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Effects of triethyllead on the neurite structure of embryonic rat cells

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Effects of triethyllead on the neurite structure of embryonic rat cells
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Wardle, Karen
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English
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viii, 137 leaves : illustrations ; 29 cm

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Lead -- Toxicology ( lcsh )
Neurotoxic agents ( lcsh )
Lead -- Toxicology ( fast )
Neurotoxic agents ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 134-137).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Biology.
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Department of Integrative Biology
Statement of Responsibility:
by Karen Wardle.

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|University of Colorado Denver
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Auraria Library
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Full Text
EFFECTS OF TRIETHYLLEAD ON THE NEURITE
STRUCTURE OF EMBRYONIC RAT CELLS
by
Karen Wardle
B.A., University of Colorado
at Denver, 1990
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1994


This thesis for the Master of Arts
degree by
Karen Wardle
has been approved for the
Graduate School
By
Gerald Audesirk
Date /y/y


Wardle, Karen (M.A., Biology)
Effects of Triethyllead on the Neurite Structure of
Embryonic Rat Cells
Thesis directed by Associate Professor Teresa Audesirk
ABSTRACT
Triethyllead (TEL) is the toxic, lipophilic metabolite of the nontoxic
compound tetraethyllead of which the major source is leaded gasoline. TELs
presence inside the brain causes a number of detrimental effects including
alterations in neurite growth; however, the mechanism by which TEL causes these
alterations is unclear. My thesis looked into some of the mechanisms by which
TEL might alter neurite growth of E18 rat hippocampal and cortex neurons. I
knew from previous experiments that 5uM TEL increased intracellular calcium
(Ca+ +) levels; therefore, I began my research by looking to see if the increase in
intracellular Ca++ could be related to the alterations in neurite growth. I used


patch clamping techniques to find out if TEL was affecting voltage-gated cation
channels, and Ca+ + imaging techniques to see if the intracellular increase in
Ca++ seen with uM concentration of TEL could be chelated by using the Ca+ +
chelator BAPTA-AM. The latter experiments were done in conjunction with
culture experiments to see if BAPTA-AM could decrease the alterations in neurite
growth seen after TEL exposure. I also did western blot techniques to see if TEL
was affecting the neurite constituents tubulin and MAP2. The results of my
experiments show that the Ca+ + increase seen after exposure to TEL is not
related to the alterations in neurite growth; however, preliminary western blot
experiments show that TEL may decrease phosphorylation of both tubulin and
MAP2.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
Sign
Teresa Audesirk
IV


CONTENTS
Chapter
1. Introduction....................1
1.1 Toxic Effects of TEL................2
1.2 Background.......................5
1.2.1 Neurons.........................5
1.2.2 Hippocampal Neurons................11
1.3 Research Goals.................... 12
1.3.1 Background Research................12
1.3.2 My Research......................15
1.4 Thesis Organization................16
2. Patch Clamp Experiments...............17
2.1 Introduction...................17
2.1.1 Membrane Structure................17
2.1.2 Membrane Potential................21
2.1.3 Action Potential..............24
2.1.4 Properties of Ion Channels........25
v


2.1.5 Patch Clamping..................36
2.2 Methods.........................40
2.2.1 Dissection....................40
2.2.2 Culturing.....................42
2.2.3 Patch Clamping..................43
2.3 Results.....................57
2.3.1 Ca++ Channels...................57
2.3.2 Na+Channels.....................59
2.3.3 K+ Channels.....................61
2.4 Discussion......................63
3. Ca++ Imaging Experiments..............65
3.1 Introduction....................65
3.1.1 Intracellular Ca++..............66
3.1.2 Intracellular Ca++ and TEL.........72
3.1.3 Ca++ Imaging....................75
3.2 Methods.........................77
3.2.1 Dissection....................77
3.2.2 Perfusion Experiments...........77
3.2.3 BAPTA-AM Experiments................82
vi


3.3 Results
84
3.3.1 TEL Perfusion Experiments...........84
3.3.2 BAPTA-AM Experiments...............86
3.4 Discussion.....................88
4. Western Blot Experiments...........92
4.1 Introduction...................92
4.1.1 Neurite Growth..................92
4.1.2 Growth Cone.....................93
4.1.3 Neurite Composition.............95
4.1.4 TEL and Neurite Growth..............102
4.1.5 Gel Electrophoresis............103
4.2 Methods.........................110
4.2.1 Dissection......;............110
4.2.2 Culturing.....................Ill
4.2.3 Toxicants......................112
4.2.4 Harvesting.................. ..114
4.2.5 SDS-PAGE.........................115
4.2.6 Western Blotting...............116
4.3 Results.......................119
vii


4.3.1 Tubulin........................120
4.3.2 MAP2..........................123
4.4 Discussion......................125
5. Conclusions......................130
5.1 [Ca++]in Increases.................130
5.2 Neurite Growth.....................132
References...........................134
Vlll


1. Introduction
Our use of environmental chemicals has more often than not progressed more
rapidly than our knowledge of their involvement in human health and safety.
Lead is very prevalent in the environment, and can cause neurological
problems for both men and animals. Children between the ages of 12 and 48
months of age are especially vulnerable to the neurotoxic effects of lead
(Niklowitz 1987). The reported neurotoxic effects of both inorganic and organic
lead are many, including neuroclinical changes, neuropathological changes and
biochemical changes.
The major source of organic lead is from leaded gasoline. Tetraethyllead,
('IT'LL), has been added as an antiknock agent to gasoline since 1923. TTEL
particles emitted into the atmosphere are greatly increased and concentrated in
enclosed areas such as tunnels and parking garages. A phase-out of leaded
gasoline has been mandated by the Environmental Protection Agency, (EPA) and
in addition, the advent of catalytic converters in 1975 dropped the proportion of
leaded gasoline immensely. This decline however, has yet to spread worldwide.
1


Many countries in Europe still rely heavily on leaded gasoline.
1.1 Toxic Effects of TEL
TTEL is a very lipophilic and volatile liquid at room temperature. When
mixed with gasoline, exposures to TTEL can occur through inhalation and/or skin
contact. Since TTEL is very lipophilic, it easily penetrates the lung tissue and
skin, and readily crosses the blood brain barrier. TTEL itself is a nontoxic
compound, however due to its instability, once it enters the body, it degrades to
triethyllead, (TEL). The TTEL TEL degradation takes place mainly in the
smooth endoplasmic reticulum of the liver cells (Konat, 1984; Grandjean 1987).
The conversion is an oxidative dealkylation catalyzed by cytochrome P-450
dependent monooxygenases (Grandjean, 1987). TEL is fairly stable in biological
tissues, however further degradation to diethyllead and inorganic lead may take
place. Once TEL is formed, it is distributed via the vascular system to its target
sites, which include the liver, kidneys and brain; the latter being the most
2


vulnerable (Cremer, 1959; Bolanowska, 1968; Tilson and Sparber, 1990).
Cremer (1959) suggests that it may not be the high concentration of TEL in the
brain that is responsible for the toxic effects, but the sensitivity of the brain tissue
to the toxicant.
The neurotoxic effects of TEL, both clinical and morphological, have been
studied in mammals for several decades. The most frequently reported clinical
manifestations appear in three phases: 1) initial lethargy, followed by 2) tremors,
hypermotility, hyperexcitability, aggression, and finally, 3) convulsions,
incoordination, ataxia, paralysis and death (Grandjean, 1987).
Clinical manifestations following sublethal doses of TEL can resemble deficits
induced by lesions in the limbic system. The limbic system is involved with the
regulation of behavior, motivation, learning and memory (Verity, 1990). Some of
the clinical effects reported following sublethal doses of TEL are hypermotility,
hyperexcitability, and aggression (Verity, 1990; Tilson and Sparber, 1990;
Grandjean, 1987; Toxicology Profile for Lead, 1990). These effects are
exacerbated in the developing organism and susceptibility decreases with age.
The effects described above have been observed in humans exposed to TTEL
in high concentrations via occupational accidents, gasoline sniffing, and in
laboratory animals under experimental conditions. There have been many studies
3


exposing rats to TEL, and although there seems to be a large amount of data on
the toxic effects of TEL on rats, the mechanisms by which these toxic effects
occur are poorly understood.
Following sublethal TEL poisoning, morphological changes within the brain
include interference with the normal proton gradient across the mitochondrial
membrane, hypomyelination, increased neuronal excitation, alterations in neurite
growth, transmitter release and ion concentrations. (Verity, 1990; Tilson and
Sparber, 1987; Grandjean, 1987; Konat, 1984).
My research focused on the following morphological changes in neurons: 1)
the toxic effects of TEL cause alterations in neurite growth and 2) one
mechanism by which these alterations in neurite growth occurs following TEL
exposure may be due to changes in ion concentrations, especially calcium, or 3)
another mechanism by which TEL exposure may be altering neurite growth is by
i
decreasing phosphorylation of MAP2.
My research was conducted on embryonic rat brain cells at 18 days of
gestation (El 8).
4


1.2 Background
1.2.1 Neurons
The cells that make up the central nervous system can be divided into two
distinct groups: neurons, and cells surrounding and filling the spaces between the
neurons. The function of neurons is to 1) sense information about its surrounding
environment, 2) carry the information to the brain to be processed, and 3)
generate a response to the processed information (Levitan and Kaczmarek, 1991).
The cells that surround the neurons are called neuroglia or glial cells, which
themselves can be subdivided into astrocytes and oligodendrocytes. Astrocytes are
star like in appearance, with radiating arms extending from the cell body. They
provide structural support for the central nervous system (Hall, 1992).
Oligodendrocytes also have radiating arms extending from a cell body, however
these arms are shorter and more branched than those of the astrocytes.
Ogliodendrocytes make up the myelin sheath which is an insulating sheath that
5


forms around central nervous system axons allowing for faster conduction of cell
signals. Other roles for glial cells include 1) providing structural support for
neurons, 2) acting like electrical insulators between neurons, 3) providing
nourishment for neurons, 4) participating in the metabolism of neurotransmitters,
5) buffering the neurons from ions in the extracellular matrix, and 6) participating
in memory storage (Levitan and Kaczmarek, 1991).
1.2.1.1 Neuronal Structure
Neurons are composed of cellular constituents that are similar to any other cell
in the body. The neuron is surrounded by a lipid bilayer called the plasma
membrane. The plasma membrane acts to separate the intracellular constituents
from the extracellular matrix, and also acts as an electrical insulator keeping the
intracellularly charged ions separate from those in the extracellular matrix. It also
contains embedded in its lipid bilayer proteins that control cell signaling processes
by allowing the movement of ions across the membrane.
6


