RGC spike post epiretinal stimulation was 24.3+/-0.94 ms (mean+/- SEM; n=10)

-0.5 0 0.5Time (sec)

0

40

80

Time(sec)

Counts/bin

100ms Bin width:2ms

100ms

40 counts/bin

10V

Spontaneous activity

38

(a)

(b)

Fig 14: Above figure (a) shows high pass(50Hz) filtered spike waveform, evoked electrically from RGCs of rd1 retina via sub-retinal stimulation. The arrow in black indicates the biphasic pulse stimulus and the red circle encircles the RGCs activity post stimulus application. Fig (b) is the generated peri-event raster of the spike response in (a), the green arrow indicates the spontaneous activity prior to stimulation; the red arrow shows clear stimulus correlated activity of the retinal ganglion cells. The rasters depicts typical oscillatory activity, of around 10Hz, which lasts for around 350ms (as above) and then returns to baseline activity. Oscillatory pattern suggests the inhibitory effects on the excitatory pathway thereby shaping the RGCs signals. The mean latency of the first RGC spike post subretinal stimulation was 27+/-3.2 ms (mean+/- SEM; n=4)

-0.5 0 0.5Time (sec)

0

20

40

Time(sec)

Counts/bin

100ms Bin width:3ms

100ms

20 counts/bin

10V

Spontaneous activity

39

Summary of latencies of RGCs of rd1 retina to epi-retinal and sub-retinal stimulations

Rd1 Retina(P60)

Epi-retinal stimulation

Sub-retinal stimulation

Mean Latency (ms)

24.3+/-0.94

27+/-3.2

No. of stimulus correlated responses(n)

10

5

Total no. of responses(N)

50

40

Value of latency: mean+/-SEM Table 1: Above table illustrates the mean response latency of stimulus correlated activity for epi-retinal and sub-retinal stimulation. It depicts also the successful number of stimulus correlated activity (electrical stimulations) out of the total number of RGCs responses (spontaneous activity). Table suggests no significant difference in latency of both the stimulation suggesting that both epi-retinal and sub-retinal stimulations stimulate the same neural element.

40

Fig 15: Peri-event raster plot showing suppression of spontaneous activity of RGCs in rd1 retina to local exogenous application of glutamate. Suppression is followed by excitation and increased firing rate. The black arrow indicates the glutamate application (2mM) and the red arrow indicates the increased excitation of RGCs post suppression. The blue arrow indicates the suppression(notch) post glutamate stimulation (n=5/10).

0 0.5 10

20

40

ch_36_unit_0

Time(sec)

Counts/bin

100ms Bin width:5ms

20 counts/bin

41

Fig 16: Peri-event raster plot shows oscillatory pattern of activity of RGCs post application of glutamate stimulation. Focal application of glutamate (2mM) is indicated by the black arrow. The blue arrow indicates the suppression post glutamate stimulation(notch) and the red arrow indicates the increased excitation of RGCs post suppression. The brown arrow shows the oscillatory activity over a period of time. The oscillations (around 10Hz) could be a result of activation of glutamate receptors on amacrine cells which in turn release GABA or glycine inhibiting the excitatory activity of RGCs(n=2/5).

0 0.5 1Time (sec)

0

5

10

ch_41_unit_0Ch_41_unit_0

Time(sec)

10 0 1

5

10

Counts/bin

100ms Bin width:5ms

5 counts/bin

0

42

Summary of response latencies of RGCs with respect to the distance from glutamate ejection site of rd1 retina.

Distance from ejection site(m) Rd1

Retina(P60)

Site of puff application,

30m, electrode diameter

Inter electrode distance,

200m

Diagonal inter electrode distance,

282m

>=400m

Latency (ms)

16+/-3

20+/-4.2

25+/-2.25 No prominent

effect of glutamate

No. of stimulus

correlated responses(n)

5

5

5

5

Total no. of responses(N)

10

10

10

10

Value of latency: mean+/-SEM Table 2: Above table illustrates the response latencies of the RGCs activity with respect to distance from the site of ejection of glutamate. There is no significant difference between the latencies with the interelectrode distance (200m) and diagonal interelectrode distance (282m) of the MEA. No prominent effect of glutamate was seen at >=400m. It depicts also the successful number of stimulus correlated activity( glutamate stimulation) out of the total number of RGCs responses (spontaneous activity)

