EPILEPTIFORM PROPAGATION IN THE HIPPOCAMPUS
AND A RECORDING ARRAY SYSTEM FOR IN-VITRO
ANALYSIS
by
ANDREW BECKER KIBLER
Submitted in partial fulfillment for the requirements
For the degree of Doctor of Philosophy
Thesis Advisor: Dominique M. Durand, Ph.D.
Department of Biomedical Engineering
CASE WESTERN RESERVE UNIVERSITY
May, 2011
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis of
Andrew Kibler
candidate for the Ph.D. degree *.
(signed) Dominique Durand
(Chair of the committee)
Christopher Wilson
Christian Zorman
Melissa Knothe Tate
(date) 03/31/2011
*We also certify that written approval has been obtained for any proprietary material contained therein.
DEDICATION
To my wife, Anna Marie V. Kibler for her selfless support and unconditional love,
and to my parents and grandparents, who inspired and encouraged my study of
science and engineering.
TABLE OF CONTENTS
DEDICATION………………………………………………………..III
TABLE OF CONTENTS…………………………………………….IV
LIST OF TABLES……………………………………………………X
LIST OF FIGURES…………………………………………………..X
ACKNOWLEDGEMENTS………………………………………….XIII
ABSTRACT…………………………………………………………..XIV
CHAPTER 1 DISSERTATION INTRODUCTION AND OBJECTIVES…………..…1
1.1 EPILEPSY…………………………………………………………...….2
1.2 ANIMAL MODELS FOR THE STUDY OF EPILEPSY……...…….3
1.3 FOUR-AMINOPYRIDINE MODEL OF EPILEPSY……….………4
1.4 IN VITRO PREPARATIONS FOR THE STUDY OF THE HIPPOCAMPUS……………………………………………………….6
1.5 PLANAR MULTI-SITE IN VITRO RECORDING…………………7
1.6 CLINICAL RELEVANCE...………………………………….……….9
1.7 THESIS OBJECTIVES AND ORGANIZATION…………….……...9
1.7.1 OBJECTIVE 1 – TO DETERMINE THE VIABILITY OF THE UNFOLDED HIPPOCAMPUS PREPARATION…………….…..11
1.7.2 OBJECTIVE 2 – TO ANALYZE ORTHOGONALLY PROPAGATING EPILEPTIFORM ACTIVITY IN THE UNFOLDED RODENT HIPPOCAMPUS………………….……..13
1.7.3 OBJECTIVE 3 – TO DEVELOP AND CONSTRUCT A MEMS- BASED PENETRATING MICRO-ELECTRODE ARRAY FOR 2-
DIMENSIONAL UNFOLDED HIPPOCAMPUS RECORDING IN VITRO………………….……………………………….…………14
CHAPTER 2 A NOVEL UNFOLDED HIPPOCAMPUS PREPARATION…….……20
2.1 ABSTRACT……………………………………………………………21
2.2 INTRODUCTION……………………………………………….…….22
2.3 MATERIALS AND METHODS……………………………….……..24
2.3.1 ANIMALS USED……………………………………………..24
2.3.2 UNFOLDED HIPPOCAMPUS PREPARATION…………….24
2.3.3 RECORDING CHAMBER……………………………………26
2.3.4 ELECTRICAL RECORDING…………………………………27
2.3.5 OPTICAL IMAGING………………………………………….27
2.4 RESULTS……………………………………………………………….29
2.4.1 ELECTROPHYSIOLOGICAL PROPERTIES OF THE PLANAR HIPPOCAMPAL PREPARATION……………………………29
2.4.2 EFFECTS OF TEMPERATURE……………………………...29
2.4.3 INTRA-HIPPOCAMPAL RECORDING AT DEPTHS………29
2.4.4 LONGITUDINAL INTERCONNECTION OF ALVEAR FIBERS..31
2.4.5 OPTICAL IMAGING………………………………………….32
2.4.6 HISTOLOGICAL IMAGING………………………………….33
2.4.5 SPONTANEOUS INTERICTAL-LIKE PROPAGATION..….33
2.5 DISCUSSION…………………………………………………………...34
2.5.1 UNIQUE FEATURES OF THE PLANAR HIPPOCAMPAL PREPARATION………………………………………………34
2.5.2 SUMMARY OF FINDINGS…………………………………..34
2.5.3 FUTURE WORK………………………………………………35
2.6 FIGURE CAPTIONS………………………………………………..…37
2.7 FIGURES………………………………………………………………..39
CHAPTER 3 ORTHOGONAL WAVE PROPAGATION OF EPILEPTIFORM ACTIVITY IN THE PLANAR MOUSE HIPPOCAMPUS IN VITRO…47
3.1 ABSTRACT…………………………………………………………..…48
3.1.1 PURPOSE……………………………………………………...48
3.1.2 METHODS…………………………………………………….48
3.2.3 RESULTS……………………………………………….……..48
3.1.4 DISCUSSION………………………………………….………48
3.2 INTRODUCTION………………………………………………………49
3.3 MATERIALS AND METHODS………………………….……………51
3.3.1 TISSUE PREPARATION………………………………………51
3.3.2 MULTI-SITE EXTRACELLULAR RECORDING AND ANALYSIS………………………………………………….….52
3.3.3 VOLTAGE SENSITIVE DYE RECORDING………………....53
3.4 RESULTS…………………………………………………………..……54
3.4.1 ORTHOGONAL WIDESPREAD ACTIVATION OF CA1-CA3 FROM A SINGLE STIMULUS IN CA1………………………54
3.4.2 ORTHOGONAL PROPAGATION FROM CA1 INTO CA3…55
3.4.3 SPONTANEOUS SELF-PROPAGATING LONGITUDINAL WAVE……………………………………………………..……57
3.4.4 SYNAPTIC DEPENDENCE OF SPONTANEOUS SELF-
PROPAGATING LONGITUDINAL WAVE IN THE CA3 REGION……………………………………………………..…58
3.4.5 EFFECT OF CA3 LESION ON LONGITUDINAL PROPAGATION………………………………………….……59
3.5 DISCUSSION…………………………………………………...………60
3.6 CONCLUSION…………………………………………………………64
3.7 GRANTS…………………………………………………………….…..65
3.8 FIGURE CAPTIONS…………………………………………………..66
3.9 FIGURES……………………………………………………………..…69
CHAPTER 4 A SUBSTRATE-TRANSPARENT MICRO-ELECTRODE ARRAY SYSTEM FOR IN VITRO RODENT HIPPOCAMPUS RECORDING…77
4.1 ABSTRACT………………………………………………………………78
4.2 INTRODUCTION…………………………………………………….…79
4.3 MATERIALS AND METHODS……………………………………..…81
4.3.1 TISSUE PREPARATION………………………………….……81
4.3.2 ELECTRICAL RECORDING………………………………..…82
4.3.3 OPTICAL RECORDING………………………………….….…83
4.3.4 MICRO-ELECTRODE ARRAY DESIGN AND CONSTRUCTION…………………………………………...….84
4.3.5 MICRO-ELECTRODE ARRAY AMPLIFIER SYSTEM………86
4.5 RESULTS………………………………………………………………....87
4.5.1 ARRAY FABRICATION……………………………….………87
4.5.1 SACRIFICIAL PILLARS……………………………….………87
4.5.1 NEURAL ACTIVITY MEASUREMENT..…………….………88
4.5.2 MULTI-CHANNEL RECORDING………………………..……89
4.6 DISCUSSION……………………………………………………..………90
4.7 CONCLUSION……………………………………………………………93
4.8 ACKNOWLEDGEMENTS……………………………………..…….…94
4.9 FIGURE CAPTIONS……………………………………………….……95
4.10 FIGURES………………………………………………………..………98
CHAPTER 5………………………………………………….………..106 5.1 SUMMARY AND CONCLUSIONS………………………….……….107
5.2 OBJECTIVE 1 – TO DETERMINE THE VIABILITY OF THE UNFOLDED HIPPOCAMPUS PREPARATION………………………..108
5.3 OBJECTIVE 2 – TO ANALYZE ORTHOGONALLY PROPAGATING EPILEPTIFORM ACTIVITY IN THE UNFOLDED RODENT HIPPOCAMPUS……………………………………………………….….109
5.4 OBJECTIVE 3 – TO DEVELOP AND CONSTRUCT A MEMS-BASED PENETRATING MICRO-ELECTRODE ARRAY FOR 2-DIMENSIONAL UNFOLDED HIPPOCAMPUS RECORDING IN VITRO…………….110
5.5 FUTURE WORK………………………………………………...……111
6 APPENDIX A SUPPLEMENTAL MATERIAL REGARDING THE UNFOLDED HIPPOCAMPUS PREPARATION AND LONGITUDINAL EPILEPTIFORM PROPAGATION………………………………….…116
6.1 DETAILS OF THE PROCEDURE OF PREPARING THE PLANAR UNFOLDED MOUSE HIPPOCAMPUS………………….…………….117
6.2 DETAILS ON THE METHODS USED IN VOLTAGE SENSITIVE DYE RECORDING………………………….…………………………….……118
6.3 DISCUSSION OF THE UNFOLDED HIPPOCAMPUS PREPARATION……………………….…………………………….……118
6.4 SYNAPTIC DEPENDENCE OF EVOKED ORTHOGONAL PROPAGATION..…………………….…………………………………..120
6.5 SUPPLEMENTAL REFERENCES..…………………….…….……121
6.6 SUPPLEMENTAL FIGURE CAPTIONS.…………………………122
6.7 SUPPLEMENTAL FIGURES…………….…………………………123
7 APPENDIX B SCHEMATICS OF THE ARRAY AMPLIFIER SYSTEM……..….…125
8 APPENDIX C FREQUENCY RESPONSE OF THE ARRAY AMPLIFIER SYSTEM………………………………………………………………..…138
8.1 FREQUENCY RESPONSE MEASUREMENT..…….…………….139
8.2 FIGURES………………………………………....…….…………….140
7 REFERENCES...... 141
LIST OF TABLES
Table 1.1………………………………………………….……………………17
LIST OF FIGURES
FIGURE 1.1…………………………………………………………………….18
FIGURE 1.2…………………………………………………………………….18
FIGURE 1.3…………………………………………………………………….19
FIGURE 1.4…………………………………………………………………….19
FIGURE 2.1…………………………………………………………………….39
FIGURE 2.2…………………………………………………………………….39
FIGURE 2.3…………………………………………………………………….40
FIGURE 2.4…………………………………………………………………….40
FIGURE 2.5…………………………………………………………………….41
FIGURE 2.6…………………………………………………………………….42
FIGURE 2.7…………………………………………………………………….43
FIGURE 2.8…………………………………………………………………….44
FIGURE 2.9…………………………………………………………………….44
FIGURE 2.10…..……………………………………………………………….45
FIGURE 2.11…..……………………………………………………………….46
FIGURE 3.1…………………………………………………………………….69
FIGURE 3.2…………………………………………………………………….70
FIGURE 3.3…………………………………………………………………….71
FIGURE 3.4…………………………………………………………………….72
FIGURE 3.5…………………………………………………………………….73
FIGURE 3.6…………………………………………………………………….74
FIGURE 3.7…………………………………………………………………….75
FIGURE 4.1…………………………………………………………………….98
FIGURE 4.2…………………………………………………………………….99
FIGURE 4.3…………………………………………………………………….99
FIGURE 4.4…………………………………………………………………….100
FIGURE 4.5…………………………………………………………………….100
FIGURE 4.6…………………………………………………………………….101
FIGURE 4.7…………………………………………………………………….102
FIGURE 4.8…………………………………………………………………….102
FIGURE 4.9…………………………………………………………………….103
FIGURE 4.10…………………………………………………………….….….103
FIGURE 4.11……………………………………………………………..…….104
FIGURE 4.12……………………………………………………………..…….105
FIGURE 6.1…………………………………………………………………….123
FIGURE 6.2………………………………………………………..…….….….123
FIGURE 6.3………………………………………………………..……..…….124
FIGURE 7.1…………………………………………………………………….126
FIGURE 7.2………………………………………………………..…….….….127
FIGURE 7.3………………………………………………………..……..…….128
FIGURE 7.4…………………………………………………………………….129
FIGURE 7.5………………………………………………………..…….….….130
FIGURE 7.6………………………………………………………..……..…….131
FIGURE 7.7………………………………………………………..……..…….132
FIGURE 7.8……………………………………………………..………..…….133
FIGURE 7.9………………………………………………………..……..…….134
FIGURE 7.10………………………………………………………..……..…...135
FIGURE 7.11………………………………………………………..……..…...135
FIGURE 7.12………………………………………………………..……..…...136
FIGURE 7.13………………………………………………………..……..…...136
FIGURE 7.14………………………………………………………..……..…...137
FIGURE 8.1…………………………………………………………………….140
FIGURE 8.2………………………………………………………..…….….….140
FIGURE 8.3………………………………………………………..……..…….140
ACKNOWLEDGEMENTS
This work would not have been possible without the help and support of many
others to whom I am extremely grateful.
I thank my advisor, Dr. Dominique Durand, for inspiration in research, for
guidance in development and communication, and for his dedication in support of our lab
and research.
I thank the members of my committee: Dr. Melissa Knothe Tate, Dr. Christian
Zorman, and Dr. Christopher Wilson for their time, help, and support during the research and information delivery process.
I thank the Neural Engineering Center and fellow in vitro neural researchers in
our lab for the friendship, good times, and commiseration during this period of growth and adventure.
Most importantly, I thank God for giving us the desire to learn, discover, and
create and in the process improve the lives of those around us and those to come.
This work was financially supported by the GAANN Neural Engineering Training
grant and by NIH Grant 5-R01-NS-040785.
Orthogonal Epileptiform Propagation in the Hippocampus and a Micro-electrode
Array System for In-vitro Recording
Abstract
by
ANDREW BECKER KIBLER
Individuals with epilepsy experience recurrent and unprovoked seizures
characterized by uncontrolled, excessive neurological activity. Seizures can be
debilitating and often resistant to available drug therapies. Temporal lobe epilepsy
commonly initiates from foci within the hippocampus, making it a potential target for
surgical treatments in cases where common anti-epileptic drugs have failed to produce
desired results. The field of neuroscience is rich with studies of the hippocampus,
commonly performed using rodent animal models. Most commonly, the hippocampal
slice preparation has been used to study the rodent hippocampus in vitro. This
preparation, although robust, severs longitudinal hippocampal networks such as the recurrent excitation network within CA3. With the unfolded hippocampus preparation described in this manuscript, entire hippocampi from young mice can now be kept alive for an extended period of time. A portion of this study examines the role of orthogonal pathways preserved in the unfolded hippocampus in the propagation of epileptiform waves across the tissue.
The unfolded hippocampus preparation presents a unique opportunity to study the planar network of pyramidal cells extending from CA1 to CA3. To facilitate this, a micro-electrode array of 8 by 8 recording channels was developed with spikes that
penetrate into the pyramidal cell layer for recording. This recording array improves on
currently available in vitro arrays by being both penetrating and transparent to light. An
amplifier was designed and built for the array with 64 individual amplifiers for the
electrodes in the array with a bandwidth of 0.5 Hz to 4 kHz.
The data presented in this manuscript suggest that the unfolded hippocampus
preparation maintains normal electrophysiology and as such is useful for the study of the
same. Evoked responses showed an excellent correlation to responses seen in slices,
indicating preservation of neurons. Experimental wave propagation data suggests that a
synaptically dependent 4-AP induced epileptiform wave is generated in the CA3 and
propagates across the pyramidal cell matrix across CA3 and into CA1. Further, this epileptiform wave propagation can be arrested by a selective local transverse lesion of the
CA3. Finally, tests done with the completed array system show it is capable of recording
epileptiform events across the CA1–CA3 in a more robust manner and with lower noise
than with traditional voltage sensitive dye RH-414. Together, this work furthers the field
of hipppocampal study as it relates to the CA3–CA1 and CA3–CA3 networks and
enables a more efficient and detailed analysis of activity in these regions through the use
of the recording array system described here.
CHAPTER 1
Dissertation Introduction and Objectives
1 1.1 EPILEPSY
According to the World Health Organization, epilepsy is a disease that affects
approximately 50 million individuals worldwide. About 8 in every 1000 world
inhabitants have epilepsy of some form, as it is the most common serious neurological
disorder. Patients suffering with epilepsy have recurrent seizures resulting from excessive
neuro-electrical activity in a portion or all of their brain. Seizures can be expressed in
many symptomatic forms including disturbances of movement, feeling or consciousness
(Brodie, 2000). Epilepsy can be initiated from physical trauma such as head injuries or
other neural damage, or it can be idiopathic, commonly passed down through genetic
defects. About 60% of new diagnoses are idiopathic (National, 2004). In fixed-population
studies, epilepsy sufferers are associated with a higher mortality rate, at 2–3 times higher
than the normal population after a median follow-up of 6.9 years (Cockerell, 1994).
Seizures are classified into two basic groups — focal and generalized. Focal seizures
occur in only a part of the brain, and can spread to become secondarily generalized
seizures, affecting the entire brain, including the motor cortex. Generalized or secondarily
generalized seizures can result in six symptoms: tonic-clonic, which is characterized by
unconsciousness, convulsions, and muscle rigidity; absence, characterized by a brief loss
of consciousness; myoclonic, characterized by sporadic jerking movements; clonic, characterized by repetitive jerking movements; tonic, characterized by muscle rigidity;
and atonic which is characterized by loss of muscle tone. Epilepsy is typically diagnosed
with blood tests (to look for metabolic factors), EEG recordings (to detect characteristic
abnormal waveforms), and MRI (to search for damaged tissue). By using Functional MRI
and by looking for areas of sclerosis, neurosurgeons are able to determine areas of the
2 brain that are affected most by the seizures. The two primary regions of the brain that are
involved in temporal lobe epilepsy are the cerebral neocortex and the hippocampus
(Walter, 1969).
Three basic questions asked in fundamental epilepsy research are: How do seizures
begin? How do seizures propagate from one part of the brain to another? How do the
seizures self-terminate? This study provides evidence to address the first and second
question using the intact hippocampus. First, in chapter two a new unfolded hippocampus
preparation is described which allows for the study of epileptiform generation and
propagation in transverse and longitudinal directions. Secondly, in chapter three this
propagation is analyzed, revealing that longitudinal synaptically mediated interconnections play a significant role in epileptiform propagation. Thirdly, in chapter four, a recording array system is developed to more efficiently analyze epileptiform propagation in the hippocampus.
1.2 ANIMAL MODELS FOR THE STUDY OF EPILEPSY
In most scientific research, animal models aid researchers in analyzing complex
or out-of-reach systems, and provide a basis for theoretical experimentation on these
systems. In the case of neural research on epilepsy, animal models provide access to
variables and networks that would be difficult or impossible to study in a living human
being without causing unacceptable side-effects and risks. There are a number of
methods of generating epilepsy in animals, falling into four categories: kindling, ionic,
pharmacological, and genetic. Repeated electrical or synaptic stimulation in the brain can
lead to increased activity — synchronized and desynchronized — that eventually results
3 in epileptiform discharges; this is the kindling model. Ionic models such as low calcium,
zero magnesium, and high potassium artificially alter the balance of charge species
between the intra and extra-cellular spaces. This causes the typical ionic process involved
in synaptic transmission or axonal conduction to result in seizure-like discharges in the
tissue (Bikson, Durand, 2002).
