HIPPO
THE
M.
CAMPAL
Sc.,
MB.
REGULA
A
Suez
THESIS
BCh.,
THE
NEURONS
THE
THE
Canal
TION
KHALED
We Cairo
Khaled
REQUIREMENTS
SUBMITTED
UNIVERSITY
FACULTY
accept
OF
Medical DOCTOR (Department
Medical
to
INTRA Mohammed
MOHAMMED
AND
the
December
this
required
School,
OF
THE
School, CELL
OF
IN
thesis
OF
BY
GRADUA
in
PARTIAL
of
PHILOSOPHY
FOR
INFLUENCE
BRITISH ULAR
Abdel-Hamid,
Physiology)
Suez
standard
1994
ABDEL-HAMID
as
Cairo
THE
conforming
CALCIUM
TE
Canal
FULFILLMENT
DEGREE
COLUMBIA University
STUDIES
OF
University
CALCIUM
1994.
IN
OF
CUL (1982)
OF
TURED
(1989)
BUFFERS ______
In presenting this thesis in partial fulfilment of the requirements for an advanced
degree at the University of British Columbia, I agree that the Library shall make it
freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
(Signature)
(r.’ Department of 11 4 I
The University of British Columbia Vancouver, Canada
Date 9
DE-6 (2/88)
competition the
Such
secondary
buffering to
in
hypothesis,
expressing
or
enhancing
loading
Manipulation
responses,
in
expression
(CaBP).
load
difference
subject
central
Neuronal
of
pathological
has
the
cultured
glutamate-induced
by
calcium
prolongation
been
handling
a
loading
enhancement
Excessive
prolongation
role
neurons
to
This
capacity
populations
blamed
to
neuronal this
has,
CaBP
hippocampal
and
(Ca 2 i,
of
were
between
in
situations
the
of
neurons
work
capacity
the
the
form
controversially,
on
neuronal
with
of
or
impairment
activation
more
as
high-capacity
are
triggering
the
the
aims
and
loaded
Ca 2 +
of
may
of
a
the
BAPTA-like
neuronal
differ
likely
with
causative
vulnerability
neuronal
of
vulnerable excessive
via
recovery
the
neurons
at
recovery
be
Ca 2
neurons.
buffering
a
with
BAPTA-like
studying
of
in
to
consequent
a
of
process
of
their
result
the
include
death
a
buffering
been
Ca 2 -mediated
calcium-binding
loss an
phase
mechanism
on series
Ca 2
activation
to
major
mechanisms
ABSTRACT
artificial
vulnerability
of
capacity,
The
agonist-
of
the
under
excitotoxicity.
in
attributed
known
these
an
at
the
buffers,
vitro.
buffers.
of
loss
latter
neurotransmitter
potential
capacity
increase
the
events
increased conditions
Ca 2 +
have
neurons
as
of in
and
end either
We
may
a
to
on
excitotoxicity.
Ca 2 +
will
inhibition
to
proteins
We
large
depolarization-induced
been
ending
influence
of
buffer,
the
found
could
in
one
excitotoxicity
possibly
render
by
The
tested
the
the
to
Ca 2 +
homeostasis
of
variability
number
suggested
side,
the
excitotoxic
also
that
excitotoxic
increases
mechanism(s)
net
such
in
contrary
of
glutamate
them
expression
influx
of
neuronal
the
reflect
Ca 2
and
influx
be
neurons
CaBP
as
of
Excessive
hypothesis
achieved
in
more
the
and
calbindin-D28K
neuro
and
to
to
influx,
Ca 2 + the
in
of
the
stimuli.
stimulus. receptors
expression
play
large
death.
our
neurons
this
Ca 2 ,
resistant
of
calcium-
level
involved
Ca 2 +
CaBP
and
a
influx
by
that of III number of high affinity 2Ca binding sites of the buffer on the other. In neurons that express CaBP, an added vulnerability factor may be the consistently higher peak 2+Ca responses to glutamate receptor agonists and to depolarization observed in these neurons when compared to responses in neurons lacking CaBP. The biological effects of enhanced 2Ca buffering capacity, particularly using fast and mobile buffers, would be subject to the cell-specific nature of the spatial and temporal pattern of the 2Ca signal. Thus, the influence of 2Ca buffering on neuronal vulnerability may be difficult to predict on a theoretical basis particularly in view of our results indicating the large impact of many factors in the culture environment on the vital properties of neurons in vitro. iv
TABLE OF CONTENTS Pages
ABSTRACT ii
TABLE OF CONTENTS iv
LIST OF TABLES ix
LIST OF FIGURES x
ACKNOWLEDGMENTS xiii
DEDICATION xiv
1. INTRODUCTION 1
GENERAL INTRODUCTION 2
1A. INTRANEURONAL CALCIUM AND ITS REGULATION 4 I. 2+Ca influx through neuronal membranes 5 A. Voltage-gated 2Ca channels 5 B. Ligand-gated 2Ca channels 9 II. 2Ca efflux mechanisms 12 A. Ca-ATPase 12 B. 2Na/Ca2 exchanger 13 Ill. Intracellular 2Ca sequestration 15 A. Mitochondria 15
B. Endoplasmic reticulum 18
IV. Calcium-binding proteins 20 V. Intracellular role of 2Ca 20 VI. Recapitulation 21 lB. EXCITOTOXICITY 23
I. History and definition 23
II. Evidence for glutamate involvement 23 V
A. In vivo 23
B. In vitro 26
Ill. Phases of glutamate-induced neurotoxicity: time course and ionic dependence 27
IV. A three-stage model of glutamate excitotoxicity 29 V. Limitations of the excitotoxic hypothesis 30 VI. 2Ca as the common final pathway and alternative hypotheses 31 VII. Clinical implications 33
VIII. Therapeutic approaches to excitotoxic injury 34 IX. Recapitulation 35 ic. CALBINDIN-D28K 36
I. Introduction 36
II. CaBP History and distribution 36
Ill. Chemistry and molecular biology of CaBP 38
IV. CaBP expression in the central nervous system 41 V. Developmental expression of CaBP and related CBPs 42 VI. Modulation of CaBP expression 43
VII. Biological role of CaBP 45
1D. CALCIUM BUFFERS AND BIOLOGICAL SYSTEMS 47 Theoretical considerations 47
Buffers in non-biological systems 47
Buffers in biological systems 48 Microdomains and immobile buffers 48
1E. HYPOTHESIS AND OBJECTIVES OF THE STUDY 52 2. MATERIALS AND METHODS 54
I. Tissue culture 55 a. Fetal cultures 55 b. Postnatal cultures 59
3.
CHARACTERIZATION
II.
I. Sources
VII.
Preparation VI.
V.
IV.
Ill.
II.
Survival
Ca 2
Induction
Intracellular
Staining
FURA-2
Loading
Statistical
a.
d.
c.
b.
a.
a.
b.
a.
b.
f.
e.
d.
c.
b.
a.
c.
for
binding
Calbindin-D28k
Effects
Sensitivity
Age
Identification
Relocation
Viability
Exposure
Background
Kinetic
Fura-2
Test
Image
Fura-2
Principle
bFGF-treated
of
of
Materials
data
neurons
and
of
hippocampal
dependence
media
methods
Calcium
excitotoxicity
Acquisition
of
proteins
calibration
photography AM
analysis
analysis
assessment
OF
of
protocols
basic
to exposure
experiments
and
with
Loading
Fura-2
correction
HIPPOCAMPAL
of
glutamate-induced
cultures
Measurement
(CaBP)
fibroblast
BSS
neurons
in
BAPTA
of
neurons
cultured
calcium
neuronal
protocols
and
in or
growth
in
cultures
hippocampal
its
calibration
measurement
culture
NEURONAL
density
and
analogues
excitotoxicity
factor
perfusion
in
culture
(bFGF)
neurons
CULTURES
93
93
93
92
91
90
90
89
78
78
77
75
72
71
71
70 69
70
69
68
67
66
64
64
60
60
60 vi vii
b. Parvalbumin 94 c. Effects of bFGF on 2Ca binding proteins 94 Ill. 2+Ca responses in cultured hippocampal neurons 95 a. 21+][Ca in non-stimulated neurons 95 b. Effects of excitatory amino acids on 2[Ca 96 c. Depolarization-induced 2Ca fluxes j 98 d. Effects of the absence of extracellular 2Ca 100 e. Effects of other 2Ca release agonists 101 Discussion 103 4. THE EFFECTS OF ARTIFICIAL BUFFERS ON NEURONAL Ca2+ RESPONSES 134 I. BAPTA effects on short 2Ca transients 136 II. Baseline 2Ca changes during the BAPTA loading procedure 138 III. Buffer effects on extended 2+Ca responses 140 IV. BAPTA effects on 2Ca levels during excitotoxic glutamate exposure 142 Discussion 144 5. CALBINDIN-D28K EXPRESSION AND NEURONAL 2Ca RESPONSES 163
I. The Association beteen CaBP expression and resting intraneuronal [Ca ] 164 Il. The Association between CaBP expression and transient increases in 21[Ca in cultured fetal hippocampal neurons 164 a. jNMDA-induced 2Ca transients 164 b. Depolarization-induced 2Ca transients 166 Ill. The Association between CaBP expression and transient exposure of cultured postnatal hippocampal neurons to NMDA 167 IV. The Association between CaBP expression and prolonged 2+Ca responses 169 Discussion 170
ABBREVIATIONS
BIOGRAPHICAL
BIBLIOGRAPHY
7.