. Within the neuron are organelles that are common to all cells: mitochondria,
nucleus, endoplasmic reticulum, golgi complex, lysosomes, etc. Neurons carry
out cell maintenance functions common to all cells in addition to their specialized
functions such as integration of incoming neuronal signals.
What makes a neuron unique is its formation of neurites: dendrites and axons,
which carry out the signaling function of the neuron. The structure of the neuron
is shown in Fig. 1.1.
Figure 1.1
The structure of a neuron. Electrical signals are
received by the dendrites, travel through the cell body
down the axon to the synaptic terminals. The signal is
then transmitted to another neuron by actions in the
synaptic terminal.
7


Dendrites are processes that extend from the cell body and function in the
reception of neuronal signals, conversion of the incoming chemical signal into an
electrical signal, and conduction of the electrical signal to the cell body. The
dendrites are shorter, thicker and more highly branched than the axons, hence the
name dendritic tree. The extensive branching of the dendritic tree can cover an
area within the spinal cord of up to 2-3mm in diameter (Kandel et al. 1991). The
dendrite cytoskeleton also differs from the axon cytoskeleton. Extending from the
main shaft of the dendrite are specialized regions, fingerlike extensions called
dendritic spines, which increase dendritic surface area for reception of incoming
signals from other neurons (Levitan and Kaczmarek, 1991 and Kandel et al.
1991).
In contrast to the many dendrites a neuron possesses, it only possesses one
axon. The function of the axon is to transport the neuronal signal to its target,
which could be another neuron, a muscle or a gland. An axon originates from the
cell body at a point called the axon hillock or spike initiation zone. All the
incoming signals from the dendrites are integrated in the cell body, and any
outgoing signals called action potentials are initiated in the spike initiation zone
and propagated down the axon. Signal conducting axons that must travel great
lengths such as from the leg to the brain are ensheathed in myelin so they can
8


carry the electrical signal the distance in a much shorter time. In the central
nervous system, the myelin is composed of ogliodendrocyte cells; in the peripheral
nervous system, the myelin is composed of schwann cells. Along the length of
the axon, there are gaps called nodes of Ranvier in the myelin, which allow for
the electrical signal to be boosted by action potentials.
At the end of each axon is a synaptic terminal, which takes the electrical
signal coming down the axon, and converts it to a chemical signal by releasing a
neurotransmitter. A neurotransmitter is a chemical messenger that binds to
receptors and thus affects ion channels in the plasma membrane (Hall, 1992).
Neurotransmitters are released into a space between the presynaptic and
postsynaptic neurons called the synaptic cleft (Fig. 1.2).
Axons and dendrites are very sensitive areas of the cell; they are the first
components to disintegrate in a dying cell. Neurons and their axons and dendrites
in the area of the brain involving learning and memory, called the hippocampus
are some of the most sensitive neurons in the brain.
9


CLEFT
IC
Figure 1.2
The synaptic cleft. Electrical signals coming down the
presynaptic neuron are converted to chemical signals in
the synaptic cleft. The electrical signal triggers the'
release of neurotransmitters into the synaptic cleft,
which transmits the signal to the receptors on the
postsynaptic cell.
10


1.2.2 Hippocampal Neurons
Hippocampal neurons from El8 rat embryos were used in my research. The
hippocampus is located in an area of the brain called the limbic lobe. This is an
horseshoe-shaped rim of cortex that encompasses the region between the
diencephalon (thalamus and hypothalamus) and the cerebral hemispheres, and
includes the hippocampus, the olfactory tract and the cingulate gyrus (Nolte,
1993). The limbic lobe has been implicated in drive-related and emotional
behavior; the hippocampus in particular has been implicated in learning and
memory (Nolte, 1993).
The hippocampus is a C-shaped sheet of cortex containing three different
zones: the dentate gyrus, the hippocampus proper and the subiculum.
Histologically, the hippocampus consists of three cell layers: a deep polymorphic
layer, an intermediate pyramidal cell layer and a superficial molecular layer
(Nolte, 1993).
Hippocampal neurons are very sensitive to toxicants which may be one of the
reasons that learning and memory functions are clinical symptoms of many
11


toxicant exposures.
El 8 rat cortex cells were used in the preliminary SDS-PAGE studies because
they are more numerous than hippocampal cells.
1.3 Research Goals
1.3.1 Background Research
My research into the mechanisms by which TEL affects hippocampal neurons
was a continuation of previous experiments testing TELs effects on E18 rat
hippocampal neurons. These previous experiments included 1) 2 day culture
experiments in which E18 rat hippocampal cells were cultured in media including
TEL in concentrations ranging from O.lnM to 0.5uM, and 2) Fura 2 calcium
imaging experiments in which cultured El 8 rat hippocampal cells were perfused
with TEL in concentrations ranging from O.lnM to 5uM. The calcium imaging
dye, Fura 2, was used to measure any change in intracellular Ca+ + occurring
12


due to the TEL perfusion.
1.3.1.1 Culture Experiments
In previous experiments, El8 rat hippocampal cells were cultured for 2 days
in varying concentrations of TEL ranging from O.lnM to 0.5uM. After 2 days,
various parameters were measured including the survival of the neurons, the
percent of neurons initiating neurites, the number of neurites on each neuron, the
length of the neurites, and the number and length of branches on each neurite.
Results from the culture experiments show that TEL at > luM concentrations
decreased all of the parameters tested. The parameter showing a significant
decrease due to TEL exposure at nM concentrations was the number of branches
on both the axons and dendrites, the latter in particular.
13


1.3.1.2 Ca++ Imaging Experiments
Previous experiments exposing El 8 rat hippocampal cells to TEL show that
TEL exposure caused increases in the Ca+ + levels inside the neurons. The
Ca+ + imaging dye, Fura-2 was introduced into the cells and control levels of
calcium were recorded from the cells. After control Ca++ levels were recorded,
the cells were perfused with media containing TEL at concentrations ranging from
0. InM to 0.5uM for a period of 3 hrs and Ca+ + levels were recorded at 5 min
intervals.
Results from the previous Fura-2 experiments show that the cells showed a
large increase in Ca+ + levels above the control levels after 3 hrs of perfusion
with 5uM TEL.
14


1.3.2 My Research
The above experiments became the basis for my TEL experiments. I wanted
to find the mechanism by which TEL increased Ca+ + levels inside the cell, and
whether the Ca++ increase resulted in the changes in parameters, the decrease in
branching in particular, seen after culturing the cells in TEL for 2 days.
I used a combination of research techniques to answer the above questions: 1)
I used a technique called patch clamping to determine if TEL was altering voltage-
gated ion channels, Ca+ + channels in particular, which could result in the
increase in Ca+ + inside the cell after exposure to TEL, 2) I used the Ca+ +
imaging dye, Fura-2 to repeat experiments to see if TEL increased intracellular
Ca++, and if the suspected increase could be chelated using a Ca++ chelator.
These experiments were done in conjunction with culture experiments to determine
if the increase in Ca+ + was causing the decrease in branching seen after TEL
exposure, and finally 3) I used the biochemical technique called Western blotting
to try to further elucidate the cause of the decrease in branching in cells after TEL
exposure.
15


1.4 Thesis Organization
My thesis is divided into 3 sections detailing the above experiments. Each
section begins with a summary of my experimental goal, followed by the methods
by which the goal was carried out, the results from the experiments and finally a
brief discussion. Following the 3 sections, I conclude with a summary of my
research with TEL and its effects on E18 rat cells, and a direction for future
experiments.
16


2. Patch Clamp Experiments
2.1 Introduction
My experimental goal in doing patch clamp experiments was to find out if
voltage gated Ca+ + channels were altered after exposure to TEL. I used the
patch clamp technique to measure Ca++, Na+ and K+ currents before and after
TEL exposure.
2.1.1 Membrane Structure
The plasma membrane creates an impermeable lipid barrier around all cells.
17


This lipid membrane keeps the intracellular constituents and their electrical
charges separate from the extracellular matrix. The composition of the
extracellular matrix surrounding neurons is called interstitial fluid and is composed
of inorganic compounds and water. The inside of the cell consists of
compartmentalized organelles such as the mitochondria, nucleus, etc. The inside
of a neuronal cell also contains more electrically negative ions than the
surrounding interstitial fluid, which creates a membrane potential (electrical
difference) that is dependent on the lipid membrane and embedded proteins that
act as channels and pumps. The plasma membrane surrounding a neuron
functions as an insulating lipid layer between the conducting solutions on the
inside and the outside of the membrane, which causes the thin insulation lipid
layer to function as an electrical capacitor (Hille, 1992). Table 2.1 is a very
simple description of the ionic composition of a mammalian skeletal muscle cell,
the surrounding interstitial fluid, and whether or not the plasma membrane is
permeable to each particular ion.
18