There are several genetic models where animal epilepsy is induced by a specific channel
mutation. While many of the genotypic models exhibit epilepsy similar to that seen in humans, none fulfill all of the symptoms associated with any particular form of human epilepsy. Genetic models tend to target a specific channel mutation, such as the SCN-2a sodium channel mutation which can lead to early mortality, making strain sustenance and long-term study problematic (Kearney et al., 2001; Kile et al., 2008). Furthermore, specific mutations are not analogous to that seen in many human cases.
Pharmacological models involve introducing a synaptic agonist or antagonist to the solution, which can have a number of effects such as blocking NMDA or GABA receptors, activating glutamatergic receptors, or blocking or enhancing ion channels in the membrane. 4-Aminopyridine (4-AP) is a specific potassium channel blocker that is able to induce spontaneous epileptiform activity without reducing GABA inhibition. It may, therefore, provide an appropriate model for investigating the mechanisms of epilepsy where inhibition is preserved.
1.3 FOUR-AMINOPYRIDINE MODEL OF EPILEPSY
Epileptiform activity induced by the presence of 4-AP in hippocampal slices is
characterized by short recurrent discharges sometimes combined with slow field potential
4 shifts and long lasting (100ms) depolarization of cells (Perreault and Avoli, 1991). While
the precise mechanism of activation is unknown, it has been shown that, upon application
of 4-AP, the frequency and strength of EPSPs and IPSPs in CA3 cells increases
(Perreault and Avoli, 1991). Over time, these EPSPs form clusters and increase in
synchrony at which point a giant EPSP (known as paroxysmal depolarization) is formed
and the cell has a burst of action potentials. The first few action potentials may not be
synchronized with the surrounding cells, but the final potentials are, resulting in a
recordable extracellular signal. These bursts occur at a frequency of 0.61± 0.29 s and last
25–80 ms. The mechanism underlying these bursts was analyzed pharmacologically
using the glutamate NMDA receptor antagonist 3-(2-Carboxypiperazin-4-yl)propyl-1-
phosphonic acid (CPP) and the glutamate kainate receptor antagonist 6-cyano-7- nitroquinoxaline-2,3-dione (CNQX). The bursting pattern was not affected by CPP but was abolished by CNQX, while the long lasting depolarizations were not affected by either. It has been argued that the presence of 4-AP may directly affect the kinetics of
Ca++ channels responsible for glutamate release, prolonging the release and increasing the
extra-cellular concentration of the neurotransmitter (Herrero, 1991). Also, 4-AP induces
an increase in extra-cellular potassium levels during long-lasting field potential depressions as they wash across the tissue. It has been shown that long-term microdialysis of 35–70 mM concentrations 4-AP causes neuronal lesions in CA1 and
CA3 regions related to high glutamate levels in proximity to the synapses (Pena, 1999;
Murphy et al., 1989; Olney et al., 1983). Likewise, lesions in the CA1 and CA3 are the
primary hippocampal damage in mesial temporal sclerosis (MTS) (Liu et al., 1995;
Shinnar, 2003).
5 Although ionic and pharmacological models of epilepsy are not a direct model of epilepsy found in humans, studies of such models reveal unique properties of the hyper- excited state of neurons and networks. This insight is generally accepted to be of value to understanding the mechanisms of neuronal hyper-excitability found in traditional epilepsy. This work aims to reveal how potassium channel blocker 4-AP generated seizure like activity propagates in the intact planar hippocampus, which is covered in
Chapter 3.
1.4 IN VITRO PREPARATIONS FOR THE STUDY OF THE
HIPPOCAMPUS
The orderly structure of the hippocampus has been a focal point of
electrophysiological research for many years. The weight of this research has produced
an abundance of data, leading to important findings in the health and biomedically
oriented fields. There traditionally have been three ways that the hippocampus has been
studied — in vivo, extracted and sliced, and dissociated and cultured. Advantages of the
brain slice preparation include the ability to control the external medium, visualize the
tissue layers under microscope, and control pharmacological agents (Aitken et al., 1993).
The hippocampal excitatory pathway has a “lamellar” organization (Andersen, 1971),
meaning that it has physiologically differentiable layers primarily in two dimensions. The
third dimension, along the fornix, consists of repetitions of these lamellar circuits along
with some lesser-understood inhibitory and excitatory pathways. In vitro slices make use
of this organization, as they are made in a plane transverse to the long axis of the
hippocampus, and by doing so preserve the major excitatory axon tracts (Teyler 1980).
6 Another benefit of this preparation is that the unfolded hippocampus is thin enough to allow oxygen to diffuse throughout the whole tissue.
Longitudinal hippocampal slices have also been studied, but because of the loss of the major pathways their utility is limited. In either case, the complexity of the entire neural circuit including substantial longitudinal projections is lost. (Amaral and Witter,
1989, Brown and Zador, 1990). Work is progressing to overcome these limitations in vivo
(Feng and Durand, 2003) and by isolating the whole hippocampus in vitro (Khalilov,
1997; Wu and Shen, 2002). Khalilov (1997) demonstrated the Intact Hippocampal
Formation (IHF), where the entire hippocampus is removed and studied as a whole. Even though this preparation can successfully keep the intact mouse hippocampus alive for an extended period of time, the IHF preparation has an age limitation (P0 – P10) to keep the formation small enough for proper oxygen diffusion. Mice older than P10 have a thicker hippocampus, leading to the hypoxic death of deeper tissues. For some purposes, studies of older mice are preferred in neurological experiments. Wu and Shen increased the age limit to P28 by cutting off the dentate gyrus behind the hippocampal fissure (Wu and
Shen, 2002). This results in thinner and thus more oxygenated tissue.
1.5 PLANAR MULTI-SITE IN VITRO RECORDING
With many preparations, not just the unfolded hippocampus, multi-site recordings are necessary to study various distance effects and propagation. Voltage sensitive dyes have been developed that implant in the cell membrane and emit fluorescence amplitudes modulated by the trans-membrane potential. These dyes, such as Di-8-Annepps and
RH414 can be very useful in studying large areas of tissue at once, however they suffer
7 from several problems. First, deep tissues such as those found in the intact hippocampus are difficult to study due to light scattering effects. Secondly, the dyes exhibit
phototoxicity in the intensities of light necessary to record high-speed images, causing a
change in cell response and noticeable extracellular response changes. (Chang, 2003) In
particular, phototoxicity renders imaging of epilepsy models such as low calcium very
difficult, as the spontaneous discharges tend to cease during exposure. Finally, long term
(over an hour) studies are difficult due to photobleaching for the dyes, which decreases
the fluorescence amplitude for each exposure taken.
Recently the wide range of applications of microelectrode arrays (MEAs) in scientific
research has resulted in the commercial availability of a few array-based solutions. One
of the most basic and widely used designs was first developed circa 1972 by Thomas, et
al. (Thomas, 1972). This solution involves evaporating a metal conductor onto glass and
covering the traces and non-active sites with an insulating polymer, leaving the recording
sites exposed. The result is an array of flat metal electrodes that can be pressed against
tissue such as cardiac muscle, or tissue may be cultured onto the surface such as neurons.
The main drawback to this approach is that a low density of electrically active cells are in proximity to the recording sites, a problem which is exacerbated in tissue slices by a layer of dead cells caused by the tissue preparation. We hypothesize that penetrating electrode geometry will produce higher signal amplitude recordings in slices sue to the closer proximity to active tissue. Most previously reported devices, however, have had low aspect ratio (essentially pyramidal) electrodes which are not ideal for approaching active cells. They typically have a height of 50 uM. (Thiebaud, 1999) Further, previously reported penetrating devices have been fabricated on a silicon substrate. A transparent
8 substrate is preferable, in that it allows transmission microscopy for simultaneous
imaging and electrical recording from neural networks. Once completed, the proposed
MEA will be the first known device to incorporate penetrating high aspect ratio spikes on
a transparent substrate for multi-modal neural recording.
One of the goals of this effort is to design and build a microelectrode array that is
optimal for studying the hippocampus in two preparations — the slice and the intact
unfolded hippocampus. Ideally, a multi-site recording device would exhibit high signal-
to-noise ratio, high selectivity of the deep tissues of interest in the unfolded hippocampus
(200 μm from the bottom of the hippocampus), would not cause significant alterations to
the firing or activity of neurons on the short or long term time frames, would be re-
usable, and would be optically transparent. The penetrating microelectrode array is
designed to meet those goals.
1.6 CLINICAL RELEVANCE
Seizure development is multifactorial and the mechanism of seizure generation
and propagation likely varies depending on epilepsy classification. Synaptic factors and
nonsynaptic factors each play varying roles in focal seizures versus generalized seizures, and no single animal or mathematical model can properly characterize the epilepsies of a
human population. The unfolded hippocampus model, Aim1, will increase the
understanding of and ability to investigate the hippocampus, and together with our investigations into the mechanisms and propagation of 4-AP induced epileptiform activity. In Aim 2, we will improve the understanding the hyperexcited state of the hippocampus. The design, construction, and use of a penetrating multielectrode array will
9 allow high signal to noise ratio, two dimensional multisite recordings of the pyramidal cell layer of the entire unfolded hippocampus CA1, 2, and 3 areas, as well as high fidelity recordings of other preparation of the hippocampus and brain. The sought-after improvements of this array will enable faster data acquisition and better understanding of the hippocampal structures and interactions in the lateral and longitudinal directions. The microelectrode array will have uses beyond the study of epilepsy propagation into fields such as memory organization, storage, natural rhythms, and network processing.
1.7 THESIS OBJECTIVES AND ORGANIZATION
The Unfolded Hippocampus Preparation
In order to utilize the unfolded hippocampus preparation for the study of
Epileptiform activity, its viability needs to be ascertained and the known network connections of the hippocampus verified. This will be done in a series of experiments that will look at the synaptic pathways of the hippocampus and compare evoked potentials to those seen in vivo. Furthermore, the histology of the dissected and unfolded preparations will be examined for neuronal damage.
Rationale: Because the unfolded intact hippocampus is a new preparation, normal electrophysiological comparisons with accepted tissue preparations should be made to verify accuracy as a model. After verification, the normal electrophysiological properties of the preparation will be documented using the same experimental setup as that used in future experiments so as to serve as a reference baseline for tissue viability and excitability.
10
Objective I: To establish the viability of the intact unfolded hippocampus in vitro.
Hypothesis: The unfolded intact hippocampus preparation will exhibit normal electrophysiology for the purpose of studying hippocampal networks in vitro.
Experimental Protocol: In order to test the stated hypothesis,
electrophysiological tests will be performed to confirm synaptic activity and connectivity,
and histological staining will be performed to look for premature cellular degredation.
1) Orthodromic responses to cathodic stimuli in the schaffer collaterals to will be recorded at varying depths through the pyramidal layer of the hippocampus in the CA1 region. Responses will be recoded from the alvear region, through the apical dendritic tree, through the pyramidal cell body location, to the basal dendritic tree. If our
hypothesis is correct, these responses should resemble those seen in vivo experiments.
The two-dimensional propagation of evoked antidromic responses will be measured in
the CA1 region, and the extent of transverse versus longitudinal propagation will be
characterized. Paired pulse stimuli with delays of 10–100 ms will be applied to the
schaffer collaterals and the facilitation of CA1 pyramidal neurons will be measured and
compared with that of in vivo experiments. The evoked potential delay and amplitude
response to varying bath temperature will be measured and trends noted.
11 2) Antidromic evoked potentials will be recorded in the CA3 region of the
hippocampus when the Shaffer collaterals are stimulated, and the evoked response delay
and amplitude measured (Figure 3). The measured values will be statistically compared
against those found in the in vitro slice preparation.
3) Voltage sensitive dye imaging will be employed to observe simultaneous two-
dimensional activation of hippocampal regions following a single stimulus in the CA1 region. The observed propagation pattern will be compared with expected results given
known network properties and dendritic and axonal extents.
4) Cell damage will be assessed in the unfolded preparation by fixing the tissue, slicing,
and staining with cresyl violet to how the cellular structures. The condition of the primary
structures will be ascertained using photomicroscopy. Absence of cell structures will
indicate damage.
12 Epileptiform Activity in the Intact Unfolded Hippocampus Preparation
Rationale: The intact hippocampus preparation enables the longitudinal pathways
of the rodent hippocampus to be studied. These pathways may facilitate epileptiform
propagation along directions that had previously not been observed in other models.
OBJECTIVE II: To analyze orthogonally propagating epileptiform activity in the
unfolded rodent hippocampus in vitro.
Hypothesis: The longitudinal inter-neuronal processes that are preserved in the unfolded
intact hippocampus facilitate longitudinally propagating seizure like activity.
Experimental Protocol: Epileptiform activity will be studied in the unfolded intact hippocampus preparation using the 4-AP model of epilepsy. Both spontaneous activity and the response to evoked stimuli will be recorded. The spontaneous generation site will be located and the propagation directions and speed will be measured using dual glass electrodes on independent micromanipulators. The two dimensional flat top of the unfolded hippocampus including the CA1–3 regions will be imaged during activity using
RH-414 voltage sensitive dye and a map of propagation will be created for the
preparation. The amplitude of orthodromic evoked potentials will be monitored during
the experiments to verify tissue viability. Single cell recordings will be taken from CA3
13 and CA1 pyramidal cells to reveal IPSP and EPSPs corresponding to the epileptiform
field potentials recorded by the glass electrodes.
To test the hypothesis, the amplitude and timing of the field potentials and the
intracellular potentials will be assessed in the transverse and longitudinal directions along
the CA3 and CA1 regions. If there is epileptiform propagation along the longitudinal
direction of the CA3 or CA1 region, the relative time delay from the initiation site should reveal the propagation path that the epileptiform burst takes. The results will reveal in which direction and region the 4-AP induced epileptiform activity preferentially propagates in the mouse hippocampus.
MEMS-Based Penetrating 2-D Recording Array for Field Potentials
Rationale: The use of voltage sensitive dyes has many severe drawbacks already mentioned. Modern recording arrays are designed with either flat electrodes, or deep penetrating electrodes with insufficient spatial resolution and excessive height for the study of the hippocampus. Therefore an improved electrode array will be designed and built suited for whole-hippocampus recording with accompanying amplifiers and acquisition system.
Objective III: Develop and build a MEMS based penetrating microelectrode array for
2-dimensional recording of the unfolded hippocampus in vitro
14 Hypothesis: The MEMS array will allow higher signal-to-noise (SNR) values and longer term, more sensitive recording of neural activity compared to the RH414 voltage sensitive dye technique currently employed.
Experimental Protocol: The array will be designed and built (see methods section, chapter 4) using common MEMS technology for the purpose of whole- hippocampus recording. The optimal recording depth, electrode spacing, and array size will be determined within the limits of the MEMS processes. The materials used will be chosen for biocompatibility and stability when interacting with ACSF and brain tissue during the experiments. Once built, the impedance and biocompatibility will be evaluated and appropriate amplifiers built for multi-site recording. Finally, spontaneous 4-AP generated bursts in the intact unfolded hippocampus will be recorded using the array, and the signal to noise ratio, maximum recording duration, and toxicity of the microelectrode array acquisition method will be compared with the same when using the voltage sensitive dye method with RH414.
The signal to noise ratio will be measured by the ratio of evoked potentials of a healthy slice (at least 2mV p–p extracellular field response) to the background noise recorded with no artificial activity induced. The maximum recording duration will be measured as the active recording time during which the SNR remains above 2. The toxicity of the recording method will be determined by the percent amplitude change of orthodromically evoked CA1 field potentials as measured by a glass electrode over the duration of the recording session.
15
Electrode array characterization:
Impedance Measurement
The characteristic impedance of the microtips on the MEA will be quantitatively assessed using a calibrated frequency sweep electrode impedance analyzer. The tips are expected to behave as capacitors in an electrolytic solution such as artificial cerebrospinal fluid (ACSF) and phosphate buffered saline solutions. Gold plated tips will be assessed and the capacitances measured for each. The capacitance value is of interest in the building of a preamplifier for the MEA so that the parameters of the amplifier can be chosen to result in the highest signal to noise ratio for the MEA. With the impedance data for the tips and preamplifier, a full frequency response curve can be calculated, with a goal response of no less than -3dB from 1Hz to 4KHz.
16 Table 1.1: Pharmacological and Ionic models of Epilepsy
Model Primary Mechanism Penicillin Bicuculline GABA receptor antagonist Picrotoxin a L-allylglycine Tetanus toxin GABA transmitter antagonist Pentylenetetrazol (PTZ) a 4-Deoxypyridoxine decarboxylase inhibitors reduce free GABA Early transient K+ channel blocker 4-Aminopyridine (A-current) Flurothyl gas GABA-benzodiazepine receptor antagonist Kainic acid Glutamate receptor agonist Pilocarpine Muscarinic acetylcholine receptor agonist ++ ++ Increases EPSPs by removing Mg block of NMDA 0 Mg solution receptor + Increases neuronal excitability, promotes spontaneous High K solution bursts, cause neuronal and glial swelling ++ Increases excitability by decreasing synaptic inhibition – Low Ca Solution nonsynaptic epilepsy Veratridine & 0 Mg++ Induces persistent Na Conductance Pitkanen et al., Models of Seizures and Epilepsy (2005)
17
Figure 1.1: Diagram of the Hippocampus in Cross-Section (Ramón y Cajal 1911).
Record CA1
Stimulation
Figure 1.2: Orthodromic CA1 Activation
18 Record CA3
Stimulation
Figure 1.3: Antidromic CA3 Activation
Stimulation Record CA1
Figure 1.4: Antidromic CA3 Activation
19
CHAPTER 2
A Novel Unfolded Hippocampus Preparation
20 2.1 ABSTRACT
The viability of a new unfolded hippocampal preparation was investigated for the
purpose of facilitating the study of neural networks in vitro. Most commonly, the brain
slice preparation has been used to study the rodent hippocampus in vitro, however the
slicing method severs longitudinal interconnecting processes in the hippocampus such as
recurrent excitation networks in CA3. With this new preparation, the entire hippocampus
from young mice can now be kept alive for an extended period of time during
electrophysiological study. Orthodromically evoked responses showed an excellent
correlation to responses seen in slices, indicating preservation of neuronal connectivity
and viable synapses. The lamellar organization of the unfolded hippocampus was
confirmed by a strong antidromic response along the transverse direction. This
antidromic response was also found to propagate longitudinally as well as in the
transverse direction in the same preparation. Furthermore, voltage sensitive dye optical
imaging was performed to study the extent of activation. We have also used optical
imaging of the unfolded hippocampus to map propagation. Propagation across two
dimensions of the whole hippocampus was observed, providing new potential information about the two-dimensional propagation in the whole hippocampus that a slice
preparation could not offer. The vitality of the unfolded preparation depends on
decreased temperature, and this temperature dependence was studied, resulting in the
determination of an optimal in vitro bath temperature of 29C. Histological analysis using
Cresyl violet staining of tissue after use showed intact and normal pyramidal cell and
nuclei. These data support the hypothesis that the unfolded hippocampus preparation
exhibits normal electrophysiology for the purpose of studying hippocampal networks in
21 vitro. With this new preparation, two dimensional data may be taken more easily than
before, and many of the experiments involving epileptiform activity, high frequency
stimulation, and bath additives may be repeated. The mechanisms of propagation of
synaptic and nonsynaptic epilepsy may be studied more thoroughly with this preparation, as in chapter 3, as well as the properties of two-dimensional synaptic plasticity.