6.
GENERAL
EXCITOTOXICITY
VI.
Discussion
Ill.
II.
I.
Excitotoxicity
Excitotoxicity
The
Effects
a.
b.
neurotoxicity
c.
b.
a.
analogues
d.
c.
b.
a.
DISCUSSION
association
INFORMATION Fetal
Postnatal
Correlation
Fetal
Postnatal
Effects
[Ca 2 1
Fetal
Postnatal
of
neurons
bFGF
cultures
cultures
cultures
IN
in
of
in
changes
HIPPOCAMPAL
cultures
cultures
‘naive’
cultures
neurons
between
treatment antagonists
AND
with
CONCLUSIONS
Ca 2
under
neuronal
loaded
CaBP
on
changes
excitotoxic
glutamate
NEURONAL
immunoreactivity
with
cultures
BAPTA
in
BAPTA-loaded
toxicity
conditions
CULTURES
and
and
its
glutamate
259
241
238
220
201
199
198
198
198
198
197
197
196
195
195
194
193
193
192 VIII ix
LIST OF TABLES Pages 2.1 Problems associated with fura-2 fluorimetry, and their remedies 87 2.2 Outline of the cresyl violet staining method. 88 4.1 The percentage chaes in the kinetic parameters of transient NMDA-induced Ca responses in neurons loaded with BAPTA or with DMB. 162 5.1 Comparison of the kinetics of the Ca responses in fetal CaBP CaBPN 2 and neurons exposed to NMDA. 188 5.2 Comparison of the kinetics of the 2Ca responses in fetal CaBP and CaBPN neurons depolarized by exposure to 50 mM 0[K] 189 5.3 Frequencies of the rlative magnitudes of kinetic parameters. in CaBP’ and CaBP’ fetal neurons. 190 5.4 Comparison of t[’ie kinetics of he 2Ca responses in cultured 4-day postnatal CaBPH and CaBP’ neurons exposed NMDA. 191 x
LIST OF FIGURES Pages
2.1 The excitation spectrum of fura-2. 81 2.2 Changes in fura-2 fluorescence in a typical 2+Ca response. 82 2.3 Illustrated diagram of the imaging hardware. 83 2.4 Schematic diagram of the perfusion chamber. 84
2.5 Schematic diagram of a perfusion-head assembly. 85 2.6 Kinetic parameters used in the description of 2+Ca responses. 86 3.1 Postnatal neuronal cultures stained with (a) NSE and (b) GFAP. 109 3.2 Neuronal survival in vitro varies with the different culture conditions used. 110
3.3 BFGF enhances neuronal survival in vitro. 11 2 3.4 Survival and number of CaBP cultured neurons varies in different culture types. 113 3.5 BFGF increases neuronal CaBP expression in vitro. 115
3.6 In fetal face-down cultures, bFGF effect on CaBP expression is enhanced in the presence of media conditioned by postnatal neurons. 116
3.7 NMDA induces a oncentration-dependent increase in intraneuronal [Ca 1. 117 3.8 APV and 2Mg abolish completely NMDA-induced 2Ca responses. 11 9 3.9 NMDA-induced 2Ca responses are modulated by changes in the extracellular pH. 120 3.10 Quisqualate is highly potent in inducing intraneuronal 2Ca responses. 121 3.11 Quisqualate-induced 2Ca responses show that QUIS does not have a homogeneous potency on hippocam pal neurons. 122 3.12 NMDA-induced 2+Ca responses in neurons are more homogeneous than Quis-induced responses. 123 3.13 Nifedipine inhibits depolarization-induced 2Ca responses. 124 3.14 Multiple types of VGCC contribute to HK-induced 2Ca responses in cultured hippocampal neurons. 125 xi
3.15 HK- and NMDA-induced 2Ca responses are modulated by changes in extracellular pH. 126 3.16 HK-induced and EAA-induced 2Ca responses are abolished in 2Ca-free media. 127 3.17 The effects of Ca-free medium on NMDA-induced 2Ca responses. 2 128 3.18 The effect of extracellular 2+Mg on the calcium responses to NMDA and ACPD isomers. 129
3.19 MK8O1 inhibited ACPD induced rises in neuronal free calcium. 130 3.20 An example of a single cell where MK8O1 did not abolish the calcium response to 5 mM 1S,3R-ACPD. 131
3.21 Caffeine induces only a small increase in intraneuronal 132 4.1 The effects of BAPTA loading on short-duration 2Ca transients. 150 4.2 Changes in ]2[Ca during exposure to BAPTA-AM. 151 4.3 Heterogeneity of the responses to BAPTA-AM exposure. 152 4.4 Loperamide reverses BAPTA-AM induced increases in 21.[Ca 154 4.5 Ca buffering and buffer-AM induced increases in 2[Ca are independent phenomena. 155 4.6 Thei effect of Ca buffering on short- and extended- 2+ duration 2Ca responses. 157 4.7 The effect of 2+Ca buffers on ‘excitotoxic’ exposure to glutamate. 160 5.1 NMDA-induced 2+Ca responses are larger and longer-lasting in CaBP fetal hippocampal neurons. 179 5.2 Larger and longer-lasting +Ca responses are observes in CaBP 2 + neurons even in the absence of initial high resting Ca levels. 180 5.3 Comparison of the mean 2+Ca response in CaBPN and CaBP neurons following exposure to 2OpM of NMDA for 25 seconds. 181 5.4 Depolarization-inducçd increases in +][Ca are larger and recovery is slower in compared to CaBPN2 CaBP’ fetal neurons. 183 5.5 Mean [Ca in cultured 4-day postnatal hippocampal neurons (8-DIV)2 exposed to 20 pM NMDA for 25 seconds is larger in CaBP(H] CaBPN neurons than in neurons. 185 xl’
6.1 Glutamate toxicity in face-down fetal neurons: protective effect of extracellular 2Mg 210 6.2 Glutamate toxicity. in postnatal culture. 211 6.3 Changes in 2[Ca during glutamate toxicity. 212 6.4 Time course i of glutamate-induced neuronal death is widely variable in different cells. 213
6.5 Neurons hat collapse early in response to glutamate have higher peak Ca + responses. 214
6.6 BAPTA and DMB promote glutamate toxicity in fetal neurons. 215 6.7 BAPTA shifts the concentration-response curve of glutamate toxicity to the left. 216
6.8 BAPTA and DMB promote glutamate toxicity in postnatal neurons. 217 6.9 CaBP(H postnatal neurons are more sensitive to glutamate toxicity. 218
6.10 Effect of bFGF treatment on glutamate toxicity in postnatal neurons. 219 7.1 Simulated 2Ca changes in fetal neurons control, BAPTA-loaded and CaBP, in response to a short-duration exposure to NMDA. 237 XIII
ACKNOWLEDGEMENT
I would like to express my gratitude to Dr. Ken Baimbridge for his
continuous help and advice throughout all the stages of the research work I have undertaken for the last 5 years. During the numerous discussions we had, his criticism and insightful comments were invaluable in formulating the ideas presented in this work. His support, however, extends beyond the science arena and his guidance has been extremely valuable for me as a foreign student settling in and trying to understand a new society. His friendliness and considerate attitude have made my stay here a very enjoyable experience.
To Miss Stella Atmadja I owe a lot of thanks. The excellence of her technical expertise in tissue culture and histological techniques cannot be overstated. Her supportive attitude and enthusiasm for new ideas, including those that impose a lot more demand on her time, were always encouraging.
I also want to express my deep appreciation to Dr. John Church and Dr. Steve KehI for the time and effort they spent proof-reading the manuscript of the thesis and their extensive and invaluable comments and suggestions. Many thanks are also due to the members of my advisory committee for their helpful comments and appraisal of my work, from a research proposal to a completed thesis.
I am very grateful to the technical and secretarial staff of the Department of Physiology at the University of British Columbia for their continuous help and support.