The plasma membrane does not consist entirely of passive lipids separating the
cellular constituents and their electrical charges. There are transport proteins and
channels inserted in the plasma membrane that allow different ions to cross the
plasma membrane. In fact, the membrane potential is the result of the proteins in
the plasma membrane being differentially permeable to different ions, most
commonly: Na+, K+, Ca++ and C1-.
The ability of charged ions to cross the plasma membrane is attributed to two
types of special proteins that traverse the membrane: transport proteins and
channels.
The function of cellular transport proteins is to use metabolic energy to
exchange ions such as Na+ and K+ across the plasma membrane; this transport
of ions results in a ion concentration gradient across the membrane, which then
allows the neuron to use this stored energy to initiate electrical signals (Hall,
1992). Transport can be passive and consist of small molecules diffusing through
membrane channels, or large molecules requiring carrier proteins. Transport can
also be active, requiring energy. Active transport is often carried out through the
action of specialized transport proteins in the membrane called pumps. One
specialized pump is the Na+\K+ pump. At rest when the neuron is not
conducting signals, Na+ concentrations are about 10 times higher in the
19


extracellular matrix, and the cell is almost impermeable to Na+. When the
neuron is generating electrical signals, the membrane becomes permeable to Na+,
and Na+ enters the cell; at approximately the same time, K+ is leaving the inside
of the cell. After returning to its resting potential, the neuron needs to remove
any extra Na+ that comes in during the signaling process in addition to any
incoming Na+ entering through leakage. The cell uses the splitting of ATP for
the energy required to operate the Na+\K+ pump, which continuously moves
Na+ ions across the cell membrane and back into the extracellular matrix coupled
with moving K+ back into the neuron.
Channels are integral proteins in the plasma membrane that behave like
tunnels or pores allowing charged and/or polar substances through the membrane,
often at rates of 10E6 ions per second. Two important properties of channels
include: 1) they are selective in the ions they let through. 2) they behave as
molecular gates. Channels that allow Na+ to pass through, do not allow K+, Cl-
etc. to pass through. This selection property is due to differences in charge and
size of the ions.
Many channels behave as though they have a molecular gate. Ions do not just
passively move through the pores at a constant rate, but only when the channel
gate is open. Different channel gates are controlled by different forces: some
20


depend on the membrane potential or electric field to open and close them, others
depend on stretching of the membrane or mechanical stimulation, others depend
on neurotransmitter binding or chemical stimulation, and still others depend on
thermal stimulation or temperature to open and close. The opening and closing of
gates allowing ions in only when properly stimulated allows the cell to closely
govern the movement of ions across the plasma membrane, which in turn closely
governs important cell signaling functions such as action potentials.
The neuron closely governs the movement of ions across the plasma
membrane by two forces: 1) the concentration difference between the
outside/inside of the cell maintained by the pumps and 2) the electrical potential
difference between the outside/inside of the cell. Together these two forces make
up the electrochemical gradient.
2.1.2 Membrane Potential
When excitable cells are at rest (not conducting electrical signals) the value of
21


the membrane potential is called the resting potential. For most neurons, this
value is in the range of -40 to -90mV. The negative sign is expressed as a
measure of the potential inside the cell with respect to the outside or extracellular
fluid. The inside of the cell contains more anions than the outside of the cell,
which causes the inside of the cell to be more negative than the outside of the cell
at the resting potential. If the membrane potential becomes more positive than the
resting potential due to positive ions entering the cell, the cell is considered to be
depolarized, and on the other hand if the membrane potential is more negative
than the resting potential due to positive ions being transported out or negative
ions being transported in, the cell is considered hyperpolarized.
The rate at which ions will move across a plasma membrane is determined by
three factors:
1) The concentration gradient between the inside and outside of the membrane.
2) The voltage difference or membrane potential of the membrane.
3) The permeability of the membrane to the ion, ie. what gates are open.
As the ions move across the membrane in response to their chemical forces,
they give rise to the electrical difference or membrane potential. For example,
the membrane is permeable to K+, and some K+ gates are open when the cell is
at rest. K+ will move out of the cell along its chemical gradient, but since the
22


inside of the cell is more negative than the outside, this will pull K+ back into the
cell. When the system is at an equilibrium, the chemical gradient pulling K+ out
will be balanced by the electrical forces pulling K+ in. The potential at which
there is no net flux of K+ across the membrane is called the equilibrium potential
for K+. The equilibrium potentials for any ion can be calculated by the Nemst
equation: Ek = RT/F In (X+]out/[K+]in. (1.1)
The Nemst equation relates both the chemical and electrical forces acting on an
individual ion. The equilibrium potential for K+ in skeletal muscle cells is
approximately -98mV. The addition of Na+ (and other) channels to the
membrane also contributes to the resting potential of neurons, therefore making
the resting potential of the cell close but not equal to the K+ equilibrium
potential. The equilibrium potential for Na+ in skeletal muscle is +67mV, which
is approximately 165mV away from the resting potential of the cell. The
electrical difference, in addition to the Na+ concentration difference, results in a
powerful driving force for Na+ to enter the cell when the Na+ gates open.
However, at rest most of the Na+ gates are closed, which makes the membrane
more impermeable to Na+. On the other hand, the membrane is permeable to
K+, which makes the resting potential slightly more positive than the K+
equilibrium potential.
23


2.1.3 Action Potential
Collectively, the electrochemical activities of the individual ions flowing
across the plasma membrane give rise to the electrical signaling in the neuron
eventually leading to macroscopic membrane currents called action potentials.
Action potentials provide the means for the electrical signals of neurons to
travel the great distances from the leg to the brain. Neurons can influence the
actions of each other by excitation, which leads to the production of impulses in
other neurons, or by inhibition, which leads to the prevention of impulses in other
neurons. An action potential is a "brief, positive all or nothing, large electrical
signal propagating along the axon without loss of amplitude" (Matthews, 1991).
All incoming signals from the dendrites are integrated in the cell body, action
potentials are then initiated in the spike initiation zone and propagated down the
axon. Neurons rely on the membrane potential to open and close their gates. The
ions involved in excitation and inhibition of neurons are almost exclusively Na+,
K+, Ca++ and C1-.
24


2.1.4 Properties of Ion Channels
As mentioned above, the ions whose collective movements create the firing of
an action potential are Na+, K+, Ca++ and C1-. The properties of Na+, K+
and Ca++ channels are explained below. Cl- channel properties are beyond the
scope of this research.
2.1.4.1 Na+ andK+ Channels
In the early 1950s, Alan Hodgkin and Andrew Huxley developed an
experimental procedure called the voltage clamp to study ion channels. The
function of the voltage clamp procedure is to let voltage gated ion channels open
and close in response to experimentally imposed changes in the membrane
potential. The voltage clamp setup consists of two intracellular electrodes; one for
recording the membrane potential and one for passing current into the cell. The
electrodes are attached to a inverting amplifier. The inputs to the amplifier
consist of a potential equal to the membrane potential, and a command potential
25


whose value is chosen by the experimenter. The output of the amplifier is the
difference between the two inputs multiplied by the amplification factor and
multiplied again by -1. After clamping the membrane potential by applying a
voltage to the membrane, the current flowing through the membrane can be
measured by measuring the current that must be generated by the voltage clamp to
keep the membrane potential from changing (Kandel et al. 1991).
By using the voltage clamp technique, Hodgkin and Huxley found that the
current generated by action potentials was carried by two major components: Na+
current and K+ current. The action potential is generated by a transient increase
in Na+ entering the cell down its concentration gradient. This reverses the
charge difference across the membrane, bringing the membrane potential to
approximately the Na+ equilibrium potential. This is termed the activation phase
of the action potential. The current hypothesis is that Na+ channels have two
molecular gates (Fig. 2.1). The ball and chain schematic is the activation gate,
which is closed during the resting potential, and opens to let Na+ in when the
membrane depolarizes. The second gate is open during the resting potential, and
closes when the membrane is depolarized. The inactivation gate closes more
slowly than the activation gate. This delay lets Na+ enter during the depolarizing
phase, but helps to limit the duration of the action potential.
26


1S3SBS8S*
INACTIVATION
GATE "
EXTRACELLULAR'
^s^?r'SJDE
CYTOPLASMIC -
SIDE
ACTIVATION GATE
X ,
Figure 2.1
Adapted from Kandel-i- The Na+ channel has two molecular
gates: the activation gate, which is closed at resting
potentials and opens when the membrane depolarize^, and
the inactivation gate, which is open at re
potentials and closes with time after the
depolarizes.
sting
membraf$5J
27


Another factor that limits the duration of the action potential is the opening of
K+ channels. K+ channels open with some delay after the membrane is
depolarized; this delay allows time for Na+ to flow into the cell, but then limits
the duration of the action potential. The function of the K+ channel is to
repolarize the membrane following an action potential, and since it governs the
repolarization phase, it also limits the rate at which subsequent action potentials
are fired.
The depolarizing current opens both the Na+ and K+ gates,
and the two currents overlap in time. In order to study the individual currents,
Hodgkin and Huxley separated the two currents using ion substitution among other
methods. For instance, they substituted choline which is impermeant to the
plasma membrane for Na+ in the extracellular fluid. This eliminated the Na+
current, and allowed them to exclusively determine the K+ current (Kandel et al.
1991) . The discovery of pharmacological agents that block the ion channels has
made the current separation much easier.
Tetrodotoxin (TTX) is a paralytic poison found in puffer fish. TTX blocks
Na+ channels, consequently TTX blocks action potential conduction (Hille,
1992) . It does not have any effect on K+ channels. Tetraethylammonium (TEA)
on the other hand, blocks some K+ channels, but leaves Na+ channels
28


untouched. Through the use of these drugs, the currents flowing through the
plasma membrane can be separated, recorded and studied.
The currents recorded during a voltage clamp experiment are demonstrated in
Fig. 2.2. Figure 2.2a is a record of a voltage clamp depolarizing step to OmV,
showing an immediate membrane capacitance current (large spike) followed by
currents caused by both Na+ and K+ channels opening. Figure 2.2b is a record
of a depolarizing step to OmV, only this time in the presence of TTX, and again
in the presence of TEA. The inward current caused by Na+ channels opening is
separated from the outward current caused by K+ channels opening.
Figure 2.2
Currents recorded during a voltage clamp, a) A voltage
clamp depolarizing step from -70mV to OmV, resulting in
an immediate capacitance current followed by Na+ and K+
channels opening, b) A voltage clamp depolarizing step
from -70mV to OmV in the presence of TTX and TEA.
29