2.2 INTRODUCTION
The intricate structure of the hippocampus has been a focal point of
electrophysiological research for many years. There traditionally have been three ways
that the hippocampus has been studied – in vivo, extracted and sliced, and dissociated and
cultured. Advantages of brain slice preparation include the ability to control the external
medium, visualize the tissue layers under microscope, and control pharmacological
agents (Aitken et al., 1993). The hippocampal excitatory pathway has a “lamellar” organization (Andersen, 1971), meaning that it has physiologically differentiable layers
primarily in two dimensions. The third dimension, along the fornix, consists of repetitions of these lamellar circuits along with some lesser-known inhibitory and
excitatory pathways. In vitro slices make use of this organization, as they are made in a
plane transverse to the long axis of the hippocampus, and by doing so preserve the major
excitatory axonal tracts (Teyler 1980). Another benefit of this preparation is that the
unfolded hippocampus is thin enough to allow oxygen to diffuse throughout the whole
tissue. Longitudinal hippocampal slices have also been studied, but because of the loss of
the major pathways their utility is limited. In either case, the complexity of the entire
neural circuit including substantial longitudinal projections is lost. (Amaral and Witter,
22 1989, Brown and Zador, 1990). Work is progressing to overcome these limitations in vivo
(Feng and Durand, 2003) and by isolating the whole hippocampus in vitro (Khalilov,
1997; Wu and Shen, 2002). Khalilov demonstrated the intact hippocampal formation
(IHF), where the entire hippocampus is removed and studied as a whole. Even though
this preparation can successfully keep the intact mouse hippocampus alive for an
extended period of time, the IHF preparation has an age limitation (P0 – P10) to keep the
formation small enough for proper oxygen diffusion. Mice older than P10 have a thicker
hippocampus, leading to the hypoxic death of deeper tissues. For many purposes, studies
of older mice are usually preferred in many experiments. Wu and Shen increased the age
limit to P28 by cutting off the dentate gyrus behind the hippocampal fissure. This results
in a thinner tissue and thus improved oxygenation of the tissue.
Objective
This study aims to study the viability of a whole hippocampus preparation that
supports up to P24 mice and has the capability of retaining all parts of the structure. In
addition, this method should work using common electrophysiological instruments. The goal is to excise the hippocampus in a method similar to that used by Khalilov, then to create a “planar” hippocampus by unfolding the dentate gyrate outward. This unfolding reduces the thickness by approximately half and therefore allows greater oxygen perfusion. Another advantage of the Unfolded Hippocampal Preparation (UHP) over IHF is its ability to create a flat two-dimensional model that includes all CA1, CA2, CA3, and dentate gyrus. The unfolded hippocampus will be useful to study the neural activities in two-dimensional arrays via optical or microelectrode mapping. The evoked responses
23 corresponding to different vertical layers of CA1 compared favorably with Lueng’s 2001
in vivo result (Kloosterman, 2001). It was possible to evoke a healthy response
throughout this unfolded hippocampus and the use of optical imaging was demonstrated
on its surface. With this new preparation, two dimensional data may be obtained easily,
and many of the experiments involving epileptiform activity, high frequency stimulation,
and bath additives may be repeated. The mechanisms of propagation of nonsynaptic
epilepsy can be studied more thoroughly with this preparation, as well as the properties of
two-dimensional synaptic plasticity.
2.3 MATERIALS AND METHODS
Animals Used
The experiments were performed using Charles River mice. The age of these mice was 11–24 days postnatal, while the sex of the animal was considered insignificant. All animals were housed and maintained in veterinarian-monitored animal care facility at
CWRU. The animal health and welfare procedures that were performed complied with
National Institutes of Health guidelines.
Unfolded Hippocampal Preparation
The mice were anesthetized with ethyl ether and quickly decapitated. After
o o decapitation, the head was immediately immersed in ice cold (2 C – 4 C) oxygenated (O2
95%, CO2 5%) and sucrose-rich artificial cerebrospinal fluid (ACSF) with following
composition (in mM): Sucrose 124, KCl 3, NaH2PO4 1.25, CaCl2 2.0, MgSO4 2.0,
NaHCO3 26, Dextrose 10.0. Substitution of NaCl with sucrose in ACSF has been shown
24 to reduce the potential effects of cutting-induced trauma and consistently yields more
viable tissue (Aghajanian et al. 1989). The decapitated head was cooled sufficiently to
minimize bleeding and rate of cell metabolism. This cooling process was discovered to be
crucial due to latter steps when the hippocampus is dissected under a hypoxic
environment. Following the same methodology as described for brain slice preparation
(Teyler, 1980), the brain was quickly removed onto a moistened, chilled filter paper. The
brain was hemisectioned by a razor to work with one hemisphere at a time. While one
hemisphere was being dissected, the other half was kept in the ice cold oxygenated
sucrose ACSF for later use. Experiments showed that keeping one hemisphere in a cold
“sucrose” ACSF for 3–5 minutes did not affect its viability. Following the hemisection, the hippocampus was freed from the septum and entorhinal region using either dorsal or ventral approach (Teyler, 1980).
Once dissected, the hippocampus was transferred onto a moistened filter paper that was placed on top of chilled platform. The supporting material must be cold enough to maintain low temperature, and it was found that working in a recess aided in keeping the air cold above and around the specimen. Fig. 2.1a shows the series of dissecting steps.
Under a dissecting microscope, both rounded ends of the hippocampus were cut in a line perpendicular to the longitudinal axis. This aids in the unfolding process. The folding of dentate gyrus creates a fissure, which is connected by blood vessels and neuronal perforant pathways. The dentate gyrus was freed by sliding a sharp glass electrode along the line of the fissure. Care must be taken so as to not exceed the depth of the fissure and in doing so damage the Schaeffer collaterals. Sliding a round-tip glass electrode along the hippocampal fissure and simultaneously pushing it outward completes the unfolding
25 process (Fig. 2.3). During the course of these procedures, chilled oxygenated “sucrose”
ACSF was being dropped frequently onto the hippocampus to maintain moisture and allow additional oxygen perfusion. Once the hippocampus is fully unfolded, the surface of dentate gyrus adheres to the filter paper and remains perfectly flat until manually detached from filter paper. This may be useful in an interface recording chamber, as the unfolded tissue has a tendency to re-fold. In the submerged chamber used for this study
and the optical recording, the tissue was gently held flat with a fine mesh. The complete dissection procedure requires approximately 5–10 min. Under a chilled oxygenated environment this duration is not sufficient to cause the tissue to become hypoxic. Careful dissection was found to be more important than its speed.
Finally, the unfolded hippocampal preparation was then transferred into a recovery chamber filled with artificial cerebrospinal fluid (ACSF) having the following composition (in mM): NaCl 124, KCl 3.75, KH2PO4 1.25, CaCl2 2.0, MgSO4 2.0,
NaHCO3 26, Dextrose 10.0. This chamber is bubbled with 95% O2 / 5% CO2 and maintained at a room temperature (25 oC). The unfolded hippocampus is transferred along with the filter paper that lies underneath. The unfolded preparation was allowed to recover for at least 1 hour. Viability was found to be maintained in this chamber for at least 5 hours.
Recording Chamber
After at least 1 hr of recovery in the storage chamber, the unfolded hippocampus
preparation was then transferred to the submerged recording chamber. ACSF oxygenated
with 95% O2 / 5% CO2 was constantly flowing through this chamber via a high-flow
26 recirculating peristaltic pump and in-line heater. The level of liquid was adjusted to allow
for a completely submerged hippocampus. When recording the extracellular field
potentials, the unfolded preparations were placed in the chamber with the alveus side up
(Fig. 2.3).
Electrical Recording
Field potentials were recorded using low-impedance glass micropipettes (3–6 MΩ)
backfilled with 150 mM NaCl. Stimulus evoked field potentials were induced
antidromically and orthodromically to verify optimal viability. A tungsten electrode
injected 150–350 μA, 120 μs pulses into the alveus to produce an antidromic response in
CA1 pyramidal cells, and the stimulating electrode was moved into the mossy fiber pathways to measure orthodromic potentials. The pulses applied to the electrode were generated by a voltage (NEURO DATA PG4000) and converted to a current pulse by a stimulus isolation unit (NEURO DATA). When performing optical imaging of CA1 surface region, they were placed with alveus side down. The temperature adjusted from
24 to 33 degrees Celsius during certain experiments in an effort to determine the
optimum perfusion temperature for the tissue when studying the evoked response.
Optical Imaging
The unfolded preparation was stained with the fast voltage-sensitive styryl dye RH-414
(N-(3-triethylammoniumpropyl)-4-(4-(4-diethylamino)phenyl)butadienyl)-pyridium
diacetate; Molecular Probes) at 200 μMol for 15 in a darkened room. Excess stain was
27 washed away with dye-free ACSF before recording. The unfolded preparation was then transferred to a submerged recording chamber with alveus side down. A tungsten- stimulating electrode was inserted through the back of the hippocampus tissue using a dissecting microscope and inserted into the stratum radiatum. Similar current stimuli were used for optical and electrical recordings. At the same time, a glass microelectrode filled with 150 mM NaCl (resistance 2–5 MΩ) was inserted into the pyramidal cell layers, again using the dissecting stereomicroscope. Extracellular recordings were simultaneously performed during the imaging to assess the viability of the preparation and to monitor the evoked responses. Epi-illumination via an inverted Nikon Diaphot culture microscope fitted with a low noise DC-powered tungsten-halogen lamp (100W,
12 V) was used. Stimulation, shutter control, pump pause, and bias adjustments were done automatically though a custom program running on the Neuro Data Instrument.
Fluorescence evoked was measured (interface filter: 535 ± 25 nm, dichroic mirror 580 nm; barrier filter 590 nm) at each scanning point in the 16 * 16 photodiode array
(Hamamatsu C4675-102) and amplified with a custom amplifier and low pass filter with a cutoff set to 1 KHz. The imaging objective (10*, 0.5 NA) resulted in a spatial resolution of each element was 137 * 137 μm. Current signals from each photodiode were converted to voltage, amplified, filtered, digitized, and were sampled at 500–1500 Hz. Final image processing was performed in order to better reveal the characteristics of the network.
Time averaging was used along with repetitive stimuli (4 stimuli, 50ms averaging block) in order to improve the signal to noise ratio. Typically a 1–2% change in fluorescence was seen during neuronal firing. In addition, spatial subsampling was employed to give the imaged area a smoother appearance, using two-dimensional bilinear interpolation.
28
2.4 RESULTS
Electrophysiological Properties of the Unfolded Hippocampal Preparation
Effects of Temperature
In order to characterize the temperature related properties of this preparation, an
orthodromically evoked potential based viability study was performed over a temperature
range from 24 to 33 degrees Celsius. The evoked potential amplitude was measured as
well as the delay from stimulus to response. The temperature was varied at a rate of
approximately one degree every three minutes over the course of at least ½ hour, and the
temperature was fixed at least 60 seconds before a data point was taken. The normalized
curves are shown. (Fig. 2.4, 2.5). These results indicate that the preparation reached its
maximal response amplitude at 29 degrees C. This coincides with a minimum
propagation delay at that temperature, as well.
Intra-hippocampal recording at various depths
A common test of the electrophysiological viability of neural tissue is to check for a post-synaptic population spike after a stimulus is delivered to presynaptic neurons. This tests the viability of the local neuronal synapses and the ability of the postsynaptic neurons to translate excitatory post-synaptic potentials (EPSPs) into action potentials
visible extracellularly. A stimulating electrode was placed in the Schaffer collateral of the
CA1 region of the hippocampus to stimulate local presynaptic axons. A recording electrode was placed nearby in the CA1 region and passed in steps from the basal
29 dendrites through the somatic region and into the apical dendritic region at the layer of
stimulus (Fig. 2.6). Typical field excitatory postsynaptic potentials (EPSPs) were
observed in the CA1 region. Intra-hippocampal recording at various depths revealed
expected changes in amplitude that were preceded by an afferent volley, and had a
latency and duration similar to that observed in vivo and in slices.
A stimulating electrode was positioned at the CA1 alveus layer while a recording
electrode was gradually inserted from the alveus layer down to the Schaeffer collateral
region. A current of 0.15 mA evoked a single response at a regular interval of once every
5 seconds. The recording electrode was guided further down using approximately 30 μm
increments. Fig. 2.6 shows the typical change in shape of orthodromic responses with increasing depth. Starting with a small negative wave in alveus, recording electrode was
gradually inserted through the cell bodies and eventually past the dendrite layer. The
maximum response was recorded near the pyramidal cell body layer. The amplitude of
the negative going spike corresponding with cell firing was gradually diminished as the
recording electrode receded away from the pyramidal cell layer. After further insertion, a
positive wave was recorded near the Schaeffer collateral layer. These results correspond
very well to recordings in-vivo done by Kloosterman, et al. (Kloosterman 2001). During
other experiments such as optical recording, when the depth of the electrode was difficult
to estimate, these results were used in relation to recorded waveform shape in order to
determine the approximate location of the recording electrode and make appropriate
adjustments. The lamellar organization of hippocampal neurons that was shown in vivo
(Andersen, 1971) was also reproduced by this Unfolded Hippocampal Preparation (Fig.
30 2.6), supporting our hypothesis that the unfolded hippocampus exhibits normal electrophysiology.
Longitudinal Interconnection of Alvear Fibers
Transverse propagation along both lateral and longitudinal axes was observed in the course of experiments. In order to analyze this transverse verses longitudinal extent of response, the extent of response surrounding a single antidromic stimulus in the CA1 alveus was recorded. In every experiment where antidromic stimulation was used, the stimulating electrode was inserted into the alveus layer within the CA1 region, and the recording electrode was inserted into the pyramidal cell layer. In the first set of experiments, recording was performed along two different rows of tracks parallel to the longitudinal axis indicated by a – h, i – p (Fig. 2.7). These rows were separated by approximately 0.1 mm and were parallel to each other. The space between each point in a row is approximately 0.09mm. At each position, antidromic response was evoked following a single stimulation at 0.35 mA. The recording electrode was inserted gradually until the maximum response was evoked. Fig. 2.7 shows that maximum responses from each row were recorded at e and n characterized by a sharp negative population spike.
The arrow that connects stim, e, and n represents the direction of alvear fibers. The graph in Fig. 2.7 shows the size of population spike as a function of its position. There is a difference in the size of spikes between the two rows. The amplitude of antidromic response decreased as the distance between the two electrodes was increased. This experiment illustrated a strong diagonal propagation towards septum, which was consistent with the lamellar hippocampal organization suggested by Andersen (Andersen,
31 1971). Along the lamellar direction, a recording electrode could be placed as far as 1.5 –
2 mm from the stimulating electrode and still obtain a measurable response (Fig. 2.7).
Optical Imaging
It has been suggested that stimulation at a single site will induce propagation across much of the hippocampus. To determine the spatial properties of activity propagation, and to make further use of the unique two-dimensional qualities of this preparation, evoked potentials were imaged optically. This allowed the monitoring of spatial propagation of neural activities across the entire CA1 region. The high-speed imager allowed us to peer into the time delayed response characteristics of the network
(Figure 2.9). Unsurprisingly, they revealed that propagation occurred very easily in all radial directions, but not across the entire hippocampus. Single stimuli evoked a wave of activity that originated at the stimulation site and propagated away in lateral and longitudinal directions. The activity propagated across the lamella and longitudinally as far as 500μm to each side.
The optical imaging experiment served as a verification of the electrophysiological data, and as a starting point for possible future experiments. Shown in Figure 2.9 is a time sequence of data from an evoked potential. Note the location of the stimulus is easy to find, and that the stimulus propagates laterally as well as in the expected alvear directions. Imaging of low-calcium ACSF induced spontaneous activity was attempted, but the low level of the signal yielded no data. Figure 2.8 shows the relative position of the hippocampus during this optical experiment.
32 Histological Staining
Post-experiment, tissue samples were preserved for histological analysis. A cresyl violet stain was selected to indicate whether pyramidal cells survived the unfolding procedure. The unfolded hippocampus was fixed and sliced for analysis, one stained slice is shown in Figure 2.10. Although it was unfolded, the hippocampus naturally curled up again during the fixing process, as visible in the figure. Despite this, the staining indicates that the pyramidal layer of neurons survived the preparation and experiment intact, with healthy nuclei shown in blue. Cells that undergo apoptotic destruction would not show up as a defined blue region. These results support our hypothesis that the unfolded hippocampus preparation exhibits normal physiology for use in electrophysiological experiments.
Spontaneous interictal-like propagation
It has been reported that an ACSF solution containing 50 μM 4-AP will induce interictal like events (ILEs) (Avoli, 1996). In order to test the ability of the unfolded hippocampus preparation to generate these ILEs, we applied 50 μM 4-AP into the superfusion solution and recorded extracellular potential fields using sharp glass electrodes. As expected, strong ILEs were formed beginning from 1–8 minutes after application (Fig. 2.11). To investigate the transverse propagation of this spontaneous activity, microelectrodes were positioned in transverse lamella in the CA3 and CA1, and the propagation latency was measured. From the CA3, an activation wave was observed propagating along the stratum lacunosum-molecolare with a delay of 9+/-1.6 ms to reach the CA1, with a speed of
0.22+/-0.014 m/s (fig. 9). CA1 response was observed to be bimodal, having a positive potential in some cases (n=4) and a negative field potential in others (n=6). The ILE
33 longitudinal propagation and further detailed analysis of orthogonal propagation is
covered later in chapter 3.
2.5 DISCUSSION
We have developed a new hippocampus preparation for the study of networks and
associated activity. This preparation offers an ability to study the propagation of both
transverse and longitudinal response throughout the entire hippocampus. The distinction
between the IHF (intact hippocampal formation) and the unfolded hippocampal
preparation is that this preparation allows study of older mice (up to P24), the
hippocampus is planarized in the process, and experiments lasting several hours are
feasible. Also, no additional equipment is required beyond the traditional slice
preparation tools. Although the Unfolded Hippocampal Preparation does not allow
studies in the septo-hippocampal system or between the interconnected hippocami as in
IHF (Khalilov, 1997), its flat plane model creates an alternative way to study the
hippocampus. Instead of having the dentate gyrus looped underneath CA1, the dentate
gyrus is folded out to make a planar sheet that consist of uniform lateral organization of
dentate gyrus, CA3, and CA1. For techniques such as optical imaging and use with an electrode array as described in chapter 4 which capture activities in 2-Dimensional planar
surface, this preparation is better suited.