43

Fig 17: Above graph indicates the spatio-temporal effectiveness of local glutamate stimulation. The above graph shows response latencies of RGCs in rd1 retina with respect from distance from ejection site of glutamate (2mM). The ejection site is take at 30m considering the diameter of the MEA electrode. The response latencies did not change significantly over different interelectrode distances, suggesting a week spatio-temporal resolution. Beyond 400m no prominent effect of glutamate stimulation was seen. (values indicate mean latency+/-SEM, n=5)

30

25

20

15

10

5

0

Re

spo

nse

la

tenc

y(m

s)

5004003002001000Distance from ejection site(m)

No prominent effect of local glutamate

44

Fig 18: Above graph shows the excitation of RGCs (in terms of normalized counts per bin). There is increase in activity with increase in glutamate (2mM) ejection duration until 100ms (to which all the values were normalized to) in rd1 retina. With further increase in ejection duration the activity decreased (at 200ms). There was a significant increase in activity from 20ms to 100ms (P

45

(a)

(b) Fig 19: Above peri-event rasters from the same experiment (a) shows suppression(notch) of spontaneous activity of RGCs in rd1 retina to local exogenous application of glutamate. Suppression is followed by excitation and increased firing rate. Fig (b) depicts local glutamate application in a bath solution of picrotoxin (50M)and strychnine(2M)and ACSF. The raster plot shows a level of suppression after glutamate application, however there is an overall increase in activity due to blockade of GABA& glycine .The black arrow indicates the glutamate application (2mM), the blue arrow indicates the suppression and the red arrow indicates the increased excitation of RGCs post suppression.

0 0.5 1Time (sec)

0

10

20

30

40

ch_25_unit_0

0 0.5 1Time (sec)

0

10

20

30

40

ch_25_unit_0

1 0.5

0

Counts/bin

0

10

20

30

40

10 counts/bin

Time(sec)

Ch_25_unit_0

Time(sec)

100ms Bin width:5ms

1 0.5

0

Counts/bin

0

10

20

30

40

10 counts/bin

Ch_25_unit_0

8V

100ms Bin width:5ms

46

(a)

(b)

Fig 20: Above peri-event rasters from the same experiment (a) shows suppression(notch) of spontaneous activity of RGCs in rd1 retina to local exogenous stimulation to glutamate+CTZ(50M). Suppression is followed by excitation and increased firing rate. Fig (b) depicts local glutamate application in a bath solution of CTZ and ACSF. The raster plot shows a considerable amount of reduction in the suppression effect post stimulation with overall increase in activity due to CTZ due to reduced desensitization of AMPA receptors as well as blocking subtypes of GABAA receptors. The black arrow indicates the application of Glu (2mM) + CTZ(50M). The blue arrow indicates the suppression(notch) post glutamate stimulation in (a) and the red arrow indicates the increased excitation of RGCs post suppression (n=4/10).

0 0.5 1Time (sec)

0

5

10

15

ch_31_unit_0

0 0.5 1Time (sec)

0

5

10

15

ch_31_unit_0Ch_31_unit_0

Ch_31_unit_0

0.5

Time(sec)

Counts/bin

0

5

10

15

0.0

100ms Bin width:5ms

5 counts/bin

Time(sec)

Counts/bin

0 0.5 1

10

0

5

15

100ms Bin width:5ms

5 counts/bin

47

(a)

(b)

Fig 21: Above peri-event rasters from the same experiment (a) shows suppression of spontaneous activity of RGCs in rd1 retina to local exogenous application of glutamate+CTZ(50M). Suppression is followed by excitation and increased firing rate. Fig (b) depicts local glutamate application in a bath solution of CTZ(50M), picrotoxin(50M), strychnine(2M)and ACSF. The raster plot shows no more suppression effect post stimulation with overall increase in activity due to blockade of GABA& glycine along with reduced desensitization of AMPA receptors .The black arrow indicates the application of Glu (2mM) + CTZ(50M). The blue arrow indicates the suppression(notch) post glutamate stimulation and the red arrow indicates the increased excitation of RGCs post suppression(n=3/7).