2.1.4.1.1 Na+Channel Characterization
The amino acid sequence of the Na+ channel has been elucidated by studies
using rat cDNA, followed by structural analysis. (Levitan and Kaczmarek, 1991).
Analysis of the Na+ channel revealed four transmembrane domains that strongly
resemble each other. A hypothetical model for the Na+ channel is shown in Fig.
2.3. The transmembrane domains are numbered 1-4, and each transmembrane
domain contains 6 subunits. The forth subunit (S4 region) of each transmembrane
domain contains either an arginine or a lysine in every third position. It is
believed that the S4 regions of each transmembrane domain form an alpha helix,
with the positive charges being stabilized by fixed negative charges on adjacent
alpha helixes. This lead to the hypothesis that the S4 regions form the voltage
sensors for the channel. The current hypothesis is that when the membrane is
depolarized and the inside of the cell becomes more positive, the positive charges
on the S4 regions rotate outward resulting in a conformational change in the Na+
channel. When the S4 regions of all four transmembrane domains undergo this
conformational change, Na+ activation gates open, and Na+ flows into the cell
30


(Kandel et al. 1991 and Levitan and Kaczmarek, 1991).
Figure 2.3
Hypothetical model for the Na+ channel, adapted from
Kandel. The Na+ channel is proposed to have four
transmembrane domains, numbered 1-4, each with six
subunits. The fourth subunit of each domain is proposed
to make up the voltage sensor for the channel.
31


2.1.4.1.2 K+ Channel Characterization
Voltage gated K+ channels are thought to have a structure similar to Na+
channels, however the transmembrane domains of K+ channels are made up of 4
separate subunits coded either by separate genes, or alternate RNA splicing of
single genes (Shepard, 1988; Covarrubuas et al. 1991; Schwarz et al. 1988). The
hypothetical model for the K+ channels is shown in Fig. 2.4. Four separate
transmembrane domains come together to form the K+ channel. The S4 region is
homologous to the S4 region of the Na+ channel, and it is also believed to be the
voltage sensor for the channel. One interesting consequence of the separated
domains is that alternate splicing of the gene can create more than one type of K+
channel. In fact, there are several different K+ channels each with different
properties. The two K+ channels I studied were called the fast transient K+
current (la) and the delayed rectifier K+ current (Ik). Ia and Ik K+ channels are
the K+ channels responsible for repolarization of action potentials in hippocampal
cells (Segal and Barker, 1984). Pharmacologically, both Ia and Ik K+ channels
can be blocked by TEA. Ia channels function in repolarization of the action
32


potential contributing to control of slow impulse firing rates (Shepard, 1988).
They are rapidly activated at depolarizations starting at -60m V, and are inactivated
by depolarizations to -40mV.
Ik K+ channels are different in that they have more positive thresholds for
activation, -40mV, and they inactivate very slowly (Shepard, 1988). Ik K+
channels also are active in the repolarization phase of the action potential (Segal
and Barker, 1984).
r lgure c* *
Hypothetical model for the K+ channel, adapted trom
Kandel. The K+ channel is proposed to have one
transmembrane domain consisting of six subunits. Four
separate transmembrane domains come together to form a
functional channel. The fourth subunit of each domain is
proposed to make up the voltage sensor for the channel.
33


2.1.4.2 Ca-M- Channels
Ca+ + channels were my main focus in doing the patch clamp studies.
Intracellular Ca+ + levels are closely regulated in neurons because they contribute
to many of the biochemical pathways inside the neuron. Many of these
biochemical pathways are triggered by the entry of Ca+ + from voltage-gated
Ca+ + channels. The structure of Ca+ + channels is similar to Na+ channels.
They consist of four transmembrane domains, each containing six subunits, and
the voltage sensor domain is believed to be the S4 region. The characterization of
Ca+ + channels is complicated. Some Ca+ + channels are voltage gated, and
others are not; some Ca+ + channels inactivate due to prolonged depolarization,
and others inactivate due to intracellular Ca++ accumulation (Kandel, 1991 and
Shepard, 1988). Voltage gated Ca++ channels are closed at resting membrane
potentials, and open when the membrane is depolarized. When compared to Na+
channels, Ca+ + channels require a higher depolarizing step before they will open
(Hille, 1992). Entry of Ca++ into the cell via voltage gated Ca++ channels
helps regulate contraction of muscles, secretion of neurotransmitters at the
34


presynaptic terminal and regulation of action potentials (particularly in cardiac
cells) (Hille, 1992). Once inside the cell, Ca+4- acts as an intracellular
messenger to perform many different functions such as cell division and regulation
of neurite growth. There are many voltage gated Ca+ + channels in the
presynaptic terminals of neurons that open after an action potential travels to the
terminal. Ca+ + channels open and Ca+ + enters the cell causing
neurotransmitter filled vesicles to bind to the membrane and release their
neurotransmitter into the synaptic cleft.
Pharmacologically, Ca+ + channels can be blocked by a number of
inorganic and organic blockers. Inorganic blockers include divalent cations such
as Cd++, Co++, Mn++, Ni++ and La++. Organic blockers include
verapamil, diltiazem and the dihydropyridines (Shepard, 1988).
There are several different types of voltage gated Ca+ + channels, for
example, those designated L, T, and N type Ca+ + channels. L-type channels
(long-lasting) open after strong depolarizations and are noninactivating. L-Type
channels contribute to most of the voltage gated Ca+ + current in hippocampal
neurons. T-type channels (transient) depolarize at low depolarizations of -70mV
and are rapidly inactivating. N-type channels (neither) require a strong
depolarization, (-30mV) and inactivate slowly.
35


I began my search for the mechanism behind the TEL induced Ca+ +
increase in rat hippocampal cells by looking at changes in voltage gated L-type
Ca+ + channels. I felt since L-type Ca+ + channels are the most abundant
Ca+ + channel in hippocampal neurons, one method by which this intracellular
Ca+ + increase could occur was by TEL increasing ion flow through voltage
gated L-type Ca++ channels. I used a voltage clamp technique called patch
clamping to test the Ca+ + current flowing through the L-type Ca+ + channels
before and after exposure to TEL. In addition, I also measured the current
flowing through Na+ and K+ channels before and after TEL exposure.
2.1.5 Patch Clamping
The patch clamp technique was introduced in 1976 by Erwin Neher and Bert
Sakmann for which they received the 1991 Nobel Prize in physiology. Patch
clamping allows a scientist to examine ion channels on an individual basis.
Therefore, it gives more specific ionic current measurements than the macroscopic
36


measurements of the voltage clamp technique done by Hodgkin and Huxley in the
1950s.
Figure 2.5 illustrates the patch clamp technique. A glass electrode or pipette,
tapered at the end to approximately 2uM by an electrode puller and then fire
polished is slowly lowered onto the cell membrane by a manipulator. Once the
pipette is touching the membrane, suction is applied until a gigaohm seal is
achieved (10E9 Ohms) which is measured on an oscilloscope. The gigaohm seal
is a result of a covalent bond forming between the glass pipette and the membrane
of the cell, and therefore it is very stable. Once the seal is formed, several
experimental manipulations of the channels trapped under the pipette can be
performed including several variations such as whole cell and single channel
patches (Neher and Sakmann, 1992). Figure 2.5 demonstrates the variation of the
patch clamp technique I used called the whole cell patch. Once enough suction is
made to create a gigaohm seal, further suction is gently applied until the
membrane under the pipette is ruptured. Once the membrane ruptures, the
cytoplasmic fluid is exchanged with the pipette solution allowing for manipulation
of the cytoplasmic contents, and measurement of the whole cell current.
37


Figure 2.5
Whole cell patch variation of the patch clamp technique.
After a gigaohm seal is formed between the glass pipette
and a cell membrane, further suction is applied until the
membrane is ruptured. Cytoplasmic constituents can then
be exchanged with a controlled pipette solution.
38


The conductance of the channels being studied is determined by determining
the amount of current flowing through the channels. The current of a channel or
channels can be determined using an amplifier to clamp the membrane potential at
a steady level such as -80mV, and then determining the amount of current it takes
to keep the membrane potential at -80mV. The current is measured by Ohms law
as a potential drop or change in voltage across a resistor: 1= V/R (2.2).
Current records are read on the oscilloscope, and upward deflections from the
membrane potential indicate that positive charges are being passed into the cell to
keep the membrane potential at -80mV. This information indicates the cell was in
a state of hyperpolarization. Downward deflections indicate that negative charges
are being passed into the cell, therefore the cell was in a state of depolarization.
In summary, the patch clamp variant I used to study hippocampal cell exposure
to TEL was the whole cell variant. I exchanged the cytoplasm of the cell with a
pipette solution, pharmacologically blocked all channels but the type of channel
being studied, and measured the macroscopic current flowing through those
channels before and after acute exposure to TEL.
39