Summary of Findings
The results indicate that the unfolded hippocampal preparation maintained its healthy
function for an extended period of time. The orthodromic responses verified synaptic
34 communication, and the antidromic responses revealed the longitudinal pathways. The
optimal temperature for this submerged preparation was found to be approximately 29
degrees, not too far from the temperature used by Wu et al in 2002. This preparation
provides information on neural activities in both lateral and longitudinal direction, which
could not be done by the slice preparation, and it preserves the dentate gyrus. The main
disadvantage of slice preparation — the inability to study the propagation across the
entire hippocampus, can now be eliminated. The viability of this preparation in vitro
allows similarly powerful studies that has been done on hippocampal slices: control of
external medium (temperature, pH, calcium/potassium level), application of pharmacological agents, optical imaging, etc with a similar network as is available in
vivo.
Future Work
Many of the experiments that have been done on slice preparation can now be
repeated using this preparation, but more interesting possibilities are available such as the
mapping of spontaneous activity across the entire hippocampus, and the studies of full
network potentiation, to name a few. Although other intact preparation such as IHF has
been introduced to study the neonatal and incomplete hippocampus in vitro, our
preparation has extended the age limit of study of a full hippocampus to a more mature
young mouse. For many studies, it is important to study the mature hippocampus to fully
understand its mechanism. Through this work, the best operating parameters have been found and the road is now paved for more investigation into the intricacies of the intact hippocampus. In continuation of this work, study of nonsynaptic epileptiform activity has
35 been undertaken and described in the next chapter. In addition, a two-dimensional penetrating electrode array recording system has been developed in cooperation with
NASA for future research, as described in chapter 4.
36
2.6 FIGURE CAPTIONS
Figure 2.1: Anatomical Diagram of the Hippocampus in Cross-Section (Ramón y Cajal
1911).
Figure 2.2: Internal View of Hippocampus in Rat, Shown for Reference (Amaral and
Witter 1989).
Figure 2.3: Procedure for Unfolding the Hippocampus in a Planar Fashion
Cartoons Adapted From Kazuma, 2000.
Figure 2.4: Distribution of Normalized Response Amplitude With Respect to
Temperature.
Figure 2.5: Distribution of Normalized Response Delay With Respect to Temperature.
Figure 2.6: Orthodromic Response With Varying Recording Depth. A) Comparison recordings from in vivo experiment (Kloosterman et al. 2001). B) Recorded orthodromic extracellular potentials at varying depth in the unfolded hippocampus correspond well with the expected in vivo result.
37 Figure 2.7: Antidromic Response With Respect to Recording Position. The o indicates
the stimulus position and the lettered dots represent the recording locations. Row a–h is solid in the graph and row i–p is dashed. Times on individual graphs are in milliseconds.
Figure 2.8: Diagram of the Positioning of the Hippocampus With Respect to the Optical
Field.
Figure 2.9: Optical Imaging Maps, Whole Hippocampus, 2 ms between Frames.
Figure 2.10: Cresyl Violet staining of an unfolded hippocampus preparation which has been histologically preserved and sectioned. Strong blue present in the pyramidal cell layers indicates intact nuclei and good preservation of these cells.
38 2.7 FIGURES
Figure 2.1
Figure 2.2.
39 1 2
3 4
5 6
Figure 2.3.
1.2
1
0.8
0.6
0.4
0.2 Normalized Response Amplitude
0
-0.2 22 24 26 28 30 32 34 Temperature, Degrees C
Figure 2.4.
40 1.2
1
0.8
0.6
0.4
0.2 Normalized Response Delay
0
-0.2 22 24 26 28 30 32 34 Temperature, Degrees C
Figure 2.5
41
FORNIX Schaffer Alveus Collaterals CA3 CA1 Dentate Gyrus
Mossy Fibers
A B
Rec.
alv.
pyr. St i Sch.
Figure 2.6
42
5 5 5
0 0 0 m n o -5 amplitude, mV -5 -5 amplitude, mV amplitude, mV
-10 -5 0 5 10 15 20 -10 -10 -5 0 5 10 15 20 Time, ms -5 0 5 10 15 20 Time, ms Time, ms
i p a h i p a h
5 5 5
0 0 0 d e f -5 -5 -5 amplitude, mV amplitude, mV amplitude, mV
-10 -10 -10 -5 0 5 10 15 20 -5 0 5 10 15 20 -5 0 5 10 15 20 Time, ms Time, ms Time, ms
8
7
6
5
4
3 Amplitude, mV 2
1
0 0 a b c d e f g h j k l m n o p q r -1 0 2 4 6 8 10 12 Relative Recording Position
Figure 2.7
43
3mm Figure 2.8
3mm
Figure 2.9
44
FIM
CA3
Partial DG
CA1
EC
0.5mm
Figure 2.10
45
DG Rec 1 . CA 3 Rec. 2 CA 1
Initiation Site
Figure 2.11
Fig. 2.11 : Spontaneous 4-AP burst propagation from CA3 to CA1. CA1 wave potentials had a positive polarity and a propagation delay from CA3 of 9+/- 1.6ms.
46
CHAPTER 3
Orthogonal Wave Propagation of Epileptiform Activity in the Planar Mouse Hippocampus in vitro
The following chapter was accepted for publication in Epilepsia.
47 3.1 ABSTRACT
Purpose: In vitro brain preparations have been used extensively to study the
generation and propagation of epileptiform activity. Transverse and longitudinal slices of the rodent hippocampus have revealed various patterns of propagation. Yet intact connections between the transverse and longitudinal pathways should generate
orthogonal (both transverse and longitudinal) propagation of seizures involving the entire
hippocampus. This study utilizes the planar unfolded mouse hippocampus preparation to
reveal simultaneous orthogonal epileptiform propagation and to test a method of arresting
propagation.
Methods: This study utilized an unfolded mouse hippocampus preparation. It
was chosen due to its preservation of longitudinal neuronal processes which are thought
to play an important role in epileptiform hyper-excitability. 4-aminopyridine (4-AP),
micro-electrodes, and voltage sensitive dye imaging were employed to investigate tissue
excitability.
Results: In 50 µM 4-AP, stimulation of the stratum radiatum induced transverse
activation of CA3 cells but also induced a longitudinal wave of activity propagating
along the CA3 region at a speed of 0.09 m/s. Without stimulation, a wave originated at
the temporal CA3 and propagated in a temporal–septal direction and could be suppressed
with glutamatergic antagonists. Orthogonal propagation traveled longitudinally along the
CA3 pathway, secondarily invading the CA1 region at a velocity of 0.22 ± 0.024 m/s.
Moreover, a local lesion restricted to the CA3 region could arrest wave propagation.
Discussion: These results reveal a complex two-dimensional epileptiform wave
propagation pattern in the hippocampus that is generated by a combination of synaptic
48 transmission and axonal propagation in the CA3 recurrent network. Epileptiform
propagation block via a transverse selective CA3 lesion suggests a potential surgical
technique for the treatment of temporal lobe epilepsy.
3.2 INTRODUCTION
Epileptiform activity arises from large numbers of neurons bursting in a
synchronous fashion while disrupting the normal operation of neural activity. Several
animal and pharmacological models have been developed to study the properties and the
mechanisms behind generation and propagation of epileptiform activity in neuronal
networks. In particular, 4-aminopyridine (4-AP), a potassium channel blocker, has been
used extensively to study epileptiform activity generation and propagation. While there
are numerous epileptiform models which block synaptic transmission (Bikson et al.,
2002), hippocampus preparations bathed in micromolar concentrations of 4-AP can generate bursts of activity in the presence of synaptic transmission. (Perreault & Avoli,
1991 1992; Avoli, 1996; Gu et al., 2004) In this in vitro study we investigate the spatial extent of epileptiform propagation in the hyperexcitable hippocampus with intact longitudinal and transverse connections bathed in 4-AP containing solutions.
Epileptiform activity induced by the presence of 4-AP in transverse hippocampal slices is characterized by short recurrent discharges sometimes combined with slow field potential shifts and long lasting (100 ms) depolarization of cells (Perreault & Avoli,
1991). While the complete mechanism of activation is unknown, it has been shown that, upon application of 4-AP, the frequency and strength of EPSPs and IPSPs in CA3 cells increases, leading to prolonged ictal and short interictal discharges. (Gloveli, 2005)
49 Moreover, 4-AP induces an increase in extracellular potassium levels during long-lasting field potential depressions as they propagate across the network. (Avoli, 1996) It has been shown that long term application of 35–70 mM concentrations of 4-AP in vivo
generates neuronal lesions in CA1 and CA3 regions related to high glutamate levels in
proximity to the synapses (Pena & Tapia, 1999). Likewise, lesions in the CA1 and CA3
regions are the primary hippocampal damage observed in mesial temporal sclerosis
(MTS) epilepsy (Shinnar, 2003). The in vivo 4-AP model preserves synaptic transmission
and in longer term studies mimics the cell death seen in humans (Pena & Tapia, 2000).
It is also known that the longitudinal CA3 slice can support propagation of
epileptiform-like bursts along its length when GABA inhibition is suppressed (Miles et
al., 1988). Similarly, epileptiform activity is known to propagate along the length of an
intact neonatal hippocampus (Luhmann et al., 2000), and spontaneous bursts have been
found to propagate transversely in hippocampus slice preparations. However, the effect
of combined longitudinal and transverse hippocampal pathways on the propagation of 4-
AP induced epileptiform activity in young and adult animals has not been studied.
Previous work has shown that recurrent excitation is responsible for some longitudinal
propagation of neural activity. CA3 axons innervate the entire CA1 region (Ishizuka ei
al., 1990), suggesting that the combination of longitudinal and transverse pathways
should generate orthogonal waves of activity in hyperexcitable tissue which travel from
the CA3 region into the CA1 region and invade the entire hippocampus.
This study utilized an intact unfolded hippocampus preparation, multi-electrode
recording, voltage sensitive dye imaging, and synaptic transmission antagonists to
determine the nature of propagation of 4-AP induced epileptiform interictal-like bursts
50 (ILEs) and to map the spread of this spontaneous activity in the CA3–CA1 regions of the hippocampus in both the transverse and longitudinal directions. Moreover, we applied a lesion technique to the intact hippocampus and showed that a small transverse lesion in the CA3 region is capable of preventing the propagation of these orthogonal waves.
3.3 MATERIALS AND METHODS
Experiments were performed with 60 10–18 days old CD1 mice obtained from Charles
River. Although there were differences in epileptiform onset delay in 4-AP solution, no
significant variability in establishing orthogonal propagation was seen in this age range.
All animals were housed and maintained in veterinarian-monitored animal care facility at
CWRU. Health and welfare procedures complied with ALAC guidelines.
Tissue Preparation
Mice were anesthetized with ethyl ether and decapitated using a small animal guillotine.
After decapitation, the head was immediately immersed in ice cold (2oC – 4oC) oxygenated (O2 95%, CO2 5%), and sucrose-rich artificial cerebrospinal fluid (ACSF)
with following composition (in mM): Sucrose 220, KCl 3.75, NaH2PO4 1.25, MgSO4 2.0,
NaHCO2 26, CaCl2 2.0, and Dextrose 10. Substitution of NaCl with sucrose in ACSF has been shown to reduce the potential effects of cutting-induced trauma and consistently
yields more viable tissue (Aghajanian, 1989).
The planar hippocampus was prepared via our hippocampus unfolding procedure,
detailed in the supplemental materials of this manuscript, and then transferred into a
recovery chamber filled with ACSF with the following composition (in mM): NaCl 124,
51 KCl 3.75, KH2PO4 1.25, CaCl2 2.0, MgSO4 2.0, NaHCO3 26, Dextrose 10.0. Solution
o was bubbled with 95% O2 / 5% CO2 and maintained at a room temperature (25 C). The preparation was stored in this chamber for 1–5 hours before recording.
Multisite Extracellular Recording and Data Analysis
The hippocampus was placed in a submersion brain/tissue chamber system model PH1
(Warner Instruments, Hamden, CT) to provide oxygenation (O2 95%, CO2 5%), temperature (25–32ºC) and rapid exchange of superfusing fluids (normal (n) ACSF, 50
µM 4-AP ACSF solution, or ‘blocker’ solution ACSF with 50 μM NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (DAPV), 40 μM 6,7-dinitroquinoxaline-
2,3-dione non-NMDA glutamate receptor antagonist DNQX, and 100 μM picrotoxin
GABAA receptor antagonist PTX where indicated) during recording. In the case of
normal ACSF recording and 4-AP ACSF, the drug solution was switched in to circulation after nACSF tests were complete. Field recordings were made using pulled glass
micropipettes (1mm outside diameter, 0.5mm inside diameter, 2–6 MΩ) filled with 150
mM NaCl. Population spikes were evoked using a cathodic 100 μsec, 50–150 μA,
antidromic and/or orthodromic stimulus pulse with a period of 2.5 s. Current was injected
into the Schaffer collateral pathway (orthodromic to CA1), alveus (antidromic to CA1),
or CA3 pyramidal layer and recordings were obtained from the CA1 and CA3 pyramidal
cell layers. Only preparations yielding a population spike with an amplitude of ≥ one mV
were used for data analysis. Signals were amplified with an Axoprobe-2B microelectrode
amplifier (Axon Instruments, Inc., Union City, CA) and filtered with a Cygnus FLA-01
or Princeton Applied Research model 113 amplifier. Data was digitized and stored with a
52 Microdata Instruments DT-200 digital recorder. Data processing was performed in
Matlab or Spike2. Student’s t-tests were used for statistical analysis with P < 0.05.
Amplitudes were measured with a normalized baseline voltage of zero. Transmission latency was calculated using a cross correlation algorithm between two recorded channels. Results are shown as mean ± SD unless otherwise noted.
Voltage sensitive dye recording
The planar preparation was stained with the fast voltage-sensitive styryl dye RH-414
(N-(3-(triethylammonium)propyl)-4-(4-(p-diethylaminophenyl)-butadienyl)-pyridium dibromide; Molecular Probes) at 200 µM concentration in normal ACSF solution in a micro superfusion chamber for 15 minutes in a darkened room. Excess stain was washed away with dye-free ACSF before recording. The planar preparation was then transferred to a submerged recording chamber with alveus side down, superfused with nACSF or
ACSF containing 50 µM 4-AP where indicated. A tungsten stimulating electrode was inserted through the back of the hippocampus tissue into the stratum radiatum using a dissecting microscope. Extracellular field potential recordings were simultaneously performed during the imaging to assess the viability of the preparation and to monitor the amplitude of evoked responses. Further details on the equipment and methods used to record the fluorescent signal can be found in this manuscripts supplemental materials. A
0.1 to 0.4% change in fluorescence was seen during neuronal firing. In addition, 2* linear spatial interpolative subsampling was employed to improve image visualization.
53 3.4 RESULTS
Orthogonal Widespread Activation of CA1–CA3 from a Single Stimulus in CA1:
It is well known that 4-AP increases neuronal excitability, which can lead to epileptiform activity in the hippocampus. Using the planar hippocampus preparation bathed in ACSF containing 50 μM 4-AP, we investigated in two dimensions the excitability of the CA1 to CA3 regions by recording the preparations’ response to a single apical stimulus in central (longitudinally) CA1. Membrane-binding voltage sensitive dye
RH-414 was applied to the preparation, and fluorescent imaging was used to reveal the extent of propagation of evoked potentials across the entire CA1–CA3 hippocampus with and without 4-AP in solution. Stratum radiatum (SR) axons were stimulated with a 200
μA, 120 μs pulse to generate a transverse antidromic activation potential in CA3 neurons with a control solution of normal ACSF (Fig. 3-1A) . In 4-AP ACSF, field potentials continued to propagate longitudinally along the entire length of CA3 (Fig. 3-1C).
Recovery time from stimulus to return to baseline fluorescence (resting potential) in the preparation required 33–40 ms (Fig. 3-1E) which was significantly longer than the 15–
20ms recovery time in normal ACSF, each tested in the six preparations analyzed with
RH-414 that met the required orthodromic response threshold of one mV. This response was compared to a control CA1 stimulus in normal ACSF solution (Fig. 3-1A). In the absence of 4-AP, the evoked activity propagates transversely into CA3 but does not spread longitudinally. This noticeable difference in activation pattern with and without 4-
AP indicates that an orthogonal propagation from CA1 to CA3 can take place in hyper- excitable tissue but is not seen in normal solution.
54 Five contour plots of the excitation were generated to analyze propagation across the
tissue with a representative contour plot presented in Fig. 3-2. Initially, the stimulus
response travelled transversely into CA3, as indicated by the 4–5 ms contours of Fig. 3-
2A. This initial antidromic propagation followed CA3 projections into CA1. From central
CA3, activation spread longitudinally along CA3 in both directions (5–10 ms post-
stimulus). In CA1, longitudinal propagation is seen to slow, (3–6 ms post-stimulus Fig. 3-
2A) until the time CA3 has been activated, indicating that neural activation may have
traveled back into the temporal and septal CA1, most likely along the Schaffer collaterals
(SC). CA1 activation area peaks 12–14 ms post stimulus. See Supplemental material for
additional contour plots. While the data displayed are gathered from individual optical
experiments, the results are confirmed in the following sections. These data show that a
single stimulus in the presence of 4-AP could generate widespread activation of the
hippocampus.
Orthogonal propagation from CA1 into CA3
This wide-spread propagation of neural activity in 4-AP solution could be generated by
either a broad spatial arborization of CA1 axons into CA3 or by longitudinal propagation of the activity along CA3 from the plane of stimulus. We first tested the arborization hypothesis by applying bath solution containing either normal ACSF (nACSF) or ACSF with 50 µM 4-AP over the unfolded hippocampus preparation while 100 µs, 100 µA stimulus (ST) was applied at 0.3 Hz to the stratum radiatum (SR) (Fig. 3-3A). Recording electrodes were placed along CA3 spanning a distance of approximately 2.8 mm. The longitudinal extent of CA3 activation from a single central antidromic stimulus was
55 recorded with synaptic transmission antagonists, and compared to the extent of activation in a normal ACSF bath following normalization. Examples of waveforms recorded at points 1–2 are shown in Fig. 3-3B. Stimulation in the CA1 SR in normal ACSF generated an antidromic evoked potential of 1.5 ± 0.3 mV recorded in the CA3 region. However, in control solution (normal ACSF), longitudinal activation of the CA3 was mostly confined to a transverse lamella of ± 500 μm defined as the region of at least 60% maximum amplitude recorded (Fig. 3-3C) where the amplitude is shown as a function of distance.