No doubt that my greatest debt and ultimate gratitude should go to my wife, Dr Lamice El-Kholy, for her endless dedication and sacrifice, and her unlimited support I have been receiving over the years. Her effort in proof reading the manuscript and her critical comments are also highly appreciated. xiv
DEDICATION
This work is dedicated to my wife Lamice, may God bless her with happiness and success, and to the joy of my life, my sons Omar and Mostafa. 1
CHAPTER 1
INTRODUCTION 2
GENERALINTRODUCTION
The experimental work presented in this thesis will be concerned primarily with investigations of the role of artificial 2+Ca buffers and neuronal calcium- binding proteins that have been proposed to act as neuronal 2+Ca buffers, on transient or prolonged changes in 2+][Ca induced by membrane depolarization or specific ligand-receptor interactions.
Inside the cell, 2+Ca can function as a charge carrier and as an intracellular second messenger. In the latter role, changes in 21[Ca are sensed by one or more of a number of high affinity 2Ca +bindingi sites, such as those found on the ubiquitous protein calmodulin, and then translated into a cellular response. To some extent the binding of 2+Ca to these sites can also be considered as 2Ca buffering since a rise in 21[Ca will be limited by the removal of 2Ca from the ionic pool within the cytoplasm.j With proteins such as calmodulin this buffering action, while not necessarily unimportant, is essentially secondary to their enzyme modulatory actions. However, there are members of the Ca-binding protein family for which a high capacity for binding (and buffering)2 2Ca has been demonstrated and which appear not to modulate enzyme activity. It is therefore conceivable that the latter group of proteins have evolved as natural 2Ca buffers. This possibility, and the ensuing biological consequences will be discussed after first reviewing our current understanding of the mechanisms involved in the regulation of intraneuronal 2Ca The potential role of 2Ca buffering, both natural and artificial, in neurons. during periods of prolonged neuronal stimulation which, if uncontrolled, can lead to cell death will be examined. This form of neuronal 3 death, which has been termed excitotoxicity, will therefore be reviewed with an emphasis on the role played by intraneuronal 2Ca Finally, and in view of this laboratory’s interest in 2+Ca buffering, a theoretical. framework describing the concept of 2Ca buffering and its implications for biological systems will be presented. components regulating membrane binding inside individual simultaneously, Ca 2 + integrated terminals morphologically particular several of gradient, activation, and important recognized Ca 2 even In While A regulatory the order sites change intracellular binding represent [Ca 2 j 1 components cell importance cell, and system cellular must modulation vital for 1A. contributing or to death shaping the many sequestered in maintain and and in eventually INTRANEURONAL mechanisms. molecules is intracellular events is vast all specialized sequestration tightly (Choi, each functionally. not years living in that an of majority neurons. fully to long such membrane compartment intracellular regulated 1988). be as regulate with cells, intracellular inside understood term ionized an extruded as compartments Coordination of diverse coordinated important cell and Dendrites, Neurons membrane-bound the In CALCIUM balance, [Ca 2 ] via ionic the division calcium Ca 2 release ‘extra is into Ca 2 + physico-chemical the although face endowed conductances, are signal than the interplay Ca 2 + signal of somata, Ca 2 ’ AND regulation that mechanisms, and concentration homeostasis, of very such extracellular a about for differentiation, entering perform more will huge ITS with heterogeneous is an organelles. the of axons associated now REGULATION their extremely of multiple is inward an cell control properties. known diverse [Ca 2 ] 1 through and optimized be and and ([Ca 2 +1) integration. space. response discussed. a electrochemical those presynaptic influx The of enzyme with large about polarized functions is cells, many the While of has subset Ca 2 to routes, number plasma the both injury, been and of 4 5
I. Ca INFLUX THROUGH NEURONAL MEMBRANES
2+Ca influx through neuronal membranes can take place via two major classes of 2+Ca channels. The first are the voltage-gated 2+Ca channels (VGCC) that are activated by membrane depolarization, and the second are 2Ca channels which are dependent on binding of extracellular, biologically active molecules, such as neurotransmitters, for their activation; the ligand gated 2Ca channels (LGCC). A. Voltage-gated 2Ca channels Different types of voltage-gated 2Ca channels have been identified and well characterized. These include the four well established classes of VGCC: L-,
T-, N-, and P-channels as well as the recently reported Q- and R- channels. Identification of the type of 2Ca channels is based on biophysical characteristics (gating kinetics and ionic conductance) and pharmacological criteria. Q and R types have been expressed on Xenopus oocytes using variants of 2+Ca channels a1 subunits, namely alA and doe-i, and were recently also reported in cerebellar granule cells (Zhang eta!, 1993). For the purpose of this review, only a brief description of the basic pharmacological and biophysical characteristics of the more established L-, N-, P- and T channels will be presented.
I) L-type channels: In 1980, Llinas and Sugimori, identified a high-voltage activated (HVA) 2+Ca conductance in mammalian Purkinje cells. Since then, the presence of this HVA 2+Ca current, also known as L-type current, has been confirmed in many other preparations. L-type current is characterized by slow activation with a threshold of -50 mV, half-activation at -20 mV, and a large unitary conductance (25-27 pS in 100 mM 2j.Ba With 2Ba as the 6 charge carrier, current through L-type 2+Ca channels inactivates very slowly (r > 500 ms). The channel is more permeable to 2+Ba than to 2+Ca and is blocked by 2Cd and ,2Ni being more sensitive to the former. An extracellular dihydropyridine (DHP)-binding site allows for pharmacological modulation of L-channels. While Bay K8644, a DHP agonist, evokes sustained activation of the L-channels, these channels are blocked by other DHP derivatives such as nifedipine and nimodipine in the low micromolar range, and by other organic blockers such as Verapamil and flunarizine. Some neuronal L channels, e.g. in chick dorsal root ganglion (DRG) neurons, but not muscle fiber L-channels, can also be blocked by the omega-conotoxin GVIA; an antagonist generally considered to be a selective N-channel blocker (Tsien et a!, 1988).
ii) N-type 2Ca channels are also HVA channels that have been identified only in neurons. The single channel conductance is lower than that of L channels (13-15 pS in 100mM i2Ba while their activation and inactivation kinetics are faster although both conductance and kinetics of N-channels vary widely in different preparations.. For example, the inactivation time constant is
50-80 ms in chick DRG neurons and approximately 500 ms in rat sympathetic neurons (Tsien eta!, 1988). It has been suggested that N-channels are preferentially distributed in the presynaptic terminals and are believed to be, at least in part, responsible for 2+Ca flux that triggers neurotransmitter release
(Miller, 1987; but see Luebke et a!, 1993, and Takahashi and Momiyama, 1993). N-channels are resistant to DHP antagonists but are relatively selectively blocked by the marine snail polypeptide toxin, omega-conotoxin GVIA. Like L-channels, N-channels are more permeable to 2+Ba than to 2+Ca and are blocked by 2Cd and 2Ni (Tsien eta!, 1988). 7
iii) P-channels: In addition to L- and N-types of HVA voltage-gated 2Ca channels, a third type has been initially identified in Purkinje neurons using a polyamine toxin from the funnel-web spider (FWS), Agelenopsis aperta (Llinas etal, 1992). These channels, named P-channels, show little or no voltage- dependent inactivation and are insensitive to DHP antagonists and omega conotoxin GVIA but are selectively blocked by omega-Aga-IVA fraction of FWS venom with a Kd of 2-10 nM (Mintz eta!, 1992). Single channel studies in
Purkinje cells showed the existence of 3 subconductance states: 10, 14 and 19 pS in the presence of 80 mM 2Ba + (Llinas et at, 1992). P-channels are located on neuronal somata and dendrites in many other CNS regions including the dentate gyrus, CAl and CA3 region of the hippocampus as well as in spinal and dorsal root ganglion neurons (Mintz et a!, 1992; Takahashi and Momiyama, 1993). Recent reports have also suggested that P-channels may play an important role in mediating 2+Ca fluxes responsible for neurotransmitter release in spinal, cerebellar and hippocampal neurons (Luebke et a!, 1993; Takahashi and Momiyama, 1993)
iv) T-channels are low-voltage activated (LVA) 2Ca channels with an activation threshold of about -60 mV. They exist in both neuronal and non neuronal cells and have a small single channel conductance (7-9 PS). Their most prominent feature is the transient nature of their activity, hence the name
‘T’. T-channels display a strong, and purely-voltage dependent inactivation at holding potentials positive to -50 mV. In dissociated rat hippocampal CAl neurons held at -100 mV, activation of T-channels starts at -60 mV and reaches a peak at -30 mV. T-currents inactivate with r of 25-30 ms (Takahashi etal, 1989). T-channels are equally permeant to 2+Ba and 2+Ca and are very sensitive to blockade by 2Ni though less sensitive to .2Cd Like L-channels 8
T-channels are blocked by verapamil but are more sensitive to the blocking effect of phenytoin. They are also sensitive to amiloride (Tang et a!, 1988) and medium-chain alcohols (Llinas and Yarom, 1986).