0 0.5 1Time (sec)

0

10

20

0 0.5 1Time (sec)

0

10

20

100ms Bin width:5ms

10 counts/bin

1 0.5 0

0

10

20

Counts/bin

Time(sec)

1

0.5 0 Time(sec)

Counts/bin

0

20

10

100ms Bin width:5ms

10 counts/bin

Ch_21_unit_0

Ch_21_unit_0

48

(a)

(b)

Fig 22: The above bar graph (a) represents the normalized success ratio of different modes of stimulation. It is evident that biochemical stimulation (BCHEM);N=5/10 holds a greater degree of successful stimulation of the degenerated retina in comparison to electrical stimulation (both epi-retinal (EPI);N=10/50 and sub-retinal (SUB) ;N=5/40. Additionally sub-retinal stimulation has a low success ratio in comparison to epi-retinal stimulation. In bar graph (b) the mean latencies from epi-retinal stimulation (24+/-0-94 ms), sub-retinal stimulation (27+/-3.2ms) and biochemical stimulation (22+/-3ms) is shown. This shows that the mean latencies from all the stimulations are significantly comparable, with no much of difference.

49

Summary of overall spatial resolution for electrical and biochemical stimulation in

rd1 retina.

Overall spatial resolution Rd1 Retina

Electrical stimulation

Biochemical stimulation

Spatial resolution achieved

Poor

Not well defined

Focality

Random

Well defined over a given distance(282m)

Possible reasons

Random spread of retinal ganglion axons

Poor MEA resolution

Table 3: Above table illustrates the comparison between electrical and biochemical stimulation based upon the overall spatial resolution achieved. Although the above experiments could not resolve well the spatial resolution in biochemical stimulation, however the focality in biochemical stimulation was well defined in comparison to electrical stimulation. The table also shows the possible reason due to which the spatial resolution was not well achieved in both the cases. Thus, suggesting possible reasons for trouble shooting and for achieving a good spatial resolution.

50

Discussions and Implications

The present study examined the spiking activity of the retinal ganglion cells in degenerated

rd1 retina occurring spontaneously and evoked by repetitive electrical and biochemical

stimulations. The major focus intended to study the responses of the retinal ganglion cells on stimulation of bipolar cells electrically as well as biochemically. Additionally the study

included an analysis of spatiotemporal effectiveness of the retinal ganglion cells accounted

due to electrical stimulation and due local application of glutamate in rd1 retina. It aimed at

elucidating the parameters for electrical stimulation, such as, thresholds and pulse

paradigms for stimulating rd1 retina via epi-retinal and sub-retinal stimulation. It addressed

at a preliminary comparison of the epi-retinal stimulation versus sub retinal stimulation, in

order to determine, which of the two approaches would be suitable enough for electrical

stimulation. Simultaneously, exploration of biochemical stimulation was done during the

thesis. An attempt was made at devising a locally applied glutamate application onto the

degenerated retina, as an initial step to determine the feasibility of a neurotransmitter-

based retinal prosthesis. This gave way to compare two potent candidates of retinal

prostheses and to work towards developing an efficient model for the visual

neuroprosthetics that could elucidate spatio-temporal parameters to transmit maximum

visual information.

Electrical stimulation: Bionic implants fall into two categories. Sub-retinal implants and

epi-retinal implants. (Humayun et al, 1996; Chow and Chow et al, 1997). Subretinal implants are engineered with the goal of replacing the lost photoreceptors and stimulating

the surviving retinal circuits to restore natural visual information, which would notably

include information on light adaptation, object motion, contrast vision and so on. However epi-retinal implants placed on vitreal surface of retina would theoretically drive ganglion cell

51

populations, by surpassing much of the retinal circuitry. These approaches depend

explicitly upon the preservation of the neural retina. In this study I could successfully

optimize the parameters for visualizing retinal ganglion cell activity on planar

microelectrode array (MEA) which in itself was the rate limiting step of the thesis. Secondly I could stimulate rd1 degenerated retina, via epi-retinal as well as sub-retinal stimulation.

The degenerated retina responded to repetitive electrical stimulation and was evident from

the retinal ganglion cells responses using multi-electrode array. As from the above results it

was evident that the stimulation thresholds and pulse paradigms were comparable in both

types of stimulations. However, the response pattern differed, in sub-retinal stimulation

oscillatory behaviour of the ganglion cells was observed .This could be due the co-

activation of the vertical as well as the lateral pathways of the retina. The vertical pathway

involves signal transduction from the bipolar layer to the ganglion cell layer which is

excitatory and uses glutamate as a major neurotransmitter. The lateral pathway involves inhibition from the horizontal cells and the amacrine cells. The interaction of the lateral

pathway with the vertical pathway shapes the retinal ganglion cell response yielding an

oscillatory pattern ( Neuenschwander et al, 2002). Yazulla et al (Yazulla et al, 1997) have demonstrated earlier a two fold increase in GABA content of rd1 retinas which could

account for such oscillatory patterns. This pattern however was not visible for epi-retinal

stimulation possibly as epi -retinal stimulation surpassed the inner retinal circuitry and

inhibitory circuits hence yielding a sharp rise in activity post stimulation and subsequently

fading away.