This result confirms the fact that the axonal arborization of CA3 axons in the SR primarily maintains a lamellar organization. In 4-AP ACSF, evoked potentials showed little attenuation along the entire length of the CA3. Therefore, these data show that 4-AP enhances longitudinal propagation along the CA3 layer either by increasing the number of activated axons, or by inducing a wave of activation within the CA3 region, or both.
Synaptic contribution to widespread activation was tested by adding a synaptic blocker cocktail of 50 μM DAPV, 40 μM DNQX, and 100 μM PTX to the 4-AP bath (Fig. 3-3C).
The enhanced longitudinal propagation was reduced to control levels by these synaptic transmission antagonists (Fig. 3-3C). These data show that the increase in the longitudinal extent of propagation in CA3 requires synaptic transmission and cannot be attributed to the increase in the number of activated axons. Further examination of a synaptic requirement for orthogonal propagation can be found in the supplemental literature for this manuscript.
56 Spontaneous Self-propagating Longitudinal Wave:
To investigate the mechanisms of the strong longitudinal activation in CA3 in the
presence of 4-AP, the propagation of spontaneous events was analyzed. In 50 μM 4-AP
solution, spontaneously occurring potentials were observed in the CA3 and CA1 regions
of the preparation. Fig. 3-4 shows a typical set of spontaneous events recorded from the
CA3 region. These spontaneous potentials had an amplitude of 0.5–5 mV and lasted 50–
200 ms. These potentials are generally referred to as interictal-like events (ILEs) similar to potentials recorded in the transverse slice (Avoli, 1991). These 4-AP induced ILEs were recorded from multiple simultaneous locations in order to determine their focus, propagation path, and extent of coverage of the hippocampus.
Recording electrodes were placed at a depth of 200–300 micrometers for
quantifying wave activity in the somatic region of CA3 in the experiments illustrated in
Figures 3-6 through 3-9. Interictal-like events consisting of tonic activity commenced two to five minutes after application and continued for five to six hours until washout or the preparation was removed. Spontaneous activity was characterized by frequent (one to three second interval) depolarizing bursts recorded in the CA3 region (Fig. 3-4A) and synchronous potentials in the CA1 region of the intact hippocampus. While in normal
ACSF, evoked activity was confined to transverse lamellae, spontaneous activity in 4-AP
ACSF was quite different. As in the case of the voltage sensitive dye recording shown previously, the entire CA3–CA1 region of the hippocampus was found to be active.
Recordings of spontaneous ILE potentials at opposite ends of CA3 (Fig. 3-4B) were analyzed via cross-correlation to determine the propagation delay between the recording sites. Initiation site was determined by observing the timing latency between the
57 recording locations of the initial negative peaks in the ILE waveform. ILEs initiated at
the temporal pole of the hippocampus and propagated quickly (amplitude 1.6 ± 0.9 mV,
speed 0.09 ± 0.03 m/s (Fig. 3-4B,C). Typical longitudinal propagation delay lasted 19.7 ±
6.8 ms (n=8) along the CA3 region from the temporal to septal pole. Axonal conduction velocity was measured by applying synaptic transmission antagonists and measuring the latency from a stimulus response recorded in septal CA3 to a recording electrode in temporal CA3. Compared to axonal propagation along CA3 which was recorded at 0.31 ±
0.08m/s (n=3), the 4-AP induced wave traveled approximately three times more slowly.
In this hyper-excitable state, neurons in the entire CA3 region were spontaneously active
with bursts that propagated as non-attenuating waves across the entire preparation (Fig.
3-5). An evoked traveling response wave similar in shape and duration to the ILE could be induced by stimulation at either end of CA3, creating a wave traveling in a direction leading away from the site of stimulus (data not shown). Immediately following septal stimulation, occasionally the spontaneous wave would initiate at the same pole, so even
though the ILEs were typically generated at the temporal pole, the propagation path is
bidirectional, which confirms other reports (Derchansky et al., 2006).
Synaptic dependence of spontaneous self-propagating longitudinal wave in the CA3
region
A synaptic blocker cocktail consisting of 50 μM DAPV, 40 μM DNQX, and 100 μM
PTX was applied to the bath in addition to 50 μM 4-AP to determine the role of synaptic transmission in the longitudinal wave propagation in the hippocampus. Self-propagating waves were induced by applying a single stimulus to the temporal CA3 (see Fig. 3-6A).
58 Recording electrodes were positioned in three locations along CA3 to measure the response field potential to stimulus before and after the synaptic transmission antagonists were added. Fig. 3-6B shows field potential recordings from the center of CA3 before, during and following washout of the blocker cocktail. In 4-AP alone, the evoked wave traveled the length of hippocampus without significantly decreasing in amplitude (1.7 ±
0.8 mV at the stimulus site to 1.6 ± 0.5 mV at the edge). In the presence of synaptic transmission antagonists, the stimulus response amplitude, while ± 0.8 mV at the site of stimulation, decreased to background noise levels as measured in the longitudinal direction in CA3 away from the site of stimulus to 0.1 ± 0.1 mV at the far end of the CA3 region (Fig 3-6C). These data show that the self-propagating wave can occur spontaneously or via orthogonally located stimulation and that it is synaptically mediated.
Effect of CA3 lesion on longitudinal propagation:
The previous data show that the wave can propagate along the CA3 layer at a lower velocity than that of axonal propagation. We hypothesize that this wave uses the synaptic
CA3 recurrent excitation pathways for which the axons are located within or close to the
CA3 layer. To test this hypothesis, a lesion technique was applied to five preparations displaying spontaneous ILEs in 50 μM 4-AP ACSF. Using a small cutting blade, an area limited to the CA3 region was lesioned (Fig. 3-7A) and pyramidal cell spontaneous field potentials were recorded on each side of the lesion in CA3 and CA1 before and following the procedure. The spontaneous orthogonal wave traveled in the longitudinal direction (1
→ 2) and then transversely (2 → 3) (Fig 3-7B). However, propagation in the longitudinal direction ceased following the lesion. The recorded field potentials were statistically
59 analyzed for cross-correlation timing between temporal and septal lobes and the resulting correlations before and after the lesion were normalized for each experiment (Fig. 3-7C).
In all cases, the signal correlation between the septal and temporal lobes after the lesion was drastically reduced across both CA1 and CA3, from 0.87 ± 0.07 to 0.17 ± 0.06 across
CA3 and 0.95 ± 0.05 to 0.12 ±0.02 across CA1. The data show that wave propagation from temporal to septal poles was completely arrested in both CA1 and CA3 post-lesion.
Likewise, in all cases the amplitude of the spontaneous septal pole activity decreased significantly after the lesion was made. No significant decrease in spontaneous temporal pole amplitude was seen, however a decrease in frequency was observed ranging from
1/3 to 1/4 of the pre-lesion cadence. In three out of five cases, the septal hippocampus became completely silent while in the other two cases, the septal pole began generating its own low amplitude spontaneous potentials, although at a lower frequency and amplitude than the temporal pole (every 2.6 ± 1.4 s vs. 1.3 ± 0.8s). These data show that
1) the 4-AP induced wave propagation path lies exclusively in the CA3 region, 2) a CA3 cut can arrest both longitudinal and orthogonal propagation, and 3) post-lesion CA3 can generate desynchronized spontaneous activity on each side in the presence of 4-AP.
3.5 DISCUSSION
The planar unfolded hippocampus preparation was used to investigate transverse
and longitudinal propagation of spontaneous and evoked 4-AP induced interical-like
epileptiform activity in the mouse hippocampus. This experimental study has six major
conclusions: 1) An orthogonal wave can be generated traveling first transversely along
CA1 and then longitudinally into CA3 in the unfolded hippocampus in the presence of 4-
60 AP. 2) A regenerating wave propagates along the CA3 region of the hippocampus. 3)
This wave-like propagating activity is synaptically mediated along the longitudinal recurrent excitatory pathway in the CA3. 4) The wave-like activity propagates at a speed
1/2–1/3 of axonal propagation speed. 5) This longitudinal wave spreads orthogonally into
CA1, affecting much of the hippocampus. 6) Transverse lesioning of the CA3 region can inhibit the propagation of epileptiform ILEs to longitudinal lamellae while preserving the
CA3–CA1 network. Discussion on the background and benefits of utilizing the unfolded hippocampus preparation is contained in the supplemental material for this manuscript.
The potassium channel blocker 4-AP was chosen in this study as a model of epilepsy due to its ability to generate spontaneous epileptiform activity and maintain synaptic activity at low concentrations. Furthermore, the prolongation of action potential duration enhanced recording of spontaneous activity using voltage sensitive dyes. Previous transverse slice-based studies have shown foci in the CA3–CA2 region of the hippocampus with voltage sensitive dye recordings (Colom & Saggau, 1994). This work expands on previous work to include the entire CA3–CA1 region for network observations in two dimensions. While the focus of 4-AP induced spontaneous activity in
CA3 was found to be the temporal pole, the septal pole can act as an oscillator when removed from the driving temporal oscillator. It has been shown that removal of inhibition in the hippocampus can lead to the propagation of longitudinal wave-like activity (Miles et al., 1988). Propagation velocities similar to those reported above were found in the presence of GABAergic IPSPs (0.15 m/s vs 0.1 m/s noted here), a 2–3* decrease in conduction velocity compared to that of axons in the area (Soleng et al.,
2003). 4-AP increases the excitability of CA3 cells, and at high concentrations (200 μM)
61 can have a depressing effect on interneuronal inhibition (Perreault & Avoli 1991).
Depressed inhibition can lead to an excitable state where group activation on one side of
CA3 can quickly travel the length of the hippocampus. For this reason, the concentration of 50 μM was chosen as it has been shown to maintain inhibition in the networks (Traub et al., 2001). Under these conditions, a self-regenerating, orthogonally propagating wave
was generated along the CA3 throughout the recurrent excitatory synapses on groups of
local CA3 cells. 4-AP additionally causes a prolongation of the EPSP (Avoli, 1996) seen
in CA3 neurons that is likely to come (at least partially) from other excitable CA3
pyramidal cells. The strengthening of these synapses generates a positive feedback effect
that, when combined with the hyper-excitable state of the cells and the increased
occurrence of spontaneous neurotransmitter release, induces spontaneous ILE generation
and subsequent propagation to the entire CA3. It has been shown that CA3–CA3
recurrent excitatory synapses exist in rodents, (MacVicar & Dudek, 1981; Ichizuka et al.,
1990) and computer modelling has suggested a well developed network in CA3 could
play a role in epileptiform burst propagation (Traub et al. 1987, Miles et al. 1988). As
such, it is reasonable to suggest that the longitudinal extent of CA3-CA3 recurrent
arborization plays a significant role in the hyperexcitability and ILE propagation
capability of the hippocampus network.
Spontaneous ILEs occur in slices also, and these experiments demonstrate that halving
the hippocampus can result in two spontaneously firing halves, each containing an
oscillator operating at different frequencies. Similarly, spontaneous oscillations were
observed in longitudinal hippocampal slices (Gloveli et al., 2005) and multi-foci seizure
activity was seen in an intact hippocampus preparation by Derchansky et al. (2006). As
62 the septal pole oscillator tends to drive the whole CA3, and that location has a higher density of connected axons (Amaral & Witter, 1989; Luhmann et al., 2000), it is possible that the frequency of the local oscillator is affected by the local density of axons and recurrent synapses. Whereas bidirectional propagation of epileptiform activity in the planar hippocampus was observed with low Mg2+ (Derchansky et al. 2006), the
propagation seen with 4-AP could be entrained to propagate in the septal-temporal
direction, but spontaneous activity originated from the temporal pole. The relatively slow activation of the whole CA3 region in 4-AP from an antidromic stimulus is likely related to the synaptic link between persistent bursting (ILEs) in the CA3 region and the recurrent excitation pathway (Stoop et al., 2003). The frequency stability of the
spontaneous ILEs is thought to be related to NMDA receptor interactions at CA3
recurrent collateral synapses (Hellier et al., 2007), and is a likely explanation for septal
lobe network oscillation when it is removed from the temporal hippocampal lobe.
Longitudinal self-generating waves similar to those observed in the intact hippocampus
preparation have been previously recorded in ACSF with picrotoxin in longitudinal slices
(Miles et al., 1988). These waves travel longitudinally along CA3 in slices, and as shown
above they also invade the CA1 region, creating orthogonal propagation. Therefore, in
these hyperexcitable models, the whole hippocampus can be excited by a (single) input
into CA3. This activity is most likely mediated by the recurrent excitation network, since
small cuts through the CA3 region can eliminate longitudinal propagation through the
recurrent pathway while preserving the lamellar transverse circuits and longitudinal
propagation through axons outside the CA3 region. A surgical technique of multiple
subpial transection (MST) for the treatment of temporal lobe epilepsy has been proposed
63 as a treatment for intractable epilepsy in order to preserve the formation of verbal
memory (Shimizu et al., 2006). The MST technique transects the longitudinal
interneuronal connections of the hippocampus while preserving transverse connection
fibers. Lesions of sections of the hippocampus have been shown to block synchronous
propagation of epileptiform activity (Derchansky et al., 2006). Our experiments have
shown that a selective transection of the CA3 alone is sufficient to significantly decrease or eliminate trans-hippocampal epileptiform (ILE) propagation not only in the CA3 region but also orthogonal propagation to the CA1 region as well, while at the same time
decreasing spontaneous activity in the temporal hippocampal lobe and maintaining
transverse functional pathways.
3.6 CONCLUSION
While the hippocampus does have an orderly lamellar organization and as shown above,
a preferentially transverse activation direction in normal solution, the often-overlooked
longitudinal pathways of the CA3 region can have a large effect on neuronal activity in in
vitro models of epileptiform discharge or synchronization induced by 4-AP. In particular,
these results show that 4-AP can induce orthogonal waves that propagate longitudinally
along the CA3 layer and transversely into the CA1 region. In a hyperexcitable state such
as the one generated by 4-AP, the hippocampus is transformed from a primarily lamellar
organization of activity into a broadly activated structure through orthogonal propagation
into the whole hippocampus. This propagation is slower than axonal propagation and is
synaptically mediated. Furthermore, it is possible to reduce or arrest epileptiform
propagation along CA3 via a selective transverse lesion. It is expected that the unfolded
64 planar hippocampus preparation will enable further study of the importance of
orthogonally active hippocampus networks in seizure propagation and in vitro testing of novel surgical techniques.
3.7 ACKNOWLEDGEMENTS
This work has been funded by NIH Grant 5-R01-NS-040785-04 and a Department of
Education GANN Neural Engineering Training Grant.
65 3.8 FIGURE CAPTIONS
Figure 2: Representative figure of contours of activation over time from a stimulus in
CA1 of five preparations analyzed with this technique. Contour labels are in ms. Time average of four recordings in one preparation. A) Stimulus occurs at contour time 0 in the
CA1 region and activation propagates down to CA3 in 4ms. From there, activation spreads longitudinally along CA3 and into CA1, following the diagonal contours. B)
Tissue orientation for A), contour labels are in ms.
Figure 3: Extent of evoked response in CA3 from a stimulus in CA1. A) Location of stimulus and recording sites in the unfolded hippocampus preparation. B) Examples of extra cellular field recordings at two locations in normal and 4-AP solution. C) Evoked responses in 4-AP spread across the entire CA3 region while the control (Normal ACSF) responses are only seen in a 1–1.5 mm lamella. (n=6) With 4-AP and synaptic transmission antagonists, evoked response envelope is similar to NACSF control, indicating the broadened response envelope seen in 4-AP alone is caused by synaptic transmission.
Figure. 4: Spontaneous 4-AP induced activity in the CA3 region of the hippocampus. A)
Spontaneous activity recorded over a period of two minutes showing repeated bursting pattern. Bursts occurred at a frequency of 1.3 ± 0.8s. B) Field potentials recorded at far sides of CA3. Potentials recorded at location two (Rec2) are delayed on average 13.9 ms from Rec1. Cross correlation analysis for a sample of waveforms shows a propagation
66 delay of 13.9 ms, the recording distance was 1.5mm for this set, giving a propagation velocity of 0.11 m/s. C) Cross-correlation of potentials at Rec1 and Rec2, showing group delay (n=8).
Figure 5: Spontaneously generated wave propagating along the CA3 region in the unfolded mouse hippocampus in 50 µM 4-AP. Dashed line indicates propagation speed of 0.11m/s. Trace one is a recording from the temporal pole of the CA3, the trace at location six is recorded from the septal pole of the unfolded hippocampus preparation, and the traces in between are recorded along the longitudinal axis. Peak initial wave amplitude is indicated by arrows.
Figure 6: A) CA3 recording locations for evoked wave synaptic propagation blocking experiment. B) Synaptic dependence of longitudinal wave propagation: Evoked CA3 wave in 4-AP (bottom trace), 4-AP with 50 µM DAPV, 40 µM DNQX, 100 µM PTX
(center trace), and wash with 4-AP (top trace) all recordings from location 2. The presence of synaptic transmission antagonists reversibly eliminates the evoked wave in the presence of 4-AP. C) CA3–CA3 wave propagation amplitude from lateral to septal poles. The 4-AP induced wave does not significantly decrease in amplitude along CA3, whereas the evoked potential in blocker solution does not propagate to the opposite
(septal) end of CA3.
Figure 7: Transverse lesion of the CA3 region blocks longitudinal wave propagation in the hippocampus and reduces spontaneous amplitude at the septal pole. A) location of
67 lesion and recording sites. For CA3–CA3 recordings, locations one and two were used.
For CA3 to CA1 recordings, locations one and three were used. B) Top traces (Control):
Synchronous activity observed at the poles of the CA3 region in 50μM 4-AP. Top traces
(Lesion): Unsynchronized activity observed at the poles of the CA3 region after a
selective CA3 lesion (marked in red). Note the septal pole still shows spontaneous bursts,
however at a lower amplitude than before the lesion. Bottom traces: Septal CA1 activity
recorded before and after selective lesion of the CA3. Note the amplitude decrease along
with de-synchronization from the temporal CA3, indicating that a selective lesion of the
mid-CA3 blocks wave propagation along the preparation. C) Bar graph: normalized cross
correlation of temporal and septal CA3 recordings before and after CA3 lesioning. Before
the lesion, there existed strong correlation between recordings at locations one and two,
and one and three. After lesion there was a significant decrease in correlation in both
recording sets (P<0.01, bars MSE), indicating the CA3 lesion severed the 4-AP induced
wave pathway from the initiation site to the septal pole CA3 and CA1. (n=5)
68 3.9 FIGURES
3mm
B A o CA1 Sti
CA 3mm
o D C CA1 Sti
CA DG
0.3% -Δf/f E o 10ms 0.0% 0.001Δf/f
F
3mm
Fig. 3.1
69 Propagation Transverse A Longitudinal
0 2 CA1
4 6 9 CA3
B
CA1 Sti .