Modulation of VGCC 2+Ca channels are targets for modulation by a variety of neurotransmitters and metabolites. For example, noradrenaline and isoprenaline, acting via r-adrenergic receptors, enhance the activity of L- and N-channels. The path for activation involves cAMP-sensitive protein kinase (Gray and Johnston, 1987). Adenosine has been reported to inhibit N-channel activity (Madison et a!, 1987) while acetyicholine in rat sympathetic neurons, was found to inhibit N- , but not L- or T-type of 2Ca channels. In peripheral sensory neurons, many neurotransmitters (e.g., GABA, dopamine, serotonin), acting on specific subsets of receptors, have been found to inhibit the activity of both L- and T-type 2Ca channels. Cyclic-AMP, diacyl-glycerol (DAG), protein kinase-C as well as various G-protein subunits have been implicated in such modulation.
Aside from their dependence on the membrane voltage, the gating of VGCC can be profoundly modulated by the increase in 21[Ca The -2Ca dependent inactivation of 2+Ca channels can be either .]irreversible, resulting +activated from a Ca proteolysis of the channel molecules, or reversible as a result of2 channel dephosphorylation by a Ca-dependent phosphatase (Chad, 1989). In addition, a recent study presented2 evidence that a direct, non enzymatic, mechanism of 2Ca +.dependent inactivation of VGCC also exists (lmredy and Yue, 1994). 9
Spatial heterogeneity and voltage responses mediated by VGCC Several lines of evidence from electrophysiological studies performed at various developmental ages in cultured neurons and hippocampal slices suggest spatial heterogeneity of the distribution of VGCC. For example, neuronal somata may have a larger proportion of T-channels than L- and N- channels, while in neuronal processes the reverse is true. This spatial heterogeneity may also reflect the functional specialization of the different types of VGCC. Sustained HVA 2Ca currents may underlie the 2Ca dependent slow spikes while the T-currents are predominant in sub-threshold and pacemaker potentials (Brown eta!, 1990).
B. Ligand-gated 2Ca channels (LGCC)
In contrast to VGCC, LGCC have little or no sensitivity to membrane potential and are less selective for 2+Ca over monovalent cations. The two major types of LGCC are those coupled to the NMDA-subtype of glutamate receptor or to nicotinic acetylcholine receptors (nAchR). The former is more relevant to the present work and will be discussed in detail.
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Two major classes of GluRs exist: lonotropic GluRs (iGluR) with glutamate-activated cation channels, and metabotropic receptors (mGluR) that are not linked to an ion channel but activate phospholipase-C (PL C), and possibly other G-protein mediated processes. lonotropic GluRs are further classified into NMDA-, AMPA- and Kainate-receptor subtypes according to their sensitivity to the corresponding agonist. A different class of GIuR is the AP4 receptor subtype which is a presynaptic GIuR involved in the autoregulation of glutamate release (Monaghan eta!, 1989). 10
I) NMDA receptor-associated 2Ca channels: In 1986, MacDermott et a! reported that NMDA receptor-operated channels were particularly permeable to 2+Ca compared to non-NMDA receptor-linked ionophores. Later analysis using the Goldman-Hodgkin-Katz constant field equation suggested a Ca’Na of 0.15 for AMPA/Kainate receptors compared to 10.5 for NMDA receptors (Mayer and
Westbrook, 1987). However, the fractional contribution of 2Ca + to the cation current through AMPA/KA and NMDA receptor-linked ion channels has recently been estimated to be 1.5% and 7 % of the total current, respectively
(Schneggenburger eta!, 1993). The NMDA channel conductance is relatively high (45-50 pS with a 38 pS sub-conductance state) and has a unique pharmacological profile. At physiological concentrations, extracellular 2Mg is a potent blocker of NMDA receptor-linked open channel. This open channel blockade is, however, a voltage dependent one, and at depolarized membrane potentials the channel blockade by 2Mg is relieved giving the I-V relation for NMDA receptor channels in the presence of 2+Mg a region of negative slope conductance (RNSC) prominent at membrane potentials less than -30 mV. This RNSC is absent if I-V data are recorded in the absence of extracellular 2+Mg but in both cases the reversal potential for ‘NMDA is sensitive to extracellular 2Ca reflecting the high NMDA-receptor permeability to 2Ca Another class of open channel blockers bind to the PCP-site within the .pore. These include dizocilpine (MK-801), ketamine and dextromethorphan.
An interesting modulatory site on the NMDA receptor is the glycine binding site. The strychnine-insensitive binding of glycine is an absolute requirement for NMDA receptor activation (Johnson and Ascher, 1987). At physiological concentrations of glycine, the NMDA-glycine site is saturated which raises questions about the relevance of glycine to the modulation of 11
NMDA receptor activity under physiological conditions (but see Kemp and
Leeson, 1993). The NMDA receptor is also subject to modulation by a number of other factors including 2+,Zn polyamines, p1—I,the redox state, nitric oxide, arachidonic acid and protein kinase-C with evidence for allosteric interactions between different sites, e.g., glutamate and glycine binding sites (Wroblewski and Danysz, 1989). The extensive characterization of the role of NMDA receptors in various physiological and pathological situations has been aided by the early availability of selective pharmacological antagonists, including the competitive antagonists at the glutamate/NMDA binding site such as the amino phosphonovalerate and amino-phosphonoheptanoate (APV and APH; Davies et a!, 1981). At the present time, NMDA receptor activity has been implicated in a large number of major neuronal processes from growth cone development and neuronal differentiation to activity-dependent adaptation and neuronal death. In all these situations, the role of NMDA receptors is believed to be dependent on its 2+Ca conductance. ii) Non-NMDA receptor-associated 2+Ca channels: These channels have traditionally been considered impermeant to any significant 2Ca fluxes and the increases in 2[Ca observed with non-NMDA receptor activation were considered toj be secondary to depolarization of the target cells and the consequent recruitment of VGCC. However, Murphy and Miller (1989) have demonstrated that VGCC activation may not account for all the 2[Ca in ] increases induced by activation of non-NMDA receptors various preparations.
1mb eta! (1990), using cultured hippocampal neurons also found that a subset of kainate receptors was characterized by high 2+Ca permeability and an inwardly rectifying I-V relationship. The authors labelled these as type-Il kainate receptors to distinguish them from the ‘usual’ type-I kainate receptor 12
channels which are not permeable to 2+Ca and have a linear I-V relationship. Recent studies on the molecular biology of mammalian glutamate receptors are beginning to identify the molecular basis of this receptor heterogeneity (Sommer and Seeburg, 1992).
iii) Metabotropic glutamate receptors: In addition to iGluR-mediated increases in 2+],[Ca metabotropic GluRs have been reported to increase 2[Ca via activation of a G-protein/PL-C pathway with subsequent release of 2Caj from 3lP sensitive stores (Sugiyama eta!, 1987). Although quisqualate is the ‘typical’ metabotropic receptor activator, NMDA has also been linked to the mobilization of 2Ca from 31P sensitive intracellular pool (Harada eta!, 1992).