2.2 Methods
2.2.1 Dissection
2.2.1.1 Rat Hippocampal Neurons
Rat hippocampal cells (RHC)s were obtained from Sprage-Dawley embryos at
18 days of gestation. Timed pregnant rats were anesthetized with C02 and
sacrificed by cervical dislocation. The rat pups were removed, decapitated, and
their brains dissected out. The hippocampi was dissected from each brain and
placed in a Ca++ MG++ free Hanks balanced salt solution (HBSS) with
lOmM HEPES buffer and 1 % antibiotic/antimycotic (antimix) at room
temperature. Next, the hippocampal cells were incubated in HBSS containing
2mg/ml trypsin for 15 minutes at 37 degrees C, rinsed in HBSS and incubated for
15 minutes in 2mg/ml trypsin inhibitor. The cells were then triturated through a
fire polished glass pipette to dissociate the cells. The prepared hippocampal cells
were aliquoted at a density of 2 million cells/0.25 ml of Eagles minimal essential
40


medium (MEM) buffered with lOmM NaHC03, 25mM HEPES and supplemented
with 2mM 1-glutamine, 2% glucose, ImM sodium pyruvate, 10% fetal bovine
serum, 8% dimethyl sulfoxide (DMSO) and 15mM KCL (final concentration,
20mM K+). The cell suspension was placed in 2ml freezing tubes, and frozen
at -70 degrees C in containers designed to slow the freezing process.
2.2.1.2 Astrocytes
After the hippocampi were removed from the embryonic rat brains, the
remaining brain tissue was minced with sterile dissecting knives. The brain tissue
was then incubated for 15 minutes in Ca++ Mg++ free HBSS, .25% trypsin
and 1 % antimix at 37 degrees C. The trypsin solution was then aspirated off and
the cells were incubated for 5 minutes in a solution of Eagles MEM, 10% FCS,
1 % antimix and .25 % trypsin inhibitor.
The cells were then plated in 8 mL flasks at approximately 200,000 500,000
cell/flask in a solution of Eagles MEM, 10% FCS, 1% antimix. The flasks were
41


placed in an incubator at 37 degrees C in a humidified, 5% C02 atmosphere and
\
not disturbed for 2 days. After 2 days, the flasks were shaken gently to loosen
any neurons and ogliodendrocytes, leaving the astrocytes stuck to the bottom of
the flask. The medium was aspirated off and replaced with Eagles MEM, 10%
FCS and 1 % antimix and placed back in the incubator, or frozen in aliquots of
500,000 cells/.25 mis of Eagles MEM, 2,% glucose, 10% FCS, 8% DMSO and
1% antimix.
2.2.2 Culturing
For patch clamp studies, a stock of about 10, 35mm plastic culture dishes of
astrocytes was kept in the incubator. To culture astrocytes, .75 ml of Eagles
MEM, lOmM NaHC03, 25mM HEPES, 0.2% glucose, 2mM L-glutamine, ImM
sodium pyruvate, 15mM KCL, 1% antimix (modified MEM), 10% FCS was
added to the freezing vial, and cells were thawed rapidly by agitation in a 37
degrees C water bath. Astrocyte cells were triturated 5-10 times with a fire
42


polished glass pipette to break up clumps of cells. The cells were then plated onto
35mm plastic culture dishes previously coated for 15 minutes with poly D -
lysine (MW, 300,000) at approximately 3 -4 dishes/vial. After incubating for 2
days at 37 degrees C in a humidified, 5% C02 atmosphere, RHCs were plated on
top of the astrocytes.
RHCs were obtained from the -70 degrees C freezer, .75 ml modified MEM,
10% FCS was added to the freezing vial, and the cells were thawed rapidly in a
37 degrees C water bath. The cells were triturated 1-2 times with a fire polished
glass pipette to dissociate any clumps. Approximately 200,000 cells were added
to the center of each culture dish of astrocytes. The RHCs were incubated for a
minimum of two days to allow attachment to the astrocytes.
2.2.3 Patch Clamping
2.2.3.1 Ca++ Channels
43


Standard whole cell patch clamping procedures were used to test the effects of
TEL on L-type calcium channels.
2.2.3.1.1 Solutions
Pipette solution: CsCl (lOOmM), MgCl (3mM), EGTA (lOmM), MgATP
(2mM), cAMP (.25mM), glucose (25mM) and HEPES (40mM) at pH 7.3,
High Ca++ seal: NaCl (125mM), KC1 (5mM), CaCl (lOmM), MgCl
(ImM), glucose (20mM) and HEPES (lOmM) at pH 7.3.
Ba\TTX\TEA solution: NaCl (llOmM), BaCl (lOmM), MgCl (2mM),
tetraethylammonium chloride, (TEA), (20mM), glucose (lOmM),
tetrodotoxin, (TTX), (2mM) and HEPES (lOmM) at pH 7.3. Ba++ was used in
place of Ca++ because it shows larger currents than Ca++.
Ba\TEA\TEL stock solution: The TEL solution was made by measuring out 3.3
mg of triethyllead chloride, adding it to 100 mis of a Ba\TEA solution, (without
the TTX), and dissolving it; this made a 100 uM TEL solution.
44


Ba\TEA\TTX\TEL working solution: The TEL stock solution was diluted to 10
uM, by using a 9:1 mixture of Ba\TEA\TTX and Ba\TEA\TEL. The resulting
Ba\TEA\TTX\TEL solution was used for two days and then discarded to guard
against the TEL breaking down.
2.2.3.1.2 Procedure
Pipettes were manufactured from LC16 (Dagan) glass pulled on a Sutter PC-
84 electrode puller, and polished on a Narashige microforge. The electrode puller
pulls glass pipette tubing, tapering it at the end to a diameter of approximately
2uM, after which the tapered end is polished with heat to a diameter of
approximately luM. Then the pipette is filled with the pipette solution described
above at pH 7.3; the pipette resistance ranged from 1.5 to 3 megohms. The
pipette was then mounted onto the manipulator.
A dish of cultured hippocampal cells was then placed onto the microscope
stage and the perfusion tubing and capillary tube drain was inserted into the dish
45


of cells. Bathing solutions were perfused over the hippocampal cells and could be
changed by adjusting a valve on a circular plastic ring equipped with capillary
tubing running from the solution bottles to the culture dish; this apparatus kept a
steady perfusion of solutions across the cells. A siphon drain made from capillary
tubing drained off the excess solution.
A hippocampal cell was chosen for patch clamping if it met the following
criteria: 1) it was a pyramidal-like cell characterized by the presence of an axon,
2) it appeared large and healthy with healthy neurites, and 3) it was toward the
middle of the dish.
The fire polished pipette was lowered with the coarse and fine drives on the
manipulator until contact was made with the membrane of the chosen hippocampal
pyramidal cell. Contact with the cell was determined both by watching the actual
process through a microscope, and also by watching the current trace on an
oscilloscope. The current trace appeared as square waves, and after contact was
made, a height reduction of the square waves could be seen. Gigaohm seals were
made by using a syringe to apply slight suction to the membrane. Gigaohm seals
were made in Hi Ca+ + seal solution. The seal between the membrane and
pipette was considered a gigaohm seal if the square waves on the oscilloscope
screen flattened out into a straight line.
46


After a gigaohm seal was achieved, the membrane was ruptured by further
suction via the syringe until large capacitance spikes were seen on the oscilloscope
screen indicating the whole cell was being charged up.
Whole-cell voltage clamps were performed using an Axopatch 1C amplifier
(Axon Instruments). Currents were low-pass filtered at 2 kHz. The Axon pclamp
program was used to control experiments and analyze data. A P/4 protocol was
used, which digitally reduced current artifacts at the beginning and end of voltage
steps (Chad and Eckert, 1986). All voltage clamping was done at room
temperature. Currents were produced by 120 msec clamp steps from a holding
potential of -80mV. Peak inward currents and steady state inward currents at the
end of a 120 msec voltage clamp step were measured. Peak currents are mostly
the sum of currents flowing through T-type, N-type and L-type calcium channels.
Because T-type and N-type channels inactivate more rapidly, currents at the end of
the 120 msec step are approximately the slower inactivating L-type channels alone
(Audesirk and Audesirk, 1991). The end-of-step currents were used to estimate
L-type channels alone.
After the membrane was ruptured, the following experimental procedure was
followed:
1) One minute after a gigaohm seal was made, the cell was depolarized and a
47


sodium current was recorded in Hi Ca+ + seal in order to determine the health of
the cell. If the cell did not produce a large sodium trace, it was not used.
2) Following a good sodium trace, the Hi Ca+ + seal solution was changed to a
Ba\TEA\TTX solution (blocks sodium and potassium channels), to act as a
control.
3) Two minutes after the Ba\TEA\TTX solution was started, the cell was
depolarized and a control Ca+ + trace was recorded. If the sodium current
disappeared and good calcium currents were seen, the solution was changed to the
experimental solution containing Ba\TEA\TTX\TEL.
4) Two Ca+ + traces were recorded in the TEL solution, two minutes apart, to
see if calcium channels exhibited a change in current.
5) Finally, three calcium traces were recorded in the Ba\TEA\TTX control
solution, two minutes apart, to wash out the TEL and see if any TEL induced
change in the current could be reversed.
The following criteria were followed to gaurd against experimental error:
1) If the holding current exceeded 0.3mV at any time during the experiment, the
data was discarded,
2) If the cell died before the second washout run, the data was discarded, and
3) The hippocampal cultures were discarded after a period of 10 days to insure the
48


use of healthy cells.
2.2.3.1.2 Data analysis
The Axon pclamp program was used to analyze data. The end-of-step
currents mentioned above were used to estimate L-type channels. The end of step
currents were then analyzed on a Quattro Pro spreadsheet, and finally graphed
using Sigma Plot 5.
2.2.3.2 Na+ Channels
Standard, whole cell patch clamping procedures were used to test the effects
of TEL on Na+ channels.
49


2.2.3.2.1 Solutions
Pipette solution: CsCl (lOOmM), MgCl (3mM), EGTA (lOmM), MgATP
(2mM), cAMP (.25mM), glucose (25mM) and HEPES (40mM) at pH 7.3,
High Ca++ seal: NaCl (125mM), KC1 (5mM), CaCl (lOmM), MgCl (ImM),
glucose (20mM) and HEPES (lOmM) at pH 7.3.
IOOuM Cd+4- solution: Hi Ca++ seal:10mM CaCl stock in a 99:1 ratio,
Cd++ was used to block Ca++ channels.
IOOuM TEL stock: 3.3mg TEL-CL in lOOmls of Hi Ca++ seal.
IOuM TEL working solution: Hi Ca++ seal\Cd: IOOuM TEL stock in a 9:1
ratio. All TEL solutions were remade after 2 days.
50


2.2.3.2.2 Procedure
The procedure used to measure Ca+ + currents was also used to measure
Na+ currents with the following changes:
1) the peak inward current was measured for each Na+ trace instead of end of
step currents,
2) if the Na+ trace disappeared before the washout trials, the data was discarded.
2.2.3.23 Data Analysis
The Axon pclamp program was used to analyze data. The peak Na+ currents
were used to measure Na+ channels. The peak currents were then analyzed on a
Quattro Pro spreadsheet, and graphed using Sigma Plot 5.
51