In matter of temporal resolution, the response latencies were also comparable for epi-

retinal and sub-retinal stimulation, which suggests that both sub-retinal and epi-retinal

stimulation more or less activate the same neural element and utilize the same amount of

neural processing for transmitting visual information (Thomas O Hearn,et al, 2006). These spikes could be classified as fast spikes or short latency spikes which lasted for around

52

2ms and long latency spikes which was around >=15ms. In the above results only long

latency spikes could be discerned as the stimulus artifacts obscured the first few

milliseconds of the recordings thereby masking the short latency spikes. These variations

of spike latency are possibly due to different retinal ganglion cell types as well as the

different stimulation parameters. The short latency spike yielded from direct stimulation of

the retinal ganglion cells and the long latency spikes could be due to the stimulation of the

network of ganglion cells (Jensen & Rizzo, 2007). However due to the inherent lack of cellular specificity, electrical stimulation used in most

present devices is limited in spatial distribution. Additionally electrical stimulation non-

specifically stimulates somata and axons of ON & OFF retinal ganglion cells, bipolar cells

and very likely also the inhibitory cells like amacrine cells in nonphysiological sequences

that may reduce contrast and spatial localisation within the retina (Finlayson and Iezzi, 2010). Thus electrical stimulation of the rd1 retina has few limitations in encoding important sensory features used in normal central visual processing.

In this study the spatial distribution of electrical stimulation was also addressed. The

stimulus correlation observed in few channels was variable in their distribution with respect

to the stimulation site which suggests that the electrical stimulation could activate a network

of different ganglion cells. A probable reason could be due to continuous remodelling of the

retina, which on electrical stimulation spread the stimulus pulse over the entire network

activating group of cells randomly which aligns to the stimulus pulse. Another reason could

be random distribution of retinal ganglion cell axons in degenerated retina (Ryu et al, 2009). To the extent of spatial resolution limitation, one could hypothesize a more naturalistic

means of stimulating retinal ganglion cells (RGCs) for efficient retinal prosthesis that could decipher the spatial resolution. This was addressed by focal application of glutamate as the

neurotransmitter based prosthesis that would more or less mimics the natural vision.

53

Biochemical stimulation: Due to the shortcomings of the electrical stimulations in

addressing the spatial resolution, biochemical implants came into play. However several

groups are working towards this prosthesis (Noolandi et al, 2003) yet the problems in localized chemical delivery has been a major hurdle in most of the devices. To achieve the resolution to stimulate at a single cell level or few cells is what the prosthesis aims at and

thereby generating a high spatial resolution for achieving good vision. In the thesis the

major aim was to elucidate the role of focal glutamate delivery onto rd1 retinae placed on a multi-electrode array. This gave an opportunity to analyse the activity pattern over a

network and also visualize the focality of the application. From the above results it was

seen that local exogenous application of glutamate at the site of spontaneous activity

induced an initial suppression of firing activity followed by an increased activity (which looked more or less like notch). One would anticipate that the suppression was due to two major reasons. One, as there is AMPA receptors on the retinal ganglion cells and AMPA receptors on application of glutamate desensitize rapidly (Bloomfield and Dowling et al, 1985). The other reason could be that glutamate application depolarizes the glutamate receptors present on the amacrine cells which in turn release GABA or glycine which could

lead to inhibition of the excitatory pathway. For addressing these two possibilities,

application of glutamate with different bath applications was done as shown above. Bath

application of strychnine and picrotoxin (gycinergic and GABAergic blockers) increased the basal activity; however there still existed suppression in activity (presence of a notch) as shown. This suggested that although there was an increase in activity, inhibitory signals did

not contribute much to the suppression of the firing activity post glutamate stimulation,

which lead to addressing the other possibility of the suppression of activity. Bath

application of cyclothiazide alone lead to considerable decrease of suppression of

activity(diminishing the notch) , with an overall increase in basal activity. This was due to reduced desensitization effect of AMPA receptors suggesting that the desensitization of

54

AMPA receptors contributed substantially to the suppression in activity. Cyclothiazide is

believed to destabilize the desensitized state of the AMPA receptors (Yamada and Tang, 1993) .Additionally it inhibits GABAA mediated currents, thus increasing glutamatergic response (Deng & Cheng, 2003) thereby facilitating high frequency synaptic transmission over a longer period of time. Therefore a bath application of strychnine, picrotoxin and

cyclothiazide combination removed this suppression of activity thereby suggesting that

glutamate application has both direct and indirect implications on retinal ganglion cells.