II. Ca EFFLUX MECHANISMS
After a period of influx, the restoration of cellular 2+Ca homeostasis requires all the 2Ca that entered the cell be extruded. This is achieved, against a huge electrochemical gradient, at the expense of energy stores, by both primary and secondary active transport. A. 2Ca-ATPase Direct coupling between ATP and 2Ca extrusion is mediated by a uniporter pump 2(Ca +ATPase) that is a member of the plasma membrane ATPases such as Na/K ATPase and K/H ATPase which are inhibited by vanadate (Kd=2-3pM) and 3La on the cytoplasmic side. A minimum concentration of 0.2 mM 21+Mg is an absolute requirement for the function of the plasma membrane Ca-ATPase. The 2Ca pump is distinguished from other membrane ATPases2 by a higher molecular weight (140 kDa compared to 100 kDa for other pumps), and by its sensitivity to calmodulin. Calmodulin 13 binding causes a 30-fold increase in the affinity of the pump to 2[Ca and a 10-fold increase in ] (Kd=0.2pM) the Vmax. Sensitivity to calmodulin and a 1:1 2Ca:ATP stoichiometry distinguish the plasma membrane Ca-ATPase from organellar membrane 2+Ca pumps which are insensitive to calmodulin2 and have a 2:1 2Ca:ATP ratio (Carafoli, 1987). Besides calmodulin, a multitude of factors contribute to the regulation of plasma membrane 2Ca pump activity. Phosphorylation of the pump by cAMP-dependent protein kinase results in an increased affinity for 2Ca while phosphorylation by protein kinase-C increases its Vmax (James et a!, 1989; Smallwood eta!, 1987). Poly-unsaturated long chain fatty acids as well as membrane phospholipids of the inositol phosphate pathway: phosphatidyl inositol and its mono- and di-phosphate derivatives (P1, PIP and )2PIP promote Ca-ATPase activity unlike the 2PIP hydrolysis products, 31P and DAG, suggesting2 a functional linkage between Ca-ATPase activity and inositol phosphate pathway activity (Penniston, 1982).2
The sensitivity of membrane 2Ca +.ATPase to calmodulin links an increase in ]2[Ca to 2Ca pump activation. However, this process is probably too slow to contribute significantly to the recovery of fast 2+Ca transients and, in effect, pump activity may reflect the time-averaged 2Ca level in the cytoplasm rather than the instantaneous 2Ca level. Also the small capacity of the pump and its high affinity for 2Ca makes it more suitable for the fine tuning of resting intracellular 2+][Ca rather than the fast recovery required after large 2Ca loads which are probably handled better by the 2Na/Ca exchanger. B. 2+Na+/Ca exchanger The 2NaICa exchanger is a low-affinity high-capacity bi-directional
types
analogues
exchanger
displaced
from
and
Allen the
and
hundred
phosphorylation
analogues
Ca 2 ,
that
influx),
and
process
potential
the
[Ca 2 the
antiporter
electrochemical
cannot
chelation
activity
electrochemical
DiPolo,
Little
this
the
In
+1,
While
and
of
reducing
reverse
[ATP]
antiporters
nanomolars
is
transported
[Na+]
is
in
stems
Baker,
of
selective
although
and
system
3:1
not
be
symmetric
the
and
1984).
the
of
was
displaced
other
(Na+:Ca 2 j,
the
and
mode
Na 0 :Na
the
from
of
Ca 2 1
the
latter
1985).
that
the
case.
found
pharmacological
(Taglialatela
gradient
membrane
La 3 +
Kd
operating
gradient
of
Ca 2
nucleotide
the
(i.e.,
show
operation
exchanger
with
from
couples
‘regulatory’
by
This
fact
and
to
In
Ca 2 +
binding
operating
high
EGTA
enhance forward
is
selectivity
30
i.e.
of
regulatory
that
amiloride
mode
highly
potential.
ionic
pM
phosphates
Ca 2 +
etal,
the
was
Na
influx
may
the
abolishes
site.
[Ca 2 j
to
control
exchanger
(forward/reverse)
operating
movements
initially
favorable
significantly
mode.
(100
1990).
and
0.75 stoichiometry
be
for
and
can
Ca 2
Regulatory
The
required
Na+.
Na+/Ca 2 +
mM)
pM.
Na+
is
cannot
block
is
the
assumed,
dependence
required
available
site
mode
activity
(DiPolo
while
reverse
efflux),
Three
in
Non-hydrolysable
its
the
is (DiPolo
replace
a
Ca 2
of
activity
intracellular
(i.e.,
direction
exchanger
exchanger
transported
of
for
and
later
is
the
over
major
mode
a
the
electrogenic
is
exchanger
Ca 2 +
minimum
on
and
ATP
exchanger
Beauge,
evidence
not
Na+ICa 2 +
and
exchanger:
factors
membrane
activity
determined
Beauge,
indicating
transported
efflux
affinity
over
some
and
Ca 2
ATP
of
1993;
function,
determine
distinct
indicated
other transport
(Baker
even
several
and
1993).
for
can
that
by
Na+
if
be 14 15 In spite of its high capacity, the low affinity of the exchanger for 2Ca (0.75 pM) means it cannot contribute to the regulation of resting intracellular 2+Ca levels but its role in recovery after large intracellular 2+Ca loads is widely accepted. Also, there is no evidence for its operation in the reverse mode in vivo although an interesting model has been presented by Blaustein (1988a) suggesting that during an action potential, changes in membrane potential and [Na+] could result in a biphasic operation of the exchanger promoting 2Ca influx at the beginning and 2Ca extrusion during later stages of the electrical response. The net result would be to enhance the 2+Ca signal with a faster recovery of 2+].[Ca However, this interesting model has not yet received experimental support (DiPolo and Beauge, 1993).
Mill (1993), using Heilsoma neurons in vitro, reported a large variability between different neuronal types in their capacity to recover from ‘equal’ 2+Ca loads induced by exposure to the 2Ca ionophore, A23187. The author attributed this variability to the differences in the 2Na/Ca exchanger activity in those neurons and demonstrated that interneuronal variability was markedly reduced, though not completely abolished, by removing Na from the extracellular environment. The author suggested that different neuronal 2+Ca set points, also observed by Guthrie eta? (1988) during 2Ca responses, may be attributed to different exchanger activity in different neurons.
III. INTRACELLULAR 2Ca SEQUESTRATION
A. Mitochondria
Mitochondrial respiratory chain enzyme activity is associated with pumping of H from inside the mitochondrial matrix into the cytosol thus generating a high trans-mitochondrial potential gradient of 150-1 80 mV with
effect
released of
calcium/phosphate slightly
McCormack, capacity
with
[Ca 2 jm certain
cytosolic
their
between
intramitochondrial
exchanger
generation
potential
proportion
compartment
cause
in
the
1966)
1969).
Ca 2 +
the
inside
the
response
of
Once
transport
reverse
this
level
higher.
into
According
at
is
Ca 2
the
(i.e.,
the
energy
[Ca 2 j
of
saturated
that
to
to
[Ca 2
high
inside
of
mitochondria.
the
1985
properties
the
the
the
using
net
mode
to
influx
[Ca 2 +] 1 ,
has
Once
+1
electrical
of
cytosol
across
to
mitochondrion
[Ca 2 +] 1
third
accumulation
trans-mitochondrial
ICa 2 ]
the
and
complex
Ca 2 +
above
to
a
be
an
to
and
resulting
sigmoidal
cytosolic
the
mitochondria,
accompanied
power
uniporter
1990;
generate
of
the
known
as
Ca 2 +
from
and
and
the
chemiosmotic
the
(lCa 2 jm)
the
Excess
mitochondrial
whose
set
[Ca 2 jm
of
McCormack
chemical
mitochondrial
in
the
Ca 2
as
other
accumulates
response of
being
[Ca 2 ].
the
Ca 2
ATP.
point
the
Ca 2 +.
cytosol
nature
mitochondrial
dynamic
by
+.ATPase
Ca 2 +
and
Ca 2 +
exceeds
flux
set-point,
negative
respectively,
The
can
gradient
larger
model
activity
a
membrane
Under
is
equals
to
and
can
saturable
be
same
regulatory
uniporter
not
inside
the
equilibrium
changes
the
considered
Denton,
of
whose
be
(Mitchell
exchanger
drives
yet
extreme
intramitochondrial
curve
zero)
calcium
driving
ATP
set
extruded
the
causes
clear
can
capacity.
point,
and
mechanisms
activity
generation
mitochondria
although
a
in
in
1990).
and
membrane
force,
between
be
conditions,
[Ca 2 ]m.
can
(Denton
relation
as
the
activity
changes via
the
diverted
Moyle,
a
be
exchanger
increases
Ca 2 +
however,
a
The
The
exchanger
the
slowly
Na+/Ca 2 +
(Mitchell,
to
and
[Ca 2 ]
restore
can
H-ATPase
differences
net
Up
[Ca 2 ]m
as
1967
in
from
buffering all
a
cope
to
influx
the
in
in
can
a
and
ATP
and
is 16 17 2[Ca to below the set point, thus causing prolongation of the 2Ca signal andi a delay in the recovery process.
Although the set point has not been firmly established in vivo, evidence obtained from cultured neurons suggest that it is in the range of 0.75 iiM of cytosolic 2Ca corresponding to approximately 1 pM 2]m.[Ca Thayer and Miller (1990), demonstrated that the mitochondrial uncoupler CCCP caused a marked increase in 2[Ca during the plateau phase of 21[Ca observed during a 2-mm K-induced j depolarization indicating that the plateauj level was set by mitochondria and probably corresponded to the mitochondrial set-point. In
Na+free medium, the set point was reduced in accordance with the prediction that the relative activities of 2Na/Ca exchanger and Ca-ATPase determined the mitochondrial set point. The increase in22[Ca+]m may serve a metabolic purpose since at least three major mitochondrial enzymes are regulated by 2Ca with a peak activation at 1 pM 2im.[Ca These enzymes are pyruvate dehydrogenase, oxoglutarate dehydrogenase and NAD-isocitrate dehydrogenase. Thus, the increase in 2[Ca+]m, within limits, is associated with increased production of ATP. With the slow mitochondrial transport systems, compared to the 2+Ca signal, the activation of mitochondrial metabolism follows a time-averaged 2+Ca signal and is independent of changes in ADP/ATP and NAD/NADH which are the major stimuli for mitochondrial metabolism.