CA3
Fig. 3.2
70 A St
CA1 . Rec CA3 . . 1,2 ...... 3,4 ...... DG 0 1mm 2.8
Fig. 3.3
71
0.6
0.4
0.2 0.0139 0
-0.2
-0.04 -0.02 0.00 0.02 0.04 0.06 0.08 Time seconds
Fig. 3.4
72
6
5
4
3
2 2mV 1
0 0.05 0.1 0.15
Seconds
DG ...... CA3
Rec 6 Rec 1 CA1
Initiation Site
Fig. 3.5
73
A DG CA3 . . . CA1 Rec 1 Rec 3 Rec 2 Stimulus
B
4-AP, Rec 2
2mV
10ms 4-AP+Antagonists, Rec 2
4-AP Wash,Rec 2
C Longitudinal Recurrent CA3 Propagation
4-AP +Synaptic Blockers
1.4
1.2 1
0.8
0.6 Rec 1 Rec 2 Rec 3 0.4
Normalized Amplitude 0.2
0 lateral (initial) septal 0mm 1mm 2mm 3mm
Fig. 3.6
74
A lesion
2 . CA3 1 . Initiation site 3 . CA1
B 2
Control 1
2
Lesion 1
3 Control 1
3 Lesion
1
500μV 500ms
CA 3-CA 3 (1-2) CA 3-CA 1 (1-3) C 1.2
n 1
0.8
0.6
0.4
0.2 Normalized Recording Correlatio
0 Normal Lesioned
Fig. 3.7
75
Abbreviations: EC: Entorhinal Cortex 4-AP: 4-aminopyridine ILE: Interictal-like event SR: Stratum Radiatum
76
CHAPTER 4
A Substrate-Transparent Microelectrode Array System for in-vitro Rodent Hippocampus Recording
77 4.1 ABSTRACT
A novel penetrating microelectrode array was designed and fabricated for the purpose
of recording neural activity. The array allows two dimensional recording of 64 sites in
vitro with high aspect ratio penetrating electrodes. Traditional surface electrode arrays,
although easy to fabricate, do not penetrate to the viable tissue such as central
hippocampus slices and thus theoretically have a lower signal/noise ratio and lower
selectivity than a penetrating array. Furthermore, preparation of the hippocampus slice results in dead tissue in closest proximity to these traditional electrodes and the cell bodies of the CA1 region, degrading activity-based optical imaging techniques as well.
An array of 64 electrode posts each with a target height of 200 microns was fabricated in silicon and bonded to a transparent glass substrate. The impedance of the individual electrodes was measured to be approximately 1.5M Ohms ± 497kOhms. The signal to noise ratio was measured and found to be 19.4 ± 3 dB compared to 3.9 ± 0.8 dB S/N for signals obtained with voltage sensitive dye RH414. A mouse unfolded hippocampus preparation was bathed in solution containing 50 micro-molar 4-Amino Pyridine and a complex two dimensional wave of activity was recorded using the array. These results suggest that the penetrating electrode array is superior to that of voltage sensitive dye techniques for broad field two-dimensional neuronal activity recording.
78
4.2 INTRODUCTION
Multi-site extra-cellular recordings are crucial to the study of large neuronal networks
and multiple regions of activity simultaneously during an experiment. Voltage sensitive
dyes have been developed to fluoresce with amplitudes modulated by cellular trans-
membrane potential. These dyes, such as Di-8-Annepps and RH414 can be very useful in
studying large areas of tissue at once, however they suffer from several problems. First,
in the intact hippocampus deep tissues such as the somatic layer are difficult to study due
to pronounced light scattering. Secondly, dyes exhibit photo-toxicity with the high light
intensity required to record high-speed images, causing noticeable extra-cellular response
changes. In particular, phototoxicity renders imaging of epileptiform activity models such
as low calcium and 4-AP very difficult, as the spontaneous discharges can cease during
high intensity light exposure. Finally, long term (over an hour) studies are difficult due to
photo-bleaching, which decreases the fluorescence amplitude for each exposure taken.
The wide range of applications of microelectrode arrays (MEAs) in scientific research
has resulted in the commercial availability of several array-based solutions. One of the
most basic and widely used designs was first developed circa 1972 by Thomas, et al.
(1972). This simple solution involves evaporating a metal conductor onto glass and
covering the interconnect traces and non-active sites with an insulating polymer, leaving
the recording sites exposed. The result is an array of flat metal electrodes that can be
pressed against tissue such as cardiac muscle, or used a as substrate for tissue culture.
The main drawback to this approach is that a low density of electrically active cells are in proximity of the recording sites. It has been shown that a penetrating electrode geometry will produce higher signal amplitude recordings in slices (Nordhausen et al.,
79 1994). Previously reported devices, however, have low aspect ratio (essentially
pyramidal) electrodes which are not ideal for approaching active cells. Furthermore, the
pyramidal shape constrains their usable height and array density, a tradeoff which leads
to fabricated heights of approximately 50 µM (Thiebaud et al. 1999). In addition,
previously reported penetrating devices have been fabricated on an opaque silicon
substrate. A transparent substrate is preferable because it would allow the use of
transmission microscopy for simultaneous imaging and electrical recording from neural
networks, as well as optical stimulation protocols.
Development was undertaken to create a multi-site, penetrating recording device for in vitro brain preparations of not only hippocampal slices, but a new unfolded hippocampus preparation. The unfolded hippocampus preparation has benefits over traditional slices in that it maintains longitudinal inter-neuronal processes which are thought to play a role in activity regulation, and the resulting preparation consists of a 2-dimensional pyramidal cell layer consisting of CA1 to CA3, versus a 1 dimensional layer of pyramidal neurons obtained from traditional slices. Post preparation, this pyramidal cell layer of interest
resides 180–270 microns from the alvear surface, too deep for traditional pyramidal
penetrating electrode arrays to reach. Therefore, the array described here was designed to overcome the described challenges by exhibiting the following characteristics: 1) signal- to-noise ratio greater than 10dB, 2) high inter-electrode selectivity of local neuronal populations, 3) high aspect ratio spikes reaching a height of 200 micrometers, 5) optically transparent, and 6) re-usable. The MEA and amplifier system described below is the first known device to incorporate a penetrating high aspect ratio of 10 on a transparent substrate for multi-modal neural recording in the rodent hippocampus preparation.
80
4.3 MATERIALS AND METHODS
Tissue Preparation
All experiments were performed in the CA1 and CA3 hippocampus regions of young
(P10–P25) mice (Charles River, CD-1 strain). The experimental protocol was reviewed
and approved by the Institutional Animal Case and Usage Committee. Mice were
anesthetized using ethyl ether and decapitated. The brain was rapidly removed and
chilled in ice cold (3–4o C) sucrose-rich artificial cerebro-spinal fluid (sACSF) consisting
of (mM): Sucrose 220, KCl 3.75, NaH2PO4 1.25, MgSO4 2, NaHCO2 26, CaCl2 2, and
Dextrose 10 (pH 7.4) for approximately 10 seconds. Following removal, the brain was sectioned twice, removing the cerebellum and separating the two hemispheres by cutting midsagitally. For slice preparations, one hemisphere at a time was glued sagital face down to the tray of a microtome (VT1000S, Leica, Nusslock, Germany) with cyanoacrylate, and secured in the microtome slicing chamber filled with oxygenated (O2
95% CO2 5%), sACSF. Sucrose-based slicing medium has been shown to increase cell
viability in vitro (Aghajanian and Rasmussen 1989). The resulting transverse slices
(Skrede and Westgaard, 1971; Teyler, 1980) (350 µm) were immediately immersed in
oxygenated normal ACSF (nACSF) consisting of (mM): NaCl 124, KCl 3.75, KH2PO4
1.25, CaCl2 2, MgSO4 2, NaHCO3 26, Dextrose 10 (pH 7.4), and incubated at room
temperature for at least 60 minutes before being transferred to a submerged-tissue
perfusion chamber and discarded 6–8 hours post incubation.
For unfolded hippocampus preparations, the brain was quickly moved onto a
moistened, chilled filter paper following the same methodology as described for brain
81 slice preparations (Teyler, 1980). Chilled oxygenated sucrose rich ACSF was dripped
frequently onto the hippocampus to maintain moisture and allow some oxygenation. The
cerebellum was removed and the brain was hemisectioned by a razor. The hippocampus
was freed from the septum and entorhinal region using either dorsal or ventral approach
(Teyler, 1980). Both rounded ends of the hippocampus were trimmed in a line perpendicular to the longitudinal axis, resulting in a slab approximately 3mm wide.
Similar to Wu et al. (Wu et al., 2002), the dentate gyrus (DG) was unrolled by sliding a
sharp glass electrode along the line of the fissure and carefully unrolling the DG using a
combination of glass and tungsten wire loops. All experiments were conducted using
nACSF with 50µM 4-AminoPyradine (4-AP) obtained from Sigma.
Electrical Recording
Orthodromic, evoked field potentials were recorded in the stratum pyramidale and
basal dendritic area of in vitro mouse hippocampus slices using both glass microelectrodes (3–10MΩ) filled with 150mM NaCl solution and individual microelectrode array spikes (1–2MΩ). Square pulses (100μs, 100μA, 0.5–0.2 Hz were used to evoke orthodromic potentials in the CA1 and CA3 region of the hippocampus via a sharp-tipped Tungsten stimulating electrode. Evoked somatic and dendritic region activity was recorded and later quantified. The signal to noise ratio (SNR) was calculated as shown in (1) where RMSSig is the root mean square of the section of data immediately
following a stimulus, not including the stimulus artifact and RMSNoise is the root mean
square of the same length of data for a time window with no neural activity.
82 RMS Sig SNR(dB) = 20*log10 (1) RMS Noise
Optical Recording
The hippocampus preparation was stained with the fast voltage-sensitive styryl dye RH-
414 (N-(3-(triethylammonium)propyl)-4-(4-(p-diethylaminophenyl)-butadienyl)-pyridium dibromide; Molecular Probes) at 200 µM in a micro superfusion chamber for 15 minutes in a darkened room. Excess stain was washed away with dye-free ACSF before recording. The preparation was then transferred to a submerged recording chamber.
Similar current stimuli are to be used for optical and electrical recordings. At the same time, a glass microelectrode filled with 150 mM NaCl (resistance 3–10 MΩ) was inserted into the mossy fiber cell layer, using a dissecting stereomicroscope for guidance. Extra- cellular field potential recordings were simultaneously performed during the imaging to assess the viability of the preparation and to monitor the amplitude of evoked responses.
The tissue was illuminated via epi-illumination using an inverted Nikon Diaphot culture microscope fitted with a low noise DC-powered tungsten-halogen lamp (100W, 12 V).
Stimulation, shutter control, pump pause, and bias adjustments were done automatically though a custom program running on a digital stimulator controller (PG4000, Neuro Data
Instrument). Fluorescence evoked was measured (interface filter: 535 ± 25 nm, dichroic mirror 580 nm; barrier filter 590 nm) at each scanning point in a 16 x 16 photodiode array (Hamamatsu C4675-102) and amplified with a custom amplifier and low pass filter with a cutoff set to 1 KHz. The imaging objective (10*, 0.5 NA) results in a spatial resolution of each element of 137 * 137 μm. Current signals from each photodiode were
83 converted to voltage, amplified, filtered, digitized, and then sampled at 500–1500 Hz.
Final image processing is usually performed in order to better reveal the characteristics of
the network. Techniques such as time averaging can be used along with repetitive stimuli
in order to improve the signal to noise ratio, but these techniques were not used in the
assessment and measurement of the SNR of either the optical or electrical devices.
Typically a 1–2% change in fluorescence was seen during neuronal firing.
Microelectrode Array Design and Construction
The array was designed with thin, high aspect ratio spikes that are terminated with sharp
tips for easy and non-tearing insertion resulting in minimal tissue damage. The spike tips
have electrically active surfaces with impedance appropriate for neural recording. The
non-active areas are coated with a biocompatible insulation material, and the substrate is
transparent glass. The array is designed to provide an adequate density of recording sites, and such that the active area is large enough to cover the area of interest. Based on the structure and size of the hippocampus, the distance between spike centers is 400 microns in the x direction and 300 microns in the y direction in a pattern of 8×8 spikes, resulting
in an active area of 2.1mm by 2.8 mm and a manageable 64 recording channels. The
spikes are to be 20 microns wide and 200 microns high.
The microelectrode array was designed and fabricated at the NASA Goddard Space
Flight Center Detector Development Lab, Washington, DC. The array chip was built on a
transparent Pyrex glass substrate with thin gold leads from each spike to bond pads on the outer edges of the 10×10mm chip. The chip was then glued and wire bonded to an open- top 137 pin ceramic PGA package. Before mounting the chip in the package, a 10mm
84 diameter hole was laser cut into the center of the package, allowing inverted microscopy and fluorescence imaging of the tissue while mounted on the array.
Construction of the array began with a 10mm Pyrex wafer patterned with resist to form bonding pads, spike bottoms, and spike traces and etched in 7:1 buffered hydrofluoric acid solution to create recesses. The recesses were found to be necessary in order to prevent voids around deposited metal during the anodic bonding process. Next a highly doped silicon wafer of a conductivity of at least 0.1 Ω-Cm was thoroughly cleaned and then placed in an oxidizing furnace to build up a thermal silicon oxide of approximately
5000 Ǻ thickness. The oxide was then masked and etched in a 7:1 buffered hydrofluoric acid solution down to the silicon in the locations of the bottom of the spikes and wire bonding pads. Then a thin layer of chrome gold alloy was evaporated onto the glass and gold wafers so that it rose approximately 500 Å above the glass etch and silicon oxide depths. The excess metal was lifted off and the wafers thoroughly cleaned before being anodically bonded to each other with the gold sides facing each other. The temperature of the anodic bonding process was set at just below the eutectic temperature of the gold and silicon so that good electrical bonds were formed from the silicon to the traces etched into the Pyrex backing. The bonded wafers were then placed in a warmed 25% TMAH anisotropic etch solution until the silicon was thinned to the desired spike height of
200µm. A tip mask was then exposed on the silicon and sharp tips are etched into the silicon using an anisotropic gas etch. A photoresist pattern was exposed on the silicon and gold was again evaporated on the wafers and lifted off of the non-desired areas. Gold was left on the spike tips and wire bonding pad locations to be used as contacts and active conducting area. Finally, a 50µm thick resist was spun on the silicon and used as a deep
85 RIE mask for the final etch of the silicon wafer down to the oxidation layer. An array of 8
sacrificial pillars was then patterned encircling each electrode tip to aid in the DRIE deep
etch process. The purpose and effectiveness of these pillars is discussed below. The oxide
formed on the silicon wafer in the first step, which had been sandwiched between the
Pyrex and conducting silicon, acts as a passivation layer over the traces in the Pyrex after
they are exposed by the DRIE Bosch process. A final biocompatible insulating layer, 3–
4µm of parylene was coated onto the entire chip and laser ablated from the bonding pads
and spike tips. This conformal parylene coat helps ensure biocompatibility and insulation
during experimentation. A diagram of this entire process is shown in Fig 4.1.
Microelectrode Array Amplifier System
A 64-channel low noise amplifier circuit was designed and built to be located very
close to the recording sites. Each amplifier section was designed with a low input bias current and voltage noise, along with a low-pass Sallen-key filter with a bandpass of 0.1
Hz to 4kHz (Fig. 4.3). This low-pass post filter was designed to reduce noise, prevent sample aliasing in the acquisition process, and act as a buffer with which to drive cabling and its associated capacitances to the acquisition processor. A simulated neural recording from a glass microelectrode was passed through the model with satisfactory results. (Fig.
4.3). The bandwidth of the final constructed amplifier set was tested with a sinusoidal waveform sweep and found to be 0.5 to 3.5kHz ± 0.8%.
86 4.5 RESULTS
A. Electrode Array Fabrication
Fabrication of the array was a significant challenge, with a 40% yield of 12 chips per wafer, and of those, 36% with all spikes remaining intact, final yield 14%. The spike electrodes were stress tested by placing discarded mouse brain tissue such as the cerebellum on to the spike area and, using a micromanipulator, maneuvering the tissue in a linear motion across the spikes in a simulated worst-case-scenario. Out of 8 arrays tested, none incurred fractured electrodes as a result of the tissue stress test. Despite this, care must be taken during manipulation of the tissue to ensure that no other objects come in contact with the array’s micro spikes. It was found the spikes were readily damaged by contact with tungsten stimulating electrodes or other stiff object contact. Spike electrode impedance was measured to be 1.5M Ω ± 497 kΩ at 1 kHz. Final average electrode spike height was 244µm ± 6.2 µm SD. Within a single array, the electrode spike height varied only ± 1.6 µm SD. Spike to spike spacing variability was measured to be less than 10 µm.
Average sacrificial pillar leftover height (of all leftover pieces still attached to the substrate) was 20 µm, shallow enough to not interfere with tissue penetration of the main electrodes. On average, there were 5 sacrificial pillar pieces left per array.
B. Sacrificial Pillars
In the course of fabrication it was necessary to define a ring of 8 ‘sacrificial pillars’ surrounding the electrode spikes to protect them from negatively sloped sidewalls during the DRIE etch process. The thicknesses of the pillars along with the pillar spacing determined the amount of electrode protection as well as when the pillars would be
87 completely under-cut at the end of the DRIE etch as desired. An experimental mask was created that allowed the correct proportion of spacing and sacrificial pillar to be determined, shown in figure 4.5. If the pillars were too thin, they would fall early (Fig.
4.6A) However if the pillars were too thick they would not be sufficiently undercut by the time the desired DRIE etch depth was reached (Fig. 4.6C). An analysis of the test
samples made using the desired etch height of 200 micrometers revealed that a ring of 8
sacrificial pillars of diameter of 15 microns and spaced 55.75 microns from the center of
the electrode spike performed well to protect the central spike and at the same time, by
the end of the etch the sacrificial pillars were completely or nearly completely undercut,
to the extent that a wash agitation could wash them away (Fig. 4.6B). Such an
arrangement could nearly eliminate excessive passivation erosion of the desired spike
electrode, while maintaining etch conditions that mostly eliminate grass formation during
the etch. This step of processing is reviewed further in the discussion.
C. Neural Activity Measurement
Optical imaging of CA1–CA3 evoked potentials in 50µM 4-AP was performed as a
baseline comparison of signal quality. These were compared with evoked potential
recordings from the microelectrode array. SNR analysis showed that significant
photobleaching occurred over two minutes with the RH-414 optical imaging technique,
with the SNR decreasing from 3.9 ± 0.8 dB to 1.3 ± 0.7 dB. Comparatively, little signal
degredation was seen with the microelectrode array recording showing a 20.3 ± 2.5 dB
SNR at the onset and ending with a 19.4 ± 3 dB measurement (Fig. 4.10).