2.2.3.3 K+ Channels
Standard, whole cell patch clamping procedures were used to test the effects
of TEL on K+ channels. Two different K+ channels were tested: the fast
transient current (la), and the delayed rectifier current (Ik). The la channels
rapidly activate when the membrane is depolarized to -60mV, and inactivate at
depolarizations of -40mV. The Ik channels do not activate until depolarization of
the membrane is -40mV, and then inactivate very slowly. I separated the Ik
channels from the la channels by depolarizing the cell from a holding potential
of -80mV to measure the current flowing through the la channels, after which I
switched the holding potential to -40mV to inactivate the la channels and
depolarized the cell from -40mV to measure the current flowing through the Ik
channels.
52


2.2.3.3.1 Solutions
Pipette solution: CsCl (lOOmM), MgCl (3mM), EGTA (lOmM), MgATP (2mM),
cAMP (.25mM), glucose (25mM) and HEPES (40mM) at pH 7.3,
High Ca+ + seal: NaCl (125mM), KC1 (5mM), CaCl (lOmM), MgCl (ImM),
glucose (20mM) and HEPES (lOmM) at pH 7.3.
IOOuM Cd++ working solution: Hi Ca++ seal:10mM CdCl:500nM TTX in a
97:1:2 ratio. The solution was aliquoted into small amounts and frozen.
IOOuM TEL stock solution: 3.3mg TEL-C11 in lOOmls of Hi Ca++ seal.
IOuM TEL working solution: Hi Ca++ seal\Cd\TTX:Hi Ca++ seal\TEL stock
in a 9:1 ratio. Again, TEL solutions were remade after 2 days.
53


2.2.3.3.2 Procedure
K+ currents were measured a little differently than Ca++ and Na+ currents
in that the holding potential was switched from -80mV to -40mV in order to
differentiate between the fast transient (la) and the delayed rectifier (Ik) K+
currents.
A gigaohm seal was made on a hippocampal pyramidal cell, and the
membrane was ruptured to create the whole cell clamp, after which the following
procedure was performed:
1) At 2 minutes, (2 minutes after the membrane was ruptured) the cell was
depolarized by stepping the potential from a holding potential of -80mV to a
potential of +40mV, and a K+ (la) trace was recorded in Hi Ca+ +
seal\Cd\TTX at +40mV. The holding potential was then switched from -80mV to
-40mV on the amplifier.
2) At 4 minutes, the cell was depolarized by stepping the potential from a holding
potential of -40mV to a potential of +40mV, and a K+ (Ik) was recorded in Hi
Ca++ seal\Cd\TTX. The holding potential was changed to -80mV.
54


3) At 6 minutes, the cell was depolarized by stepping the potential from -80mV
to +40mV, and a K+ (la) trace was recorded in Hi Ca+ 4- seal\Cd\TTX\TEL.
The holding potential was changed to -40mV.
4) At 8 minutes, the cell was depolarized by stepping the potential from -40mV
to +40mV, and a K+ (Ik) trace was recorded in Hi Ca+ + seal\Cd\TTX\TEL.
The holding potential was changed to -80mV.
5) At 10 minutes, the cell was depolarized by stepping the potential from -80mV
to +40mV, and a K+ (la) trace was recorded in Hi Ca+ + seal\Cd to washout
any TEL affect. The holding potential was changed to -40mV.
6) At 12 minutes, the cell was depolarized by stepping the potential from -40mV
to +40mV, and a K+ (Ik) trace was recorded in Hi Ca++ seal\Cd to washout
any TEL affect.
The criteria to gaurd against experimental error were the same as the Ca+ +
channels.
55


2.2.3.3.3 Data Analysis
K+ currents were measured as peak outward currents in both the -80mV
and -40mV traces. The Axon pclamp program was used to analyze data. The
peak K+ currents, separately at -80mV and -40mV were used to measure K+
channels. The peak currents were then analyzed on a Quattro Pro spreadsheet,
and finally graphed using Sigma Plot 5. The ANOVA statistical test was used to
determine any changes in peak measurements between control and TEL I-V
curves.
56


2.3 Results
2.3.1 Ca++ Channels
Whole cell patch clamping of L-type Ca+ + channels in 10 hippocampal
neurons show no change in Ca+ + currents after a 4 min exposure of the cell to
lOuM TEL (Fig. 2.6a). Graphs represent the current flowing through the
channels at the voltage represented on the x axis; this is called an I-V curve.
There is no significant difference in the I-V curves for pre-TEL, TEL and
washout runs (Fig. 2.7b). The washout run does show an increase in current
flowing through the Ca+ channels, which was determined at this time not to be
significant; however, it may be interesting to look further into this increase in
Ca+ current.
57


Figure 2.7
inward currents recorded from voltage-gated ca+ channels
on rat hippocampal neurons (RHNs). a) Peak inward pre-TEL
and TEL Ca+ currents elicited by voltage steps to -lOmV
from a holding potential of -80mv.
b) Current-voltage (I-V) curve from 10 RHNs.
58


2.3.2 Na+ Channels
Whole cell patch clamping of Na+ channels in hippocampal neurons show no
change in Na+ currents after a 4 min exposure of the neuron to lOuM TEL (Fig.
2.7a). There is no significant difference in the I-V curves for pre-TEL, TEL and
washout runs (Fig. 2.7b).
59


Figure 2.7
Inward currents recorded from voltage-gated Na+ channels
on BHNs. a) Peak inward pre-TEL and TEL Na+ currents
elicited by voltage steps to -lOraV from a holding
potential of -80mV. b) Current-voltage curve from 6 RHNs.
60


2.3.3 K+ Channels
Whole cell patch clamping of K+ channels in hippocampal neurons show no
change in current through the K+ channels, either la or Ik channels after a total
of 8 min in lOuM TEL (Fig. 2.8a,b). An I-V curve was not generated for K+
channels because they were depolarized using one step from either -80mV to
+40mV or -40mV to +40mV for la and Ik channels, respectively. Therefore a
bar graph is used comparing pre-TEL to TEL plus two control runs at the same
time points as the experimental runs. The bar graphs show that there is no
significant difference between pre-TEL and TEL la or Ik current flowing through
these channels (Fig. 2.8c,d).
61


Figure 2.8
Outward currents recorded from voltage-gated K+ channels
a) fast transient and delayed rectifier (la and Ik) on
RHNs and b) Ik alone on RHNs. a) Peak outward pre-TEL
i and TEL la and Ik K+ current elicited by a voltage step
i to +40mV from a holding potential of -80mV. b) Peak
: outward pre-TEL and TEL Ik K+current elicited by a
; voltage step to +40mV from a holding potential of -40mV.
c) Bar graph representing TEL and post-TEL la and Ik K+
current expressed as percent of control for 6 RHNs. Runs
2 and 3 are control la and Ik K+ currents used as a
control for time, d) Bar graph representing TEL and post-
TEL Ik K+ current expressed as percent of control for 6
RHNs. Runs 2 and 3 are control Ik K+ currents used as a
control for time.
62


2.4 Discussion
TEL appears to have no direct effect on voltage-gated channels, Ca-H-, Na+
or K+. My experiments demonstrate that after 4 min in TEL, the Ca-tf- and Na+
channel I-V curves after exposure to TEL were not significantly different from
their respective control I-V curves, and after 8 min in TEL, K-f current after
TEL exposure was not significantly different from the controls. The increase in
Ca-H- current seen in the washout run (Fig. 2.6b) may indicate that TEL is having
a delayed effect on Ca-H-channels, or it may be interfering with intracellular
constituents pertinent to Ca-H- channel function. Whether or not TEL was
interfering with intracellular constituents could be tested using a cell attached
patch, which does not alter the cytoplasm of the cell.
Steady increases of intracellular Ca++ were shown in El 8 rat hippocampal
neurons after exposure to uMTEL concentrations in previous Ca+ + imaging
experiments using the dye fura2. These steady increases in intracellular Ca+ +
are indicative of an extracellular Ca+ + source. Since TEL does not appear to be
influencing voltage-gated channels, the next source of the intracellular Ca++
63


increase I would look into would be that TEL may be altering the extrusion of
Ca++ ions by disrupting the ATP-driven Ca++ pump.
64


3. Catetmn Imaging Experiments
3.1 Introduction
My experimental goal in doing the Ca+ + imaging experiments was to
find the TEL concentration causing an increase in intracellular Ca+ + in E18
rat hippocampal neurons. Previous experiments determining that
intracellular Ca+ + increases after TEL exposure were flawed, therefore I
wanted to determine at what TEL concentrations an increase in intracellular
Ca+ + concentrations ([Ca+ +]in) occurred. I also wanted to know if these
increases in [Ca++]in could be related to decreases in neurite branching
observed in cultured E18 rat hippocampal neurons after TEL exposure. I
used Ca+ + imaging experiments to determine if there was a relationship
between the [Ca+ +]in increases and the neurite branching decreases. A
Ca+ + chelator was added to E18 rat hippocampal neurons that were exposed
to TEL. These experiments were done in conjunction with culture
experiments exposing E18 rat hippocampal neurons to TEL and the Ca+ +
65


chelator. If the Ca+ + chelator blocked the [Ca+ +]in in the Ca+ + imaging
experiments, and also blocked the neurite branching decreases in culture
experiments, the neurite branching decreases may be occurring secondary to
the [Ca++]in increases.
3.1.1 Intracellular Ca+ +
Calcium can enter the cell from the extracellular medium via voltage
gated Ca+ + channels and/or be released from intracellular stores. The
concentration of free Ca+ + inside the cell is very low, approximately 10E-7
M. This is four orders of magnitude lower than the extracellular matrix,
which means that Ca+ + has a strong electrical gradient driving it into the
cell. In addition to the large driving force, small changes [Ca+ +]in within
the cell can result in physiological Ca+ + dependent responses. The
increase of Ca+ + inside the cells, from outside or inside sources, plays a role
in a number of cellular functions including: cell excitability, gene expression,
66