Furthermore, interestingly, in few experiments the RGCs responded to glutamate

application in an oscillatory pattern which could suggest that glutamate application could

activate the glutamate receptors of the amacrine cells which on release of GABA or glycine

gave a timely inhibition, although this requires further investigation to quote it conclusively.

The response latency accounted for the temporal dynamics. It was further observed that

the RGCs response latencies to locally applied glutamate were relatively comparable to

latencies obtained via electrical stimulation, which suggested that a naturalistic

phenomenon had a temporal effectiveness like that of electrical stimulation. However this

needs further investigation with age groups of advanced photoreceptor degeneration to

study whether the sequelae associated with progressive photoreceptor degeneration

affected the sensitivity and properties like latencies of RGCs to glutamate, inorder to draw

conclusive information. The spatial effectiveness was addressed with the resolution of the

MEA. From the above results it was seen that the RCG response latencies did not vary

significantly with the distance from the glutamate ejection site which was not as expected. This probably could be due to the poor MEA resolution or dense network of axons running

across the degenerated retina or physiologically, the spatial distribution depends on the cell

type rather than the RGC distance from the site. To resolve this issue a MEA with higher

resolution needs to be considered.

55

The spread of inhibition due to the inhibitory effects from the amacrine cells and horizontal

cells and the temporal dynamics warrants further investigation in determining an efficient

spatio-temporal resolution. Furthermore this investigation could help in determining an

optimal separation of ejection sites in a prototype retinal prosthetic device. The duration of ejection did not change the firing pattern of the RGCs or the latency of response, but, however increased the level of activity this increase in activity was due to increased

exposure of the retinal ganglion cells to a glutamate rich environment. The time duration of

ejection is indeed an important parameter to be considered as one could modulate the RGCs activity by varying the durations of the glutamate application (Finlayson and Iezzi, 2010). Apart from the above aspects the glutamate concentration plays a role in modulating the RGC activity. At higher concentration chances of excitotoxicity due to glutamate could

lead to overstimulation and death of the retina. Therefore an optimal level of glutamate

concentration is required to prevent toxicity. An application of , 2mM glutamate could elicit

RGCs response in rd1 retina, in vitro. This concentration was adapted from different

literatures one being the Finlayson et al, 2010, which suggested that a 2mM concentration

was the minimum concentration of glutamate to efficiently stimulate the RGCs in normal

retinas. Although, this concentration was indeed higher than the normal synaptic condition

in vivo (around 0.5mM)( ( Finlayson and Iezzi, Springer, In press).However in the above experiments a high perfusion rate of 6ml/min could rapidly remove the glutamate

preventing any obvious amount of toxicity. This implies that lower concentrations of locally

applied glutamate could be effective in designing an effective prosthesis in vivo. However

to design a precise prosthetic device requires a lot of considerations.

The goal of the thesis was to obtain a birds eye view comparative analysis of the electrical

stimulation versus biochemical stimulation. Apart from doing a comparative analysis the

main goal intended to elucidate different aspects of each of this prosthesis in depth.

However a substantial amount of experiments needs to be performed considering other

56

dimensions like optimal concentration levels, exact location of site of ejection, multiple application of glutamate with specific time interval and so on and so forth to draw a

concrete conclusion. However due to time limitation, this could not be accomplished. This

gave an opportunity to reason out the possibilities and shortcomings of each of these

prosthesis models. It was seen that although electrical stimulation could provide a better

temporal resolution, in matter of short latency spikes, but it could not address the spatial

distribution which indeed is an important parameter for any retinal implant. However it was

seen that the biochemical stimulation had comparable time latency as electrical stimulation.

Also the spatial distribution was addressed by biochemical stimulation to quite some extent,

implying that biochemical stimulation has an advantage over electrical stimulation at

addressing spatial complexity which electrical stimulation, could not. Additionally one could

try reasoning out that the electrical(sub-retinal) stimulation and biochemical stimulation are comparable as they use the entire machinery of the remnant retina, which suggests that

proper standardization of sub-retinal parameters (like positioning of electrode) could hel