Because of the low-affinity of the mitochondrial 2Ca buffering system, the physiological significance of this buffering has been debated. Levels of 2[Ca that are necessary for mitochondria to function as a 2+Ca buffer, i.e., 2i+][Ca above 1 pM may not be reached in neuronal somata under normal conditions. However, in presynaptic terminals, smaller dendrites and dendritic
to
and share
as thapsigargin-sensitive
the
mitochondria
as
found
have
mitochondrial
detected.
and
1980,
B.
neurotransmitter
that
work
sufficient
into
and
spines,
play
ryanodine
methyixanthine-sensitive
in
two
Endoplasmic
dantrolene
ruthenium
the
The
a
mitochondrial
a
dendrites.
In
been
many
of
in
McGraw
prolonged
role
many
systems
evidence
response
submembraneous,
Nishimura
ER
numbers
These
associated
in
characteristics
associated
and
suggesting
tissues,
the
stores
red-resistant
(Palade,
et
In
Reticulum
their
(McGraw
maximal
release
non-mitochondrial
for
modification
al, (i.e.,
Ca 2
many
(1986)
and
discussed
such
1980).
but
anatomically
cytoplasmic
Ca 2
transient
Ca 2 +
in
1987).
the
and
cases buffering
resistant
response
close
high
and
eta!,
with
perinuclear
release
Ca 2 +
release
presence
possibly
They
stores
of
Biscoe
above
they
Ca 2 +
The
sarcoplasmic
proximity
1980).
very
the
sequestration
high-affinity
may
to
with
have
and
provided
stores
can
ER
exist
are
temporal
levels
mitochondrial
intense
(Brinley,
be
and
of
and
stores contribute
the
the
be
A23187
a
some
involved
to
in
Duchen
diffuse
have
triggered
synaptic
inhibition
suggests
endoplasmic
close
the
that
reticulum
supra-maximal
can
relationship
1980;
functional
(0.1
a
mechanisms
source
releasable
mitochondria
subcellular
much
in
association
to
also
(1990)
pM
uncouplers.
terminal synaptic
by
the
of Verma
that
be
(SR)
or
of
release
high
lower
regulation
reticulum
relationship
mitochondria
led
emptied
less), Ca 2 +
between
and
Ca 2 +
et
stimulus
[Ca 2 ] 1 ,
regions,
distribution
have
plasticity.
to
with
capacity
a!,
are
by
vanadate-
ATP-dependent
the
entry).
ER
stores
1990)
been
procaine
present
(Henkart
by
of
the
stores
suggestion
translated
between
as
i.e.
exposure
than
could
stimulus
The
well
being
and
such
and
also
in
the 18 19 Ca-induced 2Ca release (McBurney and Neering, 1987). Mapping the distribution2 of ER stores using ryanodine binding proved that many peripheral and central neurons show some degree of ryanodine binding but it is exceptionally high in Purkinje cells (Ellisman eta!, 1990).
As high-affinity storage sites with specific release mechanisms, these stores can play the double role of a 2+Ca buffer and a 2+Ca signal generator depending on their state of filling. For example, their role as possible 2Ca buffers has been illustrated by the ability of caffeine treatment to increase the 2+Ca buffering capacity for subsequent 2+Ca loads in sympathetic neurons (Friel and Tsien 1990). C. 31P-sensitive intracellular 2+Ca release In addition to the caffeine-releasable intracellular 2Ca lP-sensitive intracellular 2+Ca stores have been identified in many ,cell types.3 These stores have occasionally been named calciosomes (Satoh et a!, 1990; Hashimoto et a!, 1988) and they share many characteristics with other ER associated 2Ca stores such as the presence. of calsequestrin and calreticulin as well as a vanadate-sensitive 2Ca +...ATPaSe and they, too, exist in high abundance in cerebellar Purkinje cells.
Although 31P receptors are structurally similar to ryanodine receptors (Tsien and Tsien, 1990), 1P-releasable 2+Ca stores seem to be anatomically and functionally distinct from3 the caffeine releasable stores since lP-induced release could 3 2+Ca occur after caffeine stores were depleted and they have distinct subcellular distributions (Thayer eta!, 1988). Release of 2Ca from 1P-sensitive stores normally follows generation of 31P subsequent to activation of3 phospholipase-C, (PL-C), a G-protein mediated process that has been linked allocation to mechanisms (affinity, consistent V. 1990). The Ca 2 studies, latter modulator, IV. Ca 2 + central mechanisms, 1990). activity. alpha-adrenergic to mediate many INTRACELLULAR most CALCIUM-BINDING Although three The buffers. signal A theme there large kinetics, neurotransmitter in relevant large with inside and proteins the will exemplified their is number in They lP 3 - the calbindin-D28k, various number little depend many the receptor functional of cooperation and large but are cell. ROLE these evidence of models caffeine-sensitive subcellular they PROTEINS of discussed not at Ca 2 number activation receptor Functional mechanisms OF an to capacity, only +.binding have our extreme attempting to Ca 2 (in parvalbumin of on support discussion a been in domains activation diverse positive their and detail integration etc.) by proteins involved considered metabotropic pools an inherent to intracellular but later. vital (for and feed-back explain enzyme-modulating are such also are have example functions in calmodulin, of calretinin. biophysical as the by regulated such critically spatial been muscarinic Ca 2 + many glutamate manner) regulation regulatory see [Ca 2 ] identified heterogeneity In waves on to a Lederer by properties spite known be their has different role acetyicholine, receptor of is ‘passive’ (Berridge, of been [Ca 2 +] 1 in believed spatial for et enzyme intensive neurons. a!, the a of is the 20 21 In spite of the diversity in the known functions of 21Ca the major roles of 2+Ca can be summarized as follows: ,
1. Charge carrier with direct effects on membrane potential.
2. Ion channel modulator where 2Ca can directly modify the gating of other channels (e.g., 2+Ca dependent K+conductance, 2Ca +dependent Cr-conductance or 2Ca + release activated 2+Ca conductance).
3. Enzyme, protein and gene activity modulator: e.g., phospholipases, protein kinases, genes (c-fos), proteases and endonucleases.
VI. RECAPITULATION
Increases in neuronal [Ca can be a consequence of activation of a 2+] (1) variety of calcium influx mechanisms such as VGCC, LGCC, and 2Ca +.release activated calcium channels or (2) release of intracellular calcium stores by depolarization, 3PL-C/1P pathway activation and calcium-induced calcium release. Restoration of calcium homeostasis requires the extrusion of ‘excess’ calcium across the cell membrane and the replenishment of intracellular stores. This energy-consuming restoration process must of course be appropriately adjusted by the cell so that the purpose of the calcium signal is realized, with adequate specificity/selectivity, otherwise futile calcium cycling would eventually drain the cellular energy resources. In spatially heterogeneous neurons, and possibly in most other cells, high spatial (nanometer) and temporal (sub-millisecond) resolution of the calcium signal require functional clustering of the signal source (influx and/or release), effector machinery (enzymes, genetic material, presynaptic vesicles, etc) and the recovery mechanisms (efflux/sequestration/buffering).
visualizing
regulatory are
the
as
least
mitochondrial
physiologically
of
ATPase
excess
following
then
of
dissipation
binding
Blaustein
small mitochondria
approximately
an
low-affinity
ensue
in
General
oversimplification.
Considering
calcium
restricted
proteins
can
dendrites)
0.1
(1988)
mechanisms
the
of
during
fine-tune
to
Ca 2
the
models
potential
relevant
mitochondrial
restoring
in
1
(e.g.,
has
1
diffusional
calcium
the
s,
the
where
only
ms.
uptake
the
suggested
the
of
following
restoration
parvalbumin)
under
significance
stimulation
the
Sequestration
the
Na/Ca 2
the
last
localized
However,
signal
capacity
affinities
spaces
‘natural
[Ca 2 +1 1
certain
uptake
stages
tens
that
away
of
[Ca 2 +]j
is
and
with
such
might
exchanger
has
Ca 2 +
history’
system.
and
diffusional
physiological
to
of
of
controversial.
from
into
near
the
milliseconds.
relative
made
large
the
models
be
homeostasis
can
ER-associated
recovery
the
the
of
binding
the
surface
such
may
be
a
calcium
fluxes
contribution
resting
can
fastest
calcium
or
high
a
extrude
However,
reaction
process.
pathological
be
role
Finally,
and
to
enough
following
signal
level
conceptually
mechanisms
volume
calcium
signal,
a
buffering
viable
the
of
where
constants,
over
source
the
The
to
various
bulk
of
ratios
conditions.
stores
make
large
possibility
the
involvement
necessity
Ca 2 +
by
of
with
of
helpful
Ca 2
(such
calcium
the
use
might a
+
of
r
at
in 22 23
1B. EXCITOTOXICITY
I. HISTORY AND DEFINITION
The earliest report that the acidic amino acid glutamate (GLU) was toxic to neurons was presented by Lucas and Newhouse in 1957. In their search for compounds that may be useful in the amelioration of retinal dystrophy they discovered that systemic GLU administration was associated with neuronal degeneration in their experimental preparation, the immature mouse retina. A series of pioneer studies by Olney and his colleagues (Olney, 1969; Olney and Sharpe, 1969; Olney eta!, 1971; Olney eta?, 1974; Olney, 1978) confirmed and established a phenomenon that was named excitotoxicity.