neurotransmitter release, long-term potentiation, protein synthesis, muscle
contraction, secretion by exocytosis, microtubule assembly and disassembly,
cell division, and regulation of intracellular levels of cyclic nucleotides (Becker
and Deamer, 1991).
Calcium can increase inside neurons via two main sources: extracellular
sources such as voltage-gated Ca+ + channels, and intracellular sources such
as stores in the endoplasmic reticulum.
3.1.1.1 Extracellular Ca+ + Sources
The major extracellular source of intracellular Ca+ + used for the above
cellular functions is the voltage gated Ca+ + channel. These channels let
extracellular Ca+ + in when the cell depolarizes toward a positive membrane
voltage. Several different types of Ca+ + channels have been identified,
including T (transient), L (long-lasting) and N (neither). These channels have
been characterized by their differences in conduction, pharmacology and
voltage dependency (Verity, 1990). T-type channels are classified as having a


low threshold for activation (-70mV), have a small conductance (8
picoseimens, pS), make a transient current, inactivate rapidly, and are
sensitive to blockage by transition metals such as Ni+ + and Cd+ +. L-type
channels differ from T-type channels in that they have a higher threshold,
have a larger conductance (25pS), make a long-lasting current, inactivate
slower, and are blocked by dihydropyridines such as nifedipine and BAY K
8644 in addition to the transition metals. N-type channels have a threshold,
conductance and current that fall between the T and L-type channels. N-type
channels are blocked by conotoxin in addition to transition metals, but are not
sensitive to dihydropyridines (Hille, 1992). The different channel types are
found together in the same neuron but their densities are often associated with
different stages in development and different neuronal type.
Ca+ + levels inside the neuron are low at resting potentials (around -
75mV), and as the cell depolarizes toward OmV, Ca+ + channels open and
intracellular Ca+ + levels rise until the buffering and pumping mechanisms
remove the extra Ca+ + ions. Located in the plasma membrane are A TP-
driven Ca+ + pumps and Na+/Ca+ + exchange proteins that remove the
extra Ca+ + ions. The ATP-driven pump is activated by the Ca+ + binding
protein, calmodulin and can be altered by phosphorylation, proteolysis, or by
68


calmodulin levels (Verity, 1990).
3.1.1.2 Intracellular Ca+ + Sources
Ca+ + ions are also sequestered intracellularly in the neuron: in the
endoplasmic reticulum (ER), and in the mitochondria. The two major
intracellular Ca+ + stores important in hippocampal neurons are the inositol
1,4,5, triphosphate (IP3) sensitive stores and the Ca+ + induced Ca+ +
release (CICR) stores both located in the smooth endoplasmic reticulum
(SER). Smooth endoplasmic reticulum is found throughout the neuron: in
the soma, axons, growth cones, dendrites and dendritic spines.
3.1.1.2.1 IP3 Stores


LP3 is an intracellular messenger generated in response to
neurotransmitters acting on G-protein linked receptors. After a ligand binds
to the receptor, activated G-proteins in turn activate phospholipase C (PLC).
PLC then hydrolyses the membrane bound phospholipid phosphatidylinositol
4,5-biphosphate (PIP2) forming diacylglycerol (DAG), which activates PKC,
and IP3. IP3 then acts on the IP3 receptor to release Ca+ + (Fig.3.1).
Figure 3.1
Inositol triphosphate released Ca++ stores. After a
ligand bind to the receptor (R), activated G-proteins
activate PLC. PLC hydrolyses PIP2 into DAG and IP3. IP
then acts on the IP3 receptor located on the endoplasmic
reticulum (ER) and Ca++ is released.
70


3.1.1.2.2 Calcium Induced Calcium Release Stores
Calcium induced calcium release stores (CICR) stores are found in neurons,
including hippocampal neurons. Both the CICR and EP3 stores in neurons
are channel proteins located in the endoplasmic reticulum (ER) of the neuron
body, axon, synaptic terminal and dendrites (Henzi and MacDermott, 1992).
The ryanodine receptor (RyanR), is associated with the CICR stores, and is so
named because its activity is modulated by the plant alkaloid ryanodine.
Calcium induced calcium released channels are activated when voltage gated
Ca+ + channels, ligand gated Ca+ + channels and even IP3 released Ca+ +
activate the RyanR resulting in an increase intracellular Ca+ +, hence the
name Ca++ induced Ca++ release. Once Ca++ is released into the
cytoplasm, it is then pumped back into the ER by Ca+ +-ATPases.
The traditional view of Ca+ + as an intracellular messenger, whether the
influx into the cytosol is via extracellular or intracellular means is the role of
71


an intracellular messenger. As Ca+ + levels rise in the cytosol, Ca+ +
binding proteins such as calmodulin attach to the Ca+ + ions and the
Ca+ +/calmodulin complex then interacts with other proteins inside the
neuron to alter the neurons function (Rasmussen, 1989).
3.1.2 Intracellular Ca+ + and TEL
Previous experiments testing the effects of TEL on rat hippocampal
neurons have shown that [Ca+ +]in increase significantly when the neurons
are exposed to uM concentrations of TEL. Ca+ + imaging experiments
exposing hippocampal neurons to 5uM concentrations of TEL show a large,
steady increase in [Ca+ +]in. This large, steady increase in [Ca+ +]in
suggests that the Ca+ + ions are coming into the cell from the extracellular
stores rather than intracellular stores. If the intracellular Ca+ + stores were
responsible for the Ca+ + increase, the rise would show a large transient
Ca+ + rise. In addition, preliminary TEL perfusion experiments with Ca+ +
72


free media show no increase in [Ca+ +]in after exposure to TEL. Although
my patch clamp experiments showed no increase in Ca+ + current through
the voltage gated Ca+ + channels after neurons were exposed to lOuM TEL,
TEL may be altering the extrusion of Ca+ + ions by disrupting the ATP-
driven Ca+ + pump. TEL stimulates ATP hydrolysis and prevents ATP
synthesis (Kauppinen, 1988).
Previous culture experiments exposing E18 rat hippocampal cells to
concentrations of TEL ranging from O.lnM to 5.0uM show that at TEL
concentrations > luM survival of the neurons is significantly reduced.
Neurons cultured in TEL concentrations < luM survive nicely, but culture
parameters such as the number of dendrites and dendrite and axon branching
are significantly reduced (Fig 3.2).
My research focused on the role of intracellular Ca+ + and the possible
relationship between TEL, [Ca+ +]in and the decreases in neurite branching
seen after TEL exposure.
If the [Ca+ +]in increase seen after exposure of neurons to 5uM TEL could
be blocked with the Ca+ + chelator 1,2 -bis(2-aminophenoxy)ethane-
N,N,NN-tetraacetic acid acetozymethyl ester (BAPTA-AM), and in addition,
if the decrease in neuronal survival and/or neurite branching after exposure
73


to 5uM TEL could also be blocked with BAPTA-AM, then there may be a
link between the [Ca++]in increases and survival and neurite branching
decreases seen after exposure to 5uM TEL.
b
Figure 3.2
Effects of increasing TEL concentrations on dendrite
number and neurite branching in rat hippocampal cultures,
a) Dendrite numbers are significantly decreased in
cultures exposed to uM concentrations of'TEL. b) Neurite
branching is significantly decreased after TEL exposure,
dendrites after exposure to > lOnM TEL, and axons after
exposure to > lOOnM TEL.
74


The methods I used to find out this information was to use the Ca+ +
imaging dye fura>2 to measure [Ca+ +]in levels before and after exposure to
TEL. The experiments measuring [Ca+ +]in increases after TEL exposure
were repeated because previous experiments may have shown artificial
[Ca++]in increases due to excessive UV light exposure.
In order to see if the reduction in dendrite branching and number is
related to the increase in [Ca+ +]in, I clamped [Ca+ +]in with BAPTA-AM
to see if the [Ca+ +]in increase after TEL exposure could be blocked. The
latter experiments were done in conjunction with culture experiments using
BAPTA-AM to see if the decrease in neurite branching seen after TEL
exposure could be blocked.
3.1.3 Ca++ Imaging
To measure [Ca+ +]in changes after TEL exposure, I used the fluorescent
dye fura-2. Fura-2 is a Ca+ + sensitive fluorescent dye based on the
75


structure of the Ca+ + chelator ethylene glycol-bis(B-aminoethyl ether)
N,N,N,N-tetraacetic acid (EGTA). When fura-2 binds Ca+ + in the
physiological range, its fluorescence spectrum changes. For example, when
Ca+ + is bound to fura-2, the dye fluoresces maximally at a wavelength of
340nm. In contrast, when Ca+ + is not bound to fura-2, the dye fluoresces
maximally at a wavelength of 380nm (Hall, 1992). Therefore, 340/380nm
images can detect changes in [Ca+ +]in levels.
The particular dye I used was fura-2 AM which is an acetozymethyl ester
of the fluorescent dye fura-2. The attached ester groups mask the Ca+ +
chelating carboxylate groups, thus allowing the dye to cross the plasma
membrane. Once inside the cell, cytosolic esterases hydrolyze the ester
groups activating the fura-2.
76


3.2 Methods
E18 rat hippocampal cells were used for the calcium imaging and culture
experiments.
3.2.1 Dissection
Dissection procedures were the same as in the patch clamp experiments.
3.2.2 Perfusion Experiments
77