The regional specificity of the array was studied by placing active electrode array
88 spikes in the basal dendritic tree of the CA1 region of the hippocampus slice. In this
location, the orthodromically activated extra-cellular field potential is positive
(Kloosterman et al., 2001). As shown in Fig. 4.8, this positive field potential is seen in
this region by the array electrode, and the negative field potential is seen with the glass micropipette electrode near the pyramidal cell body. Such a fine spatial differentiation
was not observable with the RH414 optical technique.
Spontaneous population bursts in 4-AP (50 µM) nACSF were recorded using the MEA
and a glass electrode (Fig. 4.9). The fluorescent RH-414 imaging method had previously been unable to resolve these low-level spontaneous bursts.
D. Multi-channel Recording
After the arrays were diced and wire bonded to a PGA package, the chip was installed in a ZIF socket in the amplifier PCB, and a Faraday shield enclosure mounted around the circuitry and outer edge of the package. The recording array system was used to record spontaneous 4-AP induced epileptiform activity in the somatic layer of the unfolded hippocampus. The tissue was positioned so that recording electrodes reached CA1–CA3 hippocampal regions along the length of the unfolded preparation. Spontaneous interictal- like wave activity was recorded in the CA3 and CA1 regions as shown in Fig. 4.11. Data was acquired at a rate of 4-5kframes/sec and spatially interpolated for clarity in the figure. Data was then filtered with a low-pass filter to 150 Hz. The data show an array response from the unfolded hippocampus preparation which reveals propagation and spontaneous activity in the CA1 and CA3 regions of the hippocampus. A wave-like ILE event can be seen traveling along CA3 in Figure 4.12. This wave-like event closely follows that seen from the previously described class micropipette control shown in
89 chapter 3, figure 3.5. Both events travel at approximately 0.1 m/s in the CA3 and initiate in the temporal hippocampal pole. Figure 4.12 reveals a complex arrangement of
Epileptiform propagation within the CA1-CA3 regions along the hippocampus. Activity initiates at the temporal pole but does not immediately travel down the CA3 in a sharp wave, instead broadening out over time by the time it reaches the septal pole. The second wave of the burst, however, maintains a somewhat sharp peak as it travels along the CA3 region. In the final burst, the extracellular potential wave maintains a sharp profile along the entire CA3. Of note is that the propagation speed for all three waves, which is
graphically represented by the dashed line in Figure 4.12C, is the same from temporal initiation to septal termination. Use of the penetrating spike recording array also reveals group activity in CA1 near its connection to the entorhinal cortex. The acquisition time for the data gathered with the array system was approximately 5 seconds, whereas traditional re-positioning of the glass recording micropipette along CA3 required about
30 minutes to ensure quality recording and proper depth, over which time the spontaneous propagation may change slightly, causing artifacts.
4.6 DISCUSSION
Described in this manuscript is a novel penetrating microelectrode array device and
amplifier system that achieves a penetration depth of 180–270 microns while having high
aspect ratio electrode spikes, a transparent substrate, and biologically relevant bandwidth
and sensitivity. During array fabrication, most processing steps such as the anodic
bonding of wafers, thinning, and tip patterning proceeded without issue after parametric
90 tuning. However, the desired high aspect ratio of electrode spikes, combined with their broad inter-spike spacing, presented significant difficulties during the DRIE Bosch process etch procedure. Tuning etch parameters such that a 200 micron etch of silicon to the silicon oxide layer could be performed without formation of silicon ‘grass’ at the etch bottom resulted in a negative sidewall angle of the electrode spikes, which by the end of the etch had significantly thinned bases, down to ½ of their original diameter. This thinning produced spikes that had an apical diameter of 20 micrometers as designed, but at the base as little as 10 microns of pillar remained. Such thinning of the electrode spikes weakened them to the point of single-use applicability. One of the goals of this project was to create an array with electrodes robust enough to withstand reuse, multiple neural tissue insertion, and repeated cleaning so the spike thinning problem had to be solved.
The cause of the negative sidewall angle etch resides in the process mechanics. A standard BOSCH etch process consists of consecutively repeating two steps — etch and passivation — many times until the desired depth is achieved. The passivation step is designed to coat the features freshly exposed sidewalls created during the etch step so that subsequent etch cycles do not etch the silicon further. A negative sidewall arises when the passivation layer is eroded away during the etch step, allowing each etch to further infiltrate the silicon sidewall. Corrections can be made to reduce sidewall passivation erosion, such as reducing the platen temperature and adjusting the mean free path length of etch ions, however with the design pattern used none of these solutions were sufficient to prevent negative a sidewall angle on the electrode pillars. In areas of the etch where silicon was more densely masked with features such as troughs or test patterns, instead of featureless planes between pillars, it was observed that the sidewalls
91 were not sloped on the same wafers that contained thinned electrode pillars. In fact,
negatively sloping walls occurred preferentially at the corners of features that lacked
nearby structure. It was theorized that the lack of sloping in some areas was caused by a
shielding effect from non-perpendicularly approaching ions of nearby structures that
formed as the etch progressed. If the desired electrode pillars could be shielded from non- perpendicularly approaching ions during the etch, perhaps they could also be spared from thinning. An experiment was devised to test the shielding effect of ‘sacrificial’ pillars arranged in a ring surrounding the desired electrode. These sacrificial pillars, in theory, would absorb excess erosion caused by non-perpendicularly approaching etch ions while allowing the desired central electrode pillar which they surround to be etched with straight sidewalls. A test DRIE resist mask was created with a matrix of varying width and spacing of eight sacrificial pillars surrounding each electrode spike. Sacrificial pillar diameter ranged from zero to thirteen microns in steps of two microns along rows and center-to-center pillar spacing from the central protected spike ranged from 21.5 micrometers to 36.4 micrometers along columns. Test DRIE etches of the desired depth were performed in order to determine the ideal sacrificial pillar arrangement which would protect the electrode spike from over-etching and also result in completely undercut or sufficiently thinned sacrificial pillars at the end of the etch so they could be washed away in a rinse, leaving only freestanding electrode spikes. Fig. 4.5 depicts an electron micrograph of one such test sample. After thickness and spacing optimization, by the end of the DRIE etch, the sacrificial pillars were completely or nearly completely undercut, to the extent that a wash agitation could wash them away (Fig. 4.6). Such an arrangement successfully protects the desired electrode spike while minimizing grass formation. In
92 addition, the silicon oxide layer that is reached in device fabrication during this step acts as an etch stop, allowing a few more cycles to cut any small grass formations and sacrificial pillars down. The methods described herein allowed for the creation of high aspect ratio, broadly spaced electrode spikes with reduced silicon grass formation and negative sidewall slope.
The electrode array described in this manuscript has several desirable features for in- vitro work and is particularly well-suited for two-dimensional recording from pyramidal cells of the unfolded rodent hippocampus. Its electrode height allows for penetration to a desired recording depth and its transparent substrate allows for optical recording and stimulation while the electrodes are being used. The combination of the recording array and the unfolded hippocampus allows detailed two-dimensional electrophysiological investigation of the hippocampus neural network which was previously difficult, unreliable, or impossible. One limitation of the array is its relatively high tip impedance averaging 1.5M Ω. This impedance is a function of the electrode tip area, and the surface roughness of gold deposition. It may be desirable to decrease the electrode impedance in the future using roughened polysilicon (Paik et al., 2003) or a sonicoplated platinum black (Desai et al., 2010) coating.
4.7 CONCLUSION
The microelectrode array shows greatly improved SNR and recording durations than seen with fluorescent optical imaging. Furthermore, spontaneous activity that was not reliably observable with the optical method is now easily recorded with high SNR and
93 temporal resolution. The described device achieved the desired goals of high aspect ratio electrodes, transparent substrate, and reusability. Its electrode placement is ideal for recording from rodent hippocampus slices and unfolded hippocampus preparations. The array electrophysiological results in 4-AP ACSF agree with those recorded using traditional glass micropipettes. Furthermore, the data is more reliable than a re- positioning micropipette technique because it is gathered in real time with all positions recorded per frame within a few tens of microseconds instead of tens of minutes. This array will be used for two-dimensional mapping of spontaneous ictal and interictal epileptiform activity in the hippocampus, and is applicable in other studies such as measuring the network response of optically-sensitive normal and transfected neural tissue, and studying whole-hippocampus synaptic plasticity reponses to local stimuli. In combination with the unfolded hippocampus preparation, this new array and amplifier system should lead to an improved understanding of hippocampal ictogenesis and network operation.
4.8 ACKNOWLEDGEMENTS
Financial support for this proposal was provided by NIH grant R01NS40785
94 4.9 FIGURE CAPTIONS
Fig. 4.1: Simplified assembly process showing a single spike electrode.
Fig. 4.2. A) Completed and mounted electrode array with perfusion chamber attached. B)
Close-up of subset of array electrodes.
Fig. 4.3. Schematic and simulation of single amplifier circuit.
Fig. 4.4. Scanning electron micrograph of electrode spike tip during fabrication.
Fig. 4.5. Scanning electron micrograph of a sacrificial pillar array test showing the progression of thin sacrificial pillars being undercut and falling early toward the top, and thicker sacrificial pillars not falling near the bottom. In between, sacrificial pillars are undercut sufficiently at the end of processing to be washed away leaving the desired electrode spike undamaged.
Fig. 4.6. Electron micrographs of various sacrificial pillar diameter and spacing tests. A)
Sacrificial pillars initially protected center electrode, but were undercut too soon, leaving it unprotected, at which time thinning rate increased on central spike. Note: short pillar to the right is created by the horizontal mask of a fallen sacrificial pillar. B) Undercut sacrificial pillars and preserved electrode (center). C) Excessively tight sacrificial pillar spacing prevents undercut at the desired etch depth and causes them to merge.
95
Fig. 4.7. Simulation of amplifier circuit with real neuronal data: Top trace: amplifier
output Bottom trace: input *100
Fig. 4.8. Comparison of a single microelectrode spike recording in the basal dendrites of
a pyramidal neuron in the CA1 region with a glass microelectrode recording near the
somatic region.
Fig. 4.9. Spontaneous 4-AP induces interictal bursts in the CA3 region of the
hippocampal slice as recorded by a single microelectrode array spike (above) and a glass
micropipette (below).
Fig. 4.10. Comparison of optical dye recording and penetrating microelectrode array
recording SNRs over time of evoked potentials in 50µM 4-AP in the CA3 of the
hippocampus slice preparation.
Fig. 4.11. A)Array potential map of 4-AP induced spontaneous inter-ictal like activity in
the hippocampus, 10ms per frame. B) Placement diagram of recording frame in A) on
unfolded hippocampus and scale color bar. C) Exemplary traces from the center of the
image displayed in A), sampled top to bottom.
Fig. 4.12. A) Array potential map of 4-AP induced spontaneous inter-ictal like complex activity in the hippocampus, 5ms per frame. Black line in first frame indicates location of
96 sampled channels for trace data in C. B) Placement diagram of recording frame in A on unfolded hippocampus and scale color bar. C) Exemplary traces from the right third of the image displayed in A, sampled top to bottom.
97 4.10 FIGURES Resist
Metal
Pyrex
1) Pattern and wet etch recess in Pyrex, then deposit metal for interconnects
Metal Resist
SOI wafer
2) Pattern and wet etch recess in oxide layer on bottom of wafer, then deposit metal
3) Anodically bond SOI and Pyrex wafers together
4) Etch back silicon wafer down to Desired spike height.
5) Pattern resist above spike.
6) Isotropically etch silicon to undercut resist and create sharp tips.
7) Deposit electrode metal onto spike tip.
8) Pattern thick resist and deep reactive ion etch to form high aspect ratio spikes. Figure 4.1.
98
A B
C
Figure 4.2.
Figure 4.3.
99
Figure 4.4
Figure 4.5
100 A
B
C
Figure 4.6
101
0.1
0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6 300ms -0.7
Figure 4.7
10ms 1mV
Figure 4.8
102 50ms
250μV
Figure 4.9
Array Optical 25 7
6 20 5
15 4
3 10 Array SNR, dB Array SNR, 2 dB SNR, Optical 5 1
0 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 Time, Minutes
Figure 4.10
103 10ms/frame
A
-2mV
B CA1 CA3 0mV
2mV
C 2mV
300ms
Figure 4.11
104 A 5 ms/frame
-1mV
B CA1 CA3 0mV
1mV
C
1mV 30ms
Figure 4.12
105
CHAPTER 5
Conclusions and Future Work
106 5.1 SUMMARY AND CONCLUSIONS
The work presented in this study encompasses a multi-disciplinary approach to
advancing the field of epilepsy research through the use of electrophysiological
techniques and engineering design. A novel rodent unfolded hippocampus preparation
was developed for the study of intact hippocampal networks in the formation and
propagation of epileptiform activity. This technique revealed orthogonally propagating, synaptically dependent waves which spontaneously originated in the CA3 region and
subsequently invaded CA1. Data presented suggest that local lesions of CA3 can block
this epileptiform wave from propagating across CA3 and into post-lesional CA1. A 64-
channel recording array system was developed and constructed with parameters tuned to
fit the study of the unfolded rodent hippocampus preparation as well as hippocampus
slices. Its unique penetrating recording spike configuration allows active tissue in slices
and the pyramidal cell body layer in unfolded preparations to be targeted for study in two
dimensions. The recording array system operates in conjunction with optical data
acquisition systems to allow the simultaneous use of optical stimulation, active fluorescopy, and 64 channel extracellular population recording.
The objectives of this research were: (1) to determine the viability of the unfolded hippocampus preparation in vitro, (2) to analyze orthogonally propagating
Epileptiform activity in the unfolded rodent hippocampus in vitro, (3) to develop and build a MEMS based penetrating microelectrode array for two-dimensional recording of the unfolded hippocampus in vitro. The objectives and their realization were accomplished through several studies, which are summarized in the following sections.
Following the summaries, future directions of this work will be discussed.
107
5.2 OBJECTIVE I: TO DETERMINE THE VIABILITY OF THE
UNFOLDED HIPPOCAMPUS PREPARATION IN VITRO
In this objective, the viability of a new planar hippocampal preparation was investigated in order to facilitate the study of neural networks in vitro. Most commonly, the brain slice preparation has been used to study the rodent hippocampus in vitro, however the slicing method severs longitudinal interconnecting processes in the hippocampus such as recurrent excitation networks in CA3. Our hypothesis is that the unfolded hippocampus preparation will exhibit normal electrophysiology for the purpose of studying hippocampal networks in vitro. Orthodromically evoked responses showed a good correlation to responses seen in vivo, indicating preservation of neuronal connectivity and viable synapses. The lamellar organization of the unfolded hippocampus was confirmed after CA1 stimulation by a strong antidromic response along the transverse direction. This antidromic response was also found to propagate to a lesser extent in the longitudinal direction, a result which cannot be observed when using the traditional slice preparation. Furthermore, voltage sensitive dye optical imaging was performed on the alvear surface to study the extent of response propagation. This experiment generated the first optical imaging to be performed on an intact unfolded hippocampus. Propagation across two dimensions of the whole hippocampus was observed, which provides information about the whole hippocampus that a slice preparation could not offer. The long-term viability of the unfolded preparation depends on decreased temperature, and this temperature dependence was studied, resulting in the determination of an optimal in vitro bath temperature of 29C. Histological analysis using
108 Cresyl violet staining of tissue after use showed intact and normal pyramidal cell and
nuclei. With this new preparation, the entire hippocampus from young mice can now be
kept alive for an extended period of time during electrophysiological study. These data
support the hypothesis that the unfolded hippocampus preparation exhibits normal
electrophysiology for the purpose of studying hippocampal networks in vitro.
5.3 OBJECTIVE II: TO ANALYZE THE PROPAGATION OF
EPILEPTIFORM ACTIVITY IN THE UNFOLDED RODENT
HIPPOCAMPUS IN VITRO
In this study, the unfolded hippocampus preparation was utilized to investigate wave propagation generated in a hyperexcited state induced by superfusion of ACSF containing 4-AP. This study utilizes the unfolded unfolded mouse hippocampus preparation to reveal simultaneous orthogonal Epileptiform wave propagation and to test a method of arresting propagation.
The unfolded rodent hippocampus preparation was chosen due to its preservation of longitudinal neuronal processes which are thought to play an important role in epileptiform hyper-excitability. Our hypothesis was that longitudinal inter-neuronal processes which are preserved in the unfolded intact hippocampus facilitate longitudinally propagating seizure-like activity. 4-aminopyridine (4-AP), micro- electrodes, and voltage sensitive dye imaging were employed to investigate tissue excitability. In 50 µM 4-AP, stimulation of the stratum radiatum induced transverse activation of CA3 cells but also induced a longitudinal wave of activity propagating along the CA3 region at a speed of 0.09 m/s. Without stimulation, a wave originated at
109 the temporal CA3 and propagated in a temporal-septal direction and could be suppressed
with glutamatergic antagonists. Orthogonal propagation traveled longitudinally along the
CA3 pathway, secondarily invading the CA1 region at a velocity of 0.22 ± 0.024 m/s.
Moreover, a local lesion restricted to the CA3 region could arrest wave propagation.
These results reveal a complex orthogonal epileptiform wave propagation pattern in
the hippocampus that is generated by a combination of synaptic transmission and axonal
propagation in the CA3 recurrent network. Epileptiform propagation block via a
transverse selective CA3 lesion suggests a potential surgical technique for the treatment
of temporal lobe epilepsy. Our hypothesis that the longitudinal inter-neuronal processes
that are preserved in the unfolded hippocampus facilitate longitudinally propagating
seizure-like activity is supported.
5.4 OBJECTIVE III: DEVELOP AND BUILD A MEMS-BASED
PENETRATING MIRO-ELECTRODE ARRAY FOR TWO-
DIMENSINOAL RECORDING OF THE UNFOLDED
HIPPOCAMPUS IN VITRO
A novel penetrating microelectrode array system was designed and fabricated for the
purpose of recording activity in the hippocampus of mice. The array allows two
dimensional recording of 64 simultaneous sites of the hippocampus, in vitro. Our
hypothesis is that The MEMS array will allow higher SNR values and longer term,
more sensitive recording of neural activity compared to the RH414 voltage sensitive dye technique currently employed. Traditional surface electrode arrays, although easy to fabricate, do not penetrate to the viable tissue of hippocampus slices and thus
110 theoretically have a lower signal/noise ratio and lower selectivity than a penetrating array. Furthermore, the structure of the hippocampus slice preparation results in dead tissue in closest proximity to these traditional electrodes and the cell bodies of the CA1 region are obscured by this layer of dead cells, degrading activity-based optical imaging techniques as well. An array of 64 electrode posts each with a target height of 200 microns was fabricated in silicon and bonded to a transparent glass substrate. The electrode array was constructed with spike electrode having a very high aspect ratio of
10, resulting in the required penetration with minimal tissue insult. The impedance of the individual electrodes was measured to be approximately 1.5M Ohms ± 497kOhms. The signal to noise ratio was measured and found to be 19.4 ± 3 dB compared to 3.9 ± 0.8 dB
S/N for signals obtained with voltage sensitive dye RH414. A mouse unfolded hippocampus preparation was bathed in solution containing 50 micro-molar 4-Amino
Pyridine and a complex two dimensional wave of activity was recorded using the array.