Excitotoxicity in principle links the excitatory properties of a compound to its neurotoxic sequels. The relationship between agonist-induced over- stimulation and cell injury is not limited to neurons or to glutamatergic synapses. For example, excessive adrenergic stimulation can cause injury of cardiac muscle fibers and the role of cholinergic agonists in the induction of muscle dystrophy has been well established (Bloom and Davies, 1972; Leonard and Salpeter, 1979).
II. EVIDENCE FOR GLUTAMATE INVOLVEMENT
A. In vivo
Glutamate is the major excitatory neurotransmitter in the central nervous system (Curtis and Johnston, 1974). In addition, it has a metabolic role and serves as an intracellular anion with an average tissue concentration of approximately 1 mM while in glutamatergic nerve endings tissue GLU levels
the after
number
Olney
substantiated
treatment.
neuroprotection
using
hypoglycemic
ligation
detected
phosphonoheptanoate
specific The
indicates
not
However,
powerful
degeneration,
variety
other
evidence,
may
ubiquitous
NMDA
removed
selectivity
the
Injection
Direct
be
different
et
endogenous
of
(Simon
of
types
as
at,
ischemic
in
that
uptake
direct CNS
specific
both
receptor
amino
high
The
CAl
injection
1989).
by
by
injury of neurodegeneration
of
even
hypoxic/ischemic
of
eta!,
diseases.
in
neuroprotective
was
as
such
injection
pyramidal the
mechanisms
a
GLU
the
acid
insult
vivo
competitive
large
excitatory
10
subtype
at
The
of
specific
evident
of
1984).
neuronal
uptake
receptors
high
mM.
(APH)
such
the
and
GLU
was
majority number
of
rat
neurons
in
concentration,
as
in
GLU
Although
into
even
NMDA
delivered
in
mechanisms
In
amino
vitro,
both
caudate
the
and
populations
glutamate
the
1985,
(GluRs).
properties
of
animal
the
of
is
analogues,
when
pathogenesis
in
experiments
non-competitive
hippocampus
a
in supports
these
acids,
antagonist,
nervous
response
neurons
rats
it
Wieloch
(McDonald
could
models.
may
the
can
studies
causes
most
exposed
are
of
damaged
antagonists
e.g.
also
the seem
system
GluR
be
and
involved
to
demonstrated
in
likely
2-amino-7-
of
In hypothesis
neurotoxic,
reduced
be widespread
concentrated the
et
kainate
different
glia
antagonists
counter-intuitive
neuronal
some
to
antagonists
a!,
ameliorated
by
fails
excessive
due
transient
(Schousboe,
in
such
1
were
987;
cases,
the
or
cell
to
to
animal
that
injury
NMDA,
induce
the
accumulating
procedures
that
neuronal
pathogenesis
damage
administered
Gill
have
on
carotid
stimulation
have
presence
by
GLU
et
since
the
species
that
1981).
neuronal
APH
been
that
at,
been
and/or
role
normally
damage.
artery
1
a
a
also
are
988;
large
of
and
of
of
of
24 a
suffered
patterns
pM
1983;
induced
hypoglycemic excitatory
high
concentration
ischemia concentrations
the
response
prerequisite
useful
following
that
AMPA
nitro-7-sulfamoyl-benzo(F)quinoxaline
pharmacological
(Honore
available
(Benveniste
use
These
enough
non-NMDA
Deafferentiation
Petito
in
receptor
of
neuronal
from
of
eta!,
it
demonstrating
to
for
ischemic
amino
in
neuronal
has
and
for
ischemic
etal,
to
vivo
this
cerebral
neuronal
increases
1988),
of
been
kill
other
etal, the
blocker
receptors
acids
tools
death.
receptor
amino
microdialysis
1987).
most
insults
induction
loss
shown
experiments
1984; insults
experiments
particularly
ischemia
(EAAs) for
damage
from
that
that
hippocampal
acids
observed
Experimental
the
(Sheardown subtype
may
that
Hagberg
intact
(Johansen
crosses
less
of
manipulation
in
in
for
also
and
and
neuronal
the
after
the
than the
have
of
in
direct
excitatory
since
status
in
mediate
pathogenesis
hippocampus
the
post-mortem
etal,
glutamatergic
neurons
recent
10
(NBQX),
the
results
et
5
et
established
pM
the
blood
measurement
damage
mm
a!,
a!,
generation
epilepticus
1985).
of
some
development
early
to
1990).
1986;
synaptic
also
of
in
non-NMDA
brain
which
30
ischemia
vitro.
in
of
bear
brains 1980’s.
during of
pM
Such
pathways
the
Onodera
the
barrier,
ischemic/hypoxic
the
of
is
(Corsellis
transmission
and
of
many
role
epilepsy-
a
hippocampus
concentrations
of
neuronal
induction
the
receptor
extracellular
of
potent
after
The
of
patients
has
2,3-dihydroxy-6-
et
similarities
GLU
have
endogenous
and
a!,
use
30
established
and
and
degeneration
subtypes
1986).
also
of
mm
is
Bruton,
of
who
selective
trauma
in
a
cerebral
new
and
been
to
are
to
500
By
the 25 26
Several characteristic features of excitotoxic lesions are evident from the accumulated experimental findings. First, excitotoxicity was found to be a primarily dendrosomatic lesion that spared axons traversing the site of the lesion. Second, the selective vulnerability of certain neuronal populations determined the final outcome of an excitotoxic lesion. This vulnerability was, amongst other factors, based on the receptor profile and the density of the synaptic inputs. Third, the induction of excitotoxic damage in vivo did not end with the termination of the primary insult (e.g., transient ischemia) possibly because excitotoxicity is, to some extent, a self-propagating process. This aspect may be of crucial value in the design of therapeutic approaches since post-insult therapeutic measures may still be capable of limiting the extent of the lesion. In other words, the therapeutic window may extend beyond the duration of the initial insult (Albers eta!, 1989).
B. In vitro Primary neuronal cultures from different brain regions (e.g., neocortex, hippocampus, striatum, spinal cord) have been used extensively to study the physiology, pharmacology and pathology of EAA in vitro. These studies have also suggested that EAA can induce neuronal injury in a concentration dependent manner, and that EAA-induced neurotoxicity can be specifically attenuated by GIuR antagonists. In addition, many elaborate in vitro models have been devised to ‘simulate’ specific in vivo situations like ischemia (Goldberg and Chol, 1993), hypoxia (Dubinsky and Rothman, 1991) and even mechanical trauma (Tecoma et a!, 1989). Though elaborate in vitro experiments cannot reproduce the complexity of the clinical phenomena, the fact remains that in the majority of such experiments, performed under diverse experimental conditions, EAAs were found to contribute to the neuronal injury and EAA antagonists were capable of alleviating, at least in part, the damage.
swelling,
distension
neurons
neuronal
phenomena.
the
during
delayed
neuronal
associated
suggesting
passive
this
could
(Rothman,
within
prolonged
is
experimental
isolation.
IONIC
III.
associated
Ca 2
swelling
PHASES
Neuronal
Removal
A
be
the
minutes
DEPENDENCE
major
death
influx
with
swelling
yet
degeneration
prevented
ionophore,
can
exposure
A
depolarization
with
1985;
that
delayed
brief
was
For
NMDA
OF
environment
with
advantage
of
of
swelling
is
itself
of
the
the
entirely
GLU
cr
GLUTAMATE-INDUCED
example,
without
Choi,
dependent
exposure
the
the
by
swelling
reversal
be
to
and
in
A23187
neuronal
or
exposure.
and
early
the
glutamate
removal
and
lethal,
1987b).
other
dependent H 2 0
of
of
the
so
a
absence
subsequent
delayed
in
of
neurons
development
of
was
brief
on
(Rothman,
that
delayed
the
(Rothman,
vitro
death
excitants
cultured
the
of
the
This
Ionic
and induced
relevance
exposure
these
relevant
swelling,
experiments
of cell on
presence
by
still
can
swelling
neuronal
Na+ 0
substitution
the
death
neurons
exposure
death
from
ions
1985).
occurred.