3.2.2.1 Culture Dishes
Special culture dishes for the perfusion experiments were made by fitting
plastic culture dishes with glass bottoms to allow for better cell attachment on
glass and better viewing of the cells. The dishes were prepared by cutting a
1.5cm x 1.5cm hole in the bottom of a 35mm plastic culture dish. A ffl glass
cover slip was sealed over the hole with Sylgard 184 (Dow Corning), after
which the dishes were soaked in 1% nituric acid for 48 hrs to neutralize
toxicants in the Sylgard. The dishes were then soaked in distilled water for
an additional 24 hrs. In preparation for each experiment, the dishes were
exposed to UV radiation for 10 min to sterilize them. The dishes were then
coated with poly-D-lysine (MW > 300,000) for 15 min and rinsed with
distilled water; this provided a negative charge on the bottom of the dish to
which the cells could adhere.
The control media I used for the perfusion experiments was modified MEM,
10% FCS; the control media was used to dissolve the TEL.
78


3.2.2.2 Solutions
TEL stock was made up by dissolving 3.3mg TEL-CL into 100ml of
modified MEM. This lOOuM stock solution was then diluted in modified
MEM, 10% FCS to make the following concentrations: 5uM, luM and lOOnM
TEL.
The Ca+ + imaging dye, fura-2AM (Molecular Probes) was used to
measure Ca+ + levels inside the cells. Fura-2AM also has an ester group
attached in order to render it membrane permeable. A lug/ml stock of fura-
2AM was prepared by dissolving it in dimethyl sulfoxide (DMSO).
3.2.2.3 Procedure
Three hundred uL of media containing 5uL/ml Fura-2AM stock was
79


pipetted into the well of a prepared culture dish. Hippocampal neurons were
thawed as previously described and 300,000 cells/dish were pipetted into the
above medium. The cells were then incubated at 37 degrees C for 30 min to
allow the fura-2AM to enter the cells. After 30 min, 2 mis of modified MEM
was added to each dish, and the cells were incubated for an additional 30 min
during which the fura2-AM de-esterified, keeping it from exiting the cells.
After incubation, the dishes were placed on a microscopic stage heated to 30
degrees C using a stage warmer. A peristaltic pump was used to perfuse the
control and experimental media over the cells at a rate of 170ul/min.
Images were taken using a Quantex imaging system at wavelengths of 340
and 380nm which were then ratioed in order to measure [Ca+ +]in
concentrations. Each cell was used as its own control, first having [Ca++]in
measured in control media followed by TEL media. Images were taken with
a Nikon microscope with an intensified CCD camera. Sixteen frames for each
the 340nm and the 380nm wavelengths were imaged with a Quantex QX-7
imaging system and averaged. A #2 Nikon neutral density filter was used to
reduce fura-2 bleaching and saturation of the camera image. Images were
taken according to the following protocol.
1) Five random fields of cells were imaged to use as UV light controls. Five
80


random fields were imaged before and after the experiment, and then
compared to the experimental field to make sure the UV light exposure was
not causing artificial [Ca+ +]in increases.
2) Cells were perfused with control media for 15 min, and images were taken
every 3 min.
3) Cells were perfused with TEL media for 45 min, and images were taken
every 3 min.
4) Cells were perfused with TEL media for 2 hrs, and images were taken
every 5 min.
5) Five random fields of cells were imaged to use as UV light controls.
A total of 10-12 cells were measured in each culture dish, and a total of 5
dishes were measured for each TEL concentration.
3.2.2.4 Data Analysis
[Ca+ +]in measurements were averaged in 15 min bins and entered into
81


a Quatro Pro spreadsheet. Each bin was then calculated as a percent of the
control bin. The averages, standard deviations and standard errors were then
graphed using Sigma Plot 5.0.
3.2.3 BAPTA-AM Experiments
The 5uM TEL imaging experiments were also repeated as described above
with the addition of the Ca+ + chelator BAPTA in the media. BAPTA is
preferred over other Ca+ chelators such as EGTA when rapid buffering of
Ca+ is needed. E18 rat hippocampal cells were perfused with 2uM BAPTA-
AM alone or 5uM TEL plus 2uM BAPTA-AM. Separate controls were
conducted with 2uM BAPTA-AM added to the control media to see if
BAPTA-AM alone had any effects on [Ca+ +]in. BAPTA-AM in 2uM
concentrations was also added to media containing 5uM TEL to see if the
increase in [Ca+ +]in seen with 5uM TEL alone could be eliminated. Since
the BAPTA stock is dissolved in DMSO, DMSO was also added to the control
82


media at concentrations that did not exceed 0.1% The above imaging protocol
was then followed with both the BAPTA-AM control and BAPTA-AM and
TEL perfusions.
83


3.3 Results
3.3.1 TEL Perfusions Experiments
TEL perfusions in concentrations of lOOnM, luM and 5uM were tested on
E18 rat hippocampal cells. Perfusions of 5uM TEL showed a steady increase
in [Ca++]in of approximately 80% over a period of 3 hr (Fig. 3.3).
Perfusion of lOOnM and luM TEL concentrations did not affect [Ca+ +]in
levels.
84


time (hrs)
Figure 3.3
Changes in intracellular Ca++ concentrations during TEL
perfusion. Five uM TEL caused a significant increase in
intracellular Ca++.
85


3.3.2 BAPTA-AM Experiments
£18 rat hippocampal cells were perfused with 2uM BAPTA-AM alone or
5uM TEL plus 2uM BAPTA-AM. Control studies using 2uM BAPTA-AM
alone showed no effect on [Ca+ +]in. When 2uM BAPTA-AM was combined
with 5uM TEL, the BAPTA-AM prevented the increase in [Ca+ +]in seen
with 5uM TEL alone (Fig. 3.4).
86


T
C
o
o
c
ID
O
i_
d)
Cl
+
CM
O
o
200
150
5 TEL I
BAPTA/TEL I 1/ /
2 /^M BAPTA

c 100
50

1 2 3
time (hrs)
Figure 3.4
Changes in intracellular Ca++ concentrations duri
and BAPTA perfusions. Two uM BAPTA prevented the
intracellular Ca++ increase seen with 5uM TEL.
t TEL
87


3.4 Discussion
Ca+ + imaging experiments show that TEL in concentrations of 5uM
cause [Ca+ +]in to increase in rat hippocampal neurons, whereas TEL in
concentrations < 5uM does not cause [Ca+ +]in to increase. The Ca+ +
chelator, BAPTA-AM was able to eliminate the increase in [Ca+ +]in see
after exposure to 5uM TEL.
Culture experiments show that neuronal exposure to TEL in
concentrations > 5uM significantly decreases survival (Fig. 3.5).
Since Ca+ + plays such major roles in neurons, both electrogenic and
regulatory, changes in Ca+ + homeostasis can be detrimental. One
hypothesis was that the [Ca+ +]in seen after TEL exposure could be linked to
the decrease in cell survival seen after exposure to 5uM TEL. Therefore,
culture experiments were done exposing neurons to TEL in concentrations of
5uM and chelating the [Ca+ +]in increase with 2uM BAPTA-AM to see if the
decrease in neuronal survival may be caused by increases in [Ca+ +]in. It
was demonstrated by 5uM TEL and 2uM BAPTA-AM perfusion experiments
88


that 2uM BAPTA-AM did prevent the Ca+ + rise seen with 5uM TEL alone.
Culture experiments done in conjunction with the perfusion experiments show
that blocking the [Ca+ +]in rise did not affect neuronal survival; therefore,
the decrease in neuronal survival caused by exposure to 5uM TEL was not
caused by increasing [Ca++]in (Fig. 3.6).
Different culture parameters were affected when neurons were exposed to
TEL in concentrations < 5uM. The most significant of these were decreases
in neurite branching, dendrite branching in particular (Fig. 3.2b). Although
the [Ca+ +]in increase did not show up in perfusion experiments with TEL
concentrations < 5uM, the increase may be there but the equipment may not
be sensitive enough show the increase, or the increase may be localized. In
order to find out if [Ca+ +]in increases had a role in the decreases in
dendrite branching, culture experiments were done in which neurons were
exposed to TEL in concentrations 5uM and 2uM BAPTA-AM to see if
chelating the [Ca+ +]in increase would restore dendrite branching to control
levels. Since chelating the [Ca+ +]in increase did not bring neurite branching
back to control levels, [Ca+ +]in increases in neurons exposed to TEL were
not causing the decreases in neurite branching seen after TEL exposure.
Since Ca+ + levels in a cell are so closely regulated, I believe the increases
89


seen in [Ca+ +]in after exposure to 5uM TEL is affecting cellular function.
However, these increases in [Ca++]in are not linked to the changes we see in
neurites after TEL exposure.
In light of the above conclusion, I chose to discontinue my experimental
goal of finding the mechanism behind the [Ca+ +]in increase after TEL
exposure and concentrate on finding the mechanism behind the decrease in
dendrite branching after TEL exposure.
Figure 3.£
Effects of increasing TEL concentrations on rat
hippocampal neuron survival in culture experiments.
Survival decreased significantly in 5uM TEL
concentrations.
90


cf)
' SURVIVAL (* CONTROL)
150
125
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50
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$ &
<* x <3* x
t)
INITIATION {% CONTROL)
150 r-
125 -
100 -


<Â¥
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c)
BRANCHES/AXON (X CONTROL)
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100 B -
75 I -
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Figure 3.5
Effects of chelating CCa++3in with 2uM BAPTA on survival,
initiation and axonal branching of RHNs in culture.
BAPTA alone did not affect any of the above culture
parameters, a) BAPTA plus luM or 5uM TEL did not negate
TEL effects on survival of RHNs, b) initiation or c)
iaxonal branching. The astricks indicate there was no
initiation or axonal branching in 5uM TEL concentrations.
91


4. Western Blot Experiments
4.1 Introduction
Because I saw no relationship between the increases in [Ca++]in seen after
perfusion with TEL and the decreases in dendritic branching seen after neurons
were cultured in TEL, I chose to look at the affects of TEL on the cytoskeletal
constitiuants tubulin and the microtubule associated protein MAP2. TEL has been
reported by Zimmerman et al. (1988) to inhibit microtubule assembly in vitro,
therefore I used western blot techniques to see if TEL was affecting tubulin and
MAP2 in rat cortex cells.
4.1.1 Neurite Growth
Neurons are specialized more than other cells in that their function is directly
92