Due to phototoxic effects and a poor signal-to-noise ratio, this spontaneous wave of activity was not previously visible using the voltage sensitive dye technique. Together, these results support our hypothesis that the penetrating electrode array is superior to that of voltage sensitive dye techniques for broad field two-dimensional neuronal activity recording.
5.5 FUTURE WORK
The work presented in this manuscript contributes to the field of neurological research through the validation of a new unfolded hippocampus preparation, the creation of a better understanding of epileptiform propagation in the hippocampus, and the
111 development of a novel electrode array for the study of the neural networks contained
within rodent hippocampus preparations.
The work presented in chapter two laid the foundation for the use of the unfolded
hippocampus preparation as a viable electrophysiological tool for the study of
hippocampal networks. The unfolded hippocampus preparation by nature preserves both
longitudinal and transverse inter-neuronal processes that otherwise would be severed in a
slice preparation, similar to in vivo preparations. Unlike in vivo preparations, however,
the entirety of the hippocampal network is easily accessible for electrophysiological recording and application of two-dimensional electrode arrays that target pyramidal cell populations in CA3 and CA1 simultaneously. Similar to slice preparation work, extracellular bath fluids may be easily modified in order to study the effects of various synaptic agonists, antagonists, and other neuro-modulation agents. One significant drawback of this preparation is that only young mice, up to 24 days old, can be used and
the perforant pathway must be cut in order unfold the dentate gyrus. However with an intact CA1–CA3 layer, it is now possible to generate novel data on the propagation of the activity in two dimensions. Further work should be undertaken to investigate specific local and network-wide effects of common anti-epileptic drugs. Use of the unfolded
hippocampus preparation could benefit other studies outside of epilepsy research as well,
such as the study of neural plasticity and the role of longitudinally oriented inter-neuronal
modulation structures such as the CA3–CA3 recurrent excitation connection field.
Intracellular recording techniques could be applied to investigate the extent of influence
longitudinally distant cells have on synaptic excitation and inhibition plasticity with
regards to action potential firing synchronicity. All of these studies could lead to
112 advances in our understanding of the role and function of the hippocampus, knowledge of which could be used to further treat related neurological diseases.
The work presented in chapter three analyzes longitudinally propagating epileptiform activity and its synaptic dependence and initiation in the CA3 region. We presented data supporting the hypothesis that longitudinal epileptiform wave propagation relies on interneuronal synaptic communication within CA3. While the traditional view of glia has been that they play mostly a maintenance role to neurons, recent work has begun to show that the role of glia in neuromodulation may be substantial (Fields, Stevens-Graham,
2002; Fields, 2006, Fields, 2010, Ortinski, 2010). Recent work has postulated that it may be possible for glial cells, typically involved in glutamatergic uptake, to release some of their stores in the presence of 4-AP, thereby providing the conditions for an unusual receptor-dependent (but not presynaptic) transmission dependent neuronal excitation
(Kang et al., 2005). Future work is warranted in this area to determine the exact dependency of longitudinal wave propagation on synaptic transmission vs. synaptic reception and its relation to glial neurotransmitter re-release. One promising result in chapter three related to the efficacy of local lesions in CA3 in blocking epileptiform propagation across the hippocampus. Noted was the fact that in some cases the isolated septal CA3 underwent its own, slower spontaneous population bursting after lesion, suggesting that the local network was sufficiently hyperexcitable to support spontaneous synchronous activity. In light of this, it would be beneficial to study the effect of multiple
CA3 lesions and thus the extent of local CA3 network populations on their spontaneous burst rates with the goal of complete suppression of epileptiform activity.
113 The work presented in chapter four laid out a micro-electrode array recording system for the study of rodent hippocampal networks. This array and amplifier system, while operational, could be improved upon in future revisions. While the recording spike electrodes are sufficiently stable to withstand tissue insertion, extraction, and lateral movements, they can bend or break when encountering common electrophysiological implements such as glass recording pipettes, tungsten stimulating electrodes, and common tissue-manipulation tools. Revisions with intent to widen the spike base for mechanical stability would improve array reusability and longevity in the field. One such method would be to simply increase the electrode diameter from 20 µm to 40 µm, with a larger pyramidal tip. Another option may be to create spikes with a pyramidal base using isotropic finishing etch after a partial DRIE etch.
Another improvement that could be made to the array system is to decrease the electrode impedance from 1.5 MΩ by electroplating platinum black to the tip surface. A reduced tip impedance would allow slower signals to be recorded because of the larger associated series capacitance of the electrode which makes a high pass filter on the amplifier input. Additionally, a lower tip impedance would allow stimulus current injection through the tip with less drive voltage required, reducing the likelihood of damage due to high local current densities.
Future work that should be undertaken using the array recording system includes a detailed study of 4-AP induced waves in the unfolded hippocampus, and the effect of lesion and low-frequency stimulus on the resulting propagation patterns. Because of the transparent substrate the array is built on, a direct and simultaneous comparison of
RH414 voltage sensitive dye imaging and array imaging is possible. Such an analysis
114 would reveal interesting correlations between the two mapping techniques. Finally, recent advances in optogenetics make it possible to stimulate hippocampal neurons with focused electromagnetic radiation instead of direct current (Zemelman et al., 2002; Boyden at al.,
2005; Aravanis et al., 2007; Llewellyn et al., 2010). Optical stimulation has the advantage of zero electrical artifact induced in the recording system and could make a helpful addition to the study of the effects of targeted stimulation on epileptiform activity propagation.
115
6 APPENDIX A
Supplemental Material Regarding the Unfolded Hippocampus Preparation and Longitudinal Epileptiform Propagation
116
6.1 Details of the Procedure of Preparing the Planar Unfolded Mouse Hippocampus
Following the same methodology as described for brain slice preparation (Teyler, 1980), the brain was quickly moved onto a moistened, chilled filter paper. Chilled oxygenated sucrose rich ACSF was dripped frequently onto the hippocampus to maintain moisture and allow some oxygenation. The cerebellum was removed and the brain was
hemisectioned by a razor. The hippocampus was freed from the septum and entorhinal
region using either dorsal or ventral approach (Teyler, 1980). Both rounded ends of the
hippocampus were trimmed in a line perpendicular to the longitudinal axis, resulting in a
slab approximately 3mm wide (Figure 6.1). Similar to Wu et al. 2002, the dentate gyrus
(DG) was unrolled by sliding a sharp glass electrode along the line of the fissure and
carefully unrolling the DG using a combination of glass and tungsten wire loops.
The unfolded intact hippocampus is a thicker preparation than the typical slice (500-
1000 µm), and showed a marked sensitivity to bath temperature in a submersion
chamber. To determine the optimal temperature for this preparation, orthodromic stimuli
(100us, 100uA, 5sec interval) in the apical dendritic CA1 region were carried out while
the bath temperature was slowly increased (25–32 °C, <1°C/m change), and the changes
in response latency and amplitude observed after 60s at a fixed temperature. The optimal
temperature in our submersion chamber was found to be 29°C based on the maximum
amplitude and minimum latency of the evoked potentials in all preparations (n=5; data
not shown). At temperatures above this level, the tissue showed progressively hypoxic
responses revealed in an eventual decrease in response amplitude and an increase in
latency, coupled with eventual spreading depression and neuronal death.
117
6.2 Details on the Methods Used in Voltage Sensitive Dye Recording
The tissue was illuminated via epi-illumination using an inverted Nikon Diaphot culture microscope fitted with a low noise DC-powered tungsten-halogen lamp (100W, 12 V).
Stimulation, shutter control, pump pause, and bias adjustments were done automatically
though a custom program running on a digital stimulator controller (PG4000, Neuro Data
Instrument). Fluorescence evoked was measured (interface filter: 535 ± 25 nm, dichroic
mirror 580 nm; barrier filter 590 nm) at each scanning point in the 16 x 16 photodiode
array (Hamamatsu C4675-102) and amplified with a custom amplifier and low pass filter
with a cutoff set to one KHz. The imaging objective (10X, 0.5 NA) resulted in a spatial
resolution of each element of 137 X 137 μm. Current signals from each photodiode were
converted to voltage signals, amplified, filtered, digitized, and then sampled at 500–1500
Hz. Final image processing was performed in order to better reveal the characteristics of
the network. Time averaging was used along with repetitive stimuli in order to improve
the signal to noise ratio, which was poorer in the unfolded preparation compared to slices
due to additional background fluorescence (Chang & Jackson 2003).
6.3 Discussion of the Unfolded Hippocampus Preparation:
Traditionally electrical neural activity from the hippocampus can be recorded
from in vivo preparations, in vitro slices, intact neonatal hippocampus preparations, and
cell cultures. The advantages of the brain slice preparation include the ability to control
the external medium, visualize the tissue layers under microscope, and control
pharmacological agents (Skrede & Westgard 1971, Aitken et al. 1995). Moreover, the
118 slice preparation maintains the “lamellar” organization (Andersen et al. 1971). However, the third dimension, (longitudinal axis), contains circuits with some lesser-known inhibitory and excitatory pathways that contain rhythmic generating circuits of their own
(Gloveli et al. 2005). Transverse in vitro slices make use of this organization, as they are
cut in a plane transverse to the long axis of the hippocampus, and by doing so preserve
the major excitatory axon tracts of the CA1 – CA3 (Teyler 1980). Longitudinal
hippocampal slices have also been studied in relation to oscillation, but because of the
loss of the major pathways their utility is limited. In either case, the complexity of the
entire neural circuit including substantial longitudinal projection is lost. (Amaral &
Witter, 1989, Brown & Zador, 1990). To overcome these limitations preparations that
involve the whole hippocampus in vitro (Khalilov et al. 1997, Wu et al. 2002,
Derchansky et. al, 2004) have been proposed. Khalilov demonstrated the feasibility of
maintaining in-vitro the intact hippocampal formation (IHF) in a specially designed
chamber, where the entire hippocampus is removed and studied as a whole. Even though
this preparation can successfully keep the entire mouse hippocampus alive for an
extended period of time, the IHF preparation has an age limitation (P0 – P10) to keep the
formation small enough for proper oxygen diffusion. Mice older than P10 have a
hippocampus thickness greater than 1mm, leading to hypoxia within the deeper tissue.
Wu et al. in 2002 cut off the Dentate Gyrus from the intact hippocampus, resulting in a
thinner tissue preparation useful in mice to P28. Derchansky et. al. in 2004 showed that
intact hippocampus and sections of the same could be studied up to P16 using a dual
perfusion chamber. In the present study, the intact hippocampus is unfolded along the
dentate gyrus to permit oxygenation of the deeper tissues, allowing the study of adult
119 mice to P25 although the range used for this particular study was P10–18. Despite the lesion of the perforant path necessary for unfolding, the intact unfolded hippocampus enables the study of combined longitudinal and transverse networks of the hippocampus while maintaining the advantages of in-vitro chamber study. Therefore, the planar
(intact) unfolded preparation is particularly suited to the study of epileptiform activity in the hippocampus of adult mice.
6.4 Synamptic Dependence of Evoked Orthogonal Propagation
In order to determine whether this orthogonal propagation generated by a stimulus in the
SR of CA1 relied upon synaptic communication, the evoked response was recorded in a lamella 1.5mm lateral to the transverse plane of stimulation in 50µM 4-AP solution with and without a blocker cocktail. With synaptic blockers, no significant field potential was recorded in the CA3 (Fig 6.2B). Without blockers, evoked activity could be observed spreading to the recording site with a delay of 22 ± 12ms post-stimulus (Fig 6.2A). This delay coupled with the absence of response in solution with synaptic blockers seen in 10 out of 10 preparations, indicates that the response is not generated by a direct antidromic activation of the CA3 axons by the stimulus. Taken together, these data show that 4-AP- induced response in locations away from the transverse plane of stimulation requires synaptic transmission.
120
6.5 Supplemental References:
Aitken PG, Breese GR, Dudek FF, Edwards F, Espanol MT, Larkman PM, Lipton P, Newman GC, Nowak TS, Panizzon KL, Raley-Susman KM, Reid KH, Rice ME, Sarvey JM, Schoepp DD, Segal M, Taylor CP, Teyler TJ, Voulalas PJ ( 1995) Preparative methods for brain slices: a discussion. J. Neurosci. Methods 59: 139-149.
Amaral DG, Witter MP (1989) The Three-Dimensional Organization of the Hippocampal Formation: A Review of Aanatomical Data. Neuroscience. 31: 571-591.
Andersen P, Bliss TV, Lomo T, Olsen LI, Skrede KK (1971) Lamellar Organization of Hippocampal Excitatory Pathways. Acta Physiol Scand. 76(1): 4A-5A.
Brown TH, Zador AM (1990) The Hippocampus. Shepherd GM (ed) The Synaptic Organization of the Brain, Oxford University Press, New York, pp. 346-388.
Chang PY, Jackson MB (2003) Interpretation and Optimization of Absorbance and Fluorescence Signals From Voltage-sensitive Dyes. J Membrane Biol. 196: 105-116.
Derchansky M, Sahar E Wennberg R A, Samoilova M, Jahromi SS, Abdelmalik, PA, Zhang L, Carlen PL (2004) Model of frequent and spontaneous seizures in the intact mouse hippocampus. Hippocampus. 14 (8): 935-947.
Gloveli T, Dugladze T, Rotstein HG, Traub, RD, Monyer H, Heinemann U, Whittington MA, Kopell NJ (2005) Orthogonal arrangement of rhythm-generating microcircuits in the hippocampus. Proc Natl Acad Sci U S A . 102(37): 13295-13300.
Khalilov I, Esclapez M, Medina I, Aggoun D, Lamsa K, Leinekugel X, Khaipov R, and Ben-Ari Y (1997) A Novel In Vitro Preparation: the Intact Hippocampal Formation. Neuron. 19: 743 –749.
Skrede KK, Westgaard RH (1971) The transverse hippocampal slice: a well-defined cortical structure maintained in vitro. Brain Res. 35: 589-593.
Teyler TJ (1980) Brain Slice Preparation: Hippocampus. Brain Res Bull. 5: 391-403.
Wu C, Shen H, Luk WP, Zhang L (2002) A Fundamental Oscillatory State of Isolated Rodent Hippocampus. J Physiol. 540.2: 509-527.
121
6.6 Supplemental Figure Captions:
Figure 6.1: Procedure for preparing the unfolded planar hippocampus. The ends are trimmed (1,2) next the perforant path and vessels are cut (3,4) finally the DG is folded back (5,6) to form a planar preparation.
Figure. 6.2: Wave delay from axonal field potential seen in 4-AP + blocker cocktail. Top trace: Response field potential in 4-AP with no synaptic blockers to a stimulus in the CA1 which is 1.5mm lateral to the lamella of the recording electrode. The stimulus elicits a strong response at the recording electrode 1.5mm lateral to the plane of stimulation. Bottom trace: Null field potential response in the same location in the presence of synaptic blockers. These data show that the wave of activity in 4-AP is not merely due to enhanced axonal response to stimulus, and that the response lasts longer than a typical orthodromic field potential.
Figure. 6.3: A) Exemplary time-wise activation contours shown in B and C out of five preparations analyzed. B) Levels of activation as a function of time along a transverse plane (B) in the hippocampus from a stimulus in CA1. This cross section is in the plane of stimulation. Stimulus occurs at contour 0ms, distance 0mm. At 4ms, the CA1 stimulus location is activated while CA3 remains near resting potential. At 8ms, CA3 is becoming active and at 12ms the CA3 response reaches its peak. This figure shows transverse antidromic propagation of the stimulus response into CA3 (propagation a to b). Contour labels are in ms. C): Levels of activation as a function of time along a transverse plane (C) in the hippocampus from a stimulus in CA1. In this cross section, activation in CA3 precedes same in CA1 in a cross-sectional lamella lateral from the stimulus. At contour time 4ms, CA3 begins to respond, and during contours 6–12ms, CA3 and CA1 activate, with CA1 following CA3. This figure shows the septal CA1 being activated after orthogonal propagation of the stimulus response along CA3 (propagation B to C). Contour labels are in ms.
122 6.7 Supplemental Figures
1 2
3 4
5 6
Fig. 6.1
Evoked 4-AP response
CA1 .ST Rec CA3 . DG Evoked response, 4-AP + blockers 1.5mm
500uV 15ms
Fig. 6.2
123
A C B
CA1 Sti .
CA3
C B CA1 CA3 CA1 CA3 Stimulus 14
14
8 8
4 4 0 0 a b c b
Fig. 6.3
124
7 APPENDIX B
Schematics Diagrams of the 64-channel Amplifier System
125
Figure 7.1: Main power regulation
126
Figure 7.2: Array ZIF Input Socket Connections
127
Figure 7.3: Mainboard I/O
128
Figure 7.4: Amplifier Sockets 1
129
Figure 7.5: Amplifier Sockets 2
130
Figure 7.6: Amplifier Wiring and Differential Control Socket
131
Figure 7.7: Amplifier Artifact Blanking Adjustment
132
Figure 7.8: Water Detect and Safety Shutdown
133
Figure 7.9: Stimulus Artifact Blanking Timing Board
134
Figure 7.10: Fuse Daughter Board
Figure 7.11: Amplifier Board (x16)
135
Figure 7.12: Mainboard Layout
Figure 7.13: Amplifier Daughter Board Layout
136
Figure 7.14 : Front of unshielded Main Board with Amplifier Daughter Cards Inserted
137
8 APPENDIX C
Frequency Response of the 64-channel Amplifier System
138 8.1 Frequency Response Measurement
In order to verify the frequency response of the 64-chanel amplifier system, a
frequency sweep generated with a function generator was applied to the electrode array in
ACSF solution. A silver-silver chloride wire was used as a stimulation return electrode,
shaped in a ring around the electrode array. A second silver-silver chloride wire was
immersed directly above the array approximately 0.5mm deep in solution. This electrode
served as the stimulus signal source. A low-amplitude sine wave (5mV) was swept in a
series of three ranges: 0.1 Hz – 10Hz, 1Hz – 150 Hz, and 60Hz – 4 kHz. An example test
series is shown in Figures 8.1 – 8.3. The channel bandwidth achieved the desired result,
nearly 0.1 Hz to 3.5 kHz. On the whole, the bandwidth of the final constructed amplifier set was 0.5 to 3.5kHz ± 0.8%. In the future, Amplifier modifications could be implemented to increase the high-end frequency response if it is determined that this is desirable. To do so, the Low-pass second stage Sallen-key filter capacitors should be targeted for modification.
139 8.2 Figures
Figure 8.1: Frequency sweep of micro electrode recording system 0.1 Hz to 10 Hz.
Figure 8.2: Frequency sweep of micro electrode recording system 1 Hz to 150 Hz.
Figure 8.3: Frequency sweep of micro electrode recording system 60 Hz to 4kHz Hz.
140
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