1985;
NEUROTOXICITY:
of
by
be
presence
parameters
to
of
neuronal
but
and
the
from
mimicked
over
the
of
appear
kainate
this
can
loss,
Choi,
to
is
extracellular
is
Cr 0
influx
bathing to
a
the
the
then
high
experiments
phase
also
Although
veratridine
period
of
while
prevented
to
swelling
1987b).
was
culture
ability
can
of
extracellular
by
concentrations
followed
be
be
medium
of
Na
exposing
treatment
associated
produced
of
be
independent
TIME
excitotoxicity
to
Na+
severe
medium
hours.
evaluated
that
followed
or
control
indicated
neuronal
by
COURSE
is
high
and
appears
Ca 2
neurons
a
usually
neuronal
of
by
This
with
delayed
cr
[K+1 0
of the
+
by
in
that
GLU
and
to
AND
the
to 27 operating dependent was However, to Tymianski death mechanism(s) development comparable brief Ca 2 development experiment the Ca 2 intracellular intense presence magnitude permeability and extracellular the the accumulation EAA-mediated prolonged glia Unlike exposure A in influx channel port unique exposure cultured in in of their eta!, on of vivo (Choi, demonstrated NMDA to the accumulation is the to space of of Ca 2 the by important. blockade an to feature (Koh excitotoxic Ca 2 delayed reverse delayed may 1993). radioactive cortical which AMPA, of of Ca 2 NMDA-induced 1992). agonists, in and entry eta!, neurons 45 Ca not vivo (MacDermott of the mode Ca 2 influx was The neurodegeneration excitotoxic allow neurons kainate the into not of and 1990). Such neuronal potency extensive tracer non-NMDA 45 Ca. latter in not NMDA only through (Choi, induces the the such vitro a effective or increase conclusion if This neuron. delayed 45 Ca group that increased eta!, injury the Specific high neuronal swelling receptor-associated to 1991). structural cell VGCC slowly GLU Ca 2 + duration in agonists [Ki 0 of 1986). is (Kurth the in death neuronal (Michaels authors doubtful, NMDA or [Ca 2 +] to is damage, or markedly influx triggered extracellular could NMDA also take support via of may eta!, are When the antagonists Na/Ca 2 death is also supported place without incapable raise and be since essential but resulted 1989; exposure when excitotoxicity performed by speculated ionophore spatially Rothman, while also or [Ca 2 1 1 medium, adjacent the the severely the Choi, by that in exchanger of for limited abolished voltage-gated is subsequent exposure the tightly the inducing to in the brief. is 1991). the that brief 1990; neurons the levels its may fact limit route linked high the and both that be time its This of 28
generating
dehydrogenase
increases
feature
perpetuation
cytoskeletal
proteases,
dependent
initial
early
responses
kinase-C,
GLU
are
and
‘positive
receptors,
of
changes
exposure
concentrations
expression.
neurotoxicity
IV.
the
triggered
diacylglycerol.
A
The
and
gene
Choi
injury
major
THREE-STAGE
of
feedback’
take
other
expression
in
calmodulin
this
determines
to
free
lipases
(1990)
expression. and
cytotoxic
is
[Ca 2 +]j
elements,
of
GluR
The
to
ambient
in
place.
not
stage
neurotransmitters,
include
oxygen
into
the
of
promote
which
induction
and
severe.
presented
subtypes: GLU
damage.
amplification
xanthine
The
which
is
phase
mechanisms
sensitive
These
the
GLU.
MODEL
endonucleases
plasma
the
accumulation
there
radicals
or
The
result
further
speed
other
involvement
result
proceeds
stage
amplification
changes
The
a
NMDA,
oxidase
were
These
membrane
OF
model
kinases,
of
(FOR).
endogenous
with
increases
effector
phase
in
such
is
and
GLUTAMATE
and
three
a
a
oxidative
AMPA/Kainate
of
with
which
reflect
for
(Olanow,
calpain-mediated
direct
are
the
even
an
High phospholipases,
of where
intracellular
stages:
the
integrity limbs
phase
involved
subsequent
generalized
accumulation
oxidative
in
a
enhance
the result
[Ca 2 ] 1
evolution
EAAs.
[Ca 2
mechanisms
set
several
1993) of
may
effects
EXCITOTOXICITY
induction,
of
+1
these
and
of
in
Ca 2 +,
mechanisms
and
primary
The
also
be
exposure
the
this
enhance
that
activation
Ca 2 +
irreversible
of
genomic
conversion
of
calpains is
more
changes
metabotropic
intensity
postsynaptic
GLU-mediated
activates
phase.
the
excessive
is
are
Na+,
amplification
sensitive
intracellular
capable
important
initiation
activated
to
the
DNA.
of
Cr,
and
in
include
A
high
damage
of
of
(CHOI,
release
all
the
major
activation
water,
this
of
xanthine
immediate
pathways
Ca 2 +
Many
of
if
and
by
protein
the
a 1990)
to
of
1P 3 29 the antagonists failure, infarcts, the that (Wieloch of 1985). receptor white cytotoxic example, major V. 1988). structural damage the dismutase, of activate membrane peroxidation phospholipase white hydroxyl insult release LIMITATIONS capacity can The matter limitations and Furthermore, White NMDA by matter be profile
nitric eta!, the in ‘pure’ mechanisms protein of lipids perpetuated peroxidases, to propagating initiated the radicals. of of with excitotoxic low-molecular-weight be matter, oxide 1988; A 2 that antagonists can cellular core the excitotoxic which (Chan effective and OF substantial which brain be is during (NO) of in THE Buchan Excessive DNA being a such explained anti-oxidant the
require eta!, models during the prerequisite ascorbic hypothesis liberates in synthase EXCITOTOXIC or ischemia, ischemic damage hypothesis have an lipid as formed and it 1985) FOR the ischemic/hypoxic the an of may production peroxidation, by not acid, Pulsinelli, the global expansion production. reperfusion oxidative which, defences iron (Olanow, of and the cannot be for lesion secondary been cytotoxic oligodendroglia of Vitamin that a oxidative complexes initiates ischemia HYPOTHESIS GLU-induced GLU-mediated unequivocally in may of explain 1990). vascular stress of excess, (glutathione-SH, 1993). the enzyme phase Increased E, the to arachidonic be lesion a stress and etc) highly chain loss too discussed the hypothesis associated However, factors (Halliwell promotes focal and promotes inactivation severe neuronal (Auer of damage and injury. reaction reactive neuroprotective [Ca 2 j Ca 2 + axons, ischemia acid and lipid and above. it for superoxide with to and the observed is This regulation damage neuronal from acidosis of FOR peroxidation can Siesjo, include the lacks possible and Gutteridge, production energy lipid damage with NMDA For also beyond major the has other in are or that the 30
general
development
unique
central
VI.
determining
HYPOTHESES
under
of
Excitotoxicity
again
reperfusion.
as
have
models,
why
thus
to
energy
focal
the
Chol
oxygen,
well
acidosis
Ca 2
main
It
Last,
hippocampal
Another rendering
a
(1991)
ischemic
contribute
such
can
for
role
high
mediator
as
depletion)
while
determinants
AS
neurons
but
the
be
even
conditions.
in
(or
the
density
of has
per
THE
the
limitation
concluded
functional
dentate
not
even
injury,
NMDA
neuronal
relative
to
if
of
suggested
se
formulation
may
CAl
least,
they
COMMON
since
the
cell
of
to
is
down
antagonists
granule
NMDA
reduce
not
dephosphorylation
of
pattern
neurons
injury
of
have
vulnerability
Rather,
all
an
capacity
injury
from
neuronal
the
enough
mammalian
that
increase
modulation
no
FINAL
in
of
receptors
excitotoxicity
the
the
cells
of
and
excitotoxicity
are
other
GluRs
the
in
cell
of
discussion
contribution
less
to
global
loss
death
are
selectively
PATHWAY
excitotoxicity
of
cell-specific
in
explain
damaged
cell
or
protective.
neurons
neurons
[Ca 2 i
spared
in
of
(Cotman
if
ischemia,
in
types. of
such
NMDA
GluRs
general.
hypothesis
NMDA
above
the
may
damaged
in of
is
in
extreme
AND
to
will
recovery
eta!,
spite
mechanisms
NMDA
The
widely
receptors
are the
hypothesis
various
be
or
that
eventually
receptors
ALTERNATIVE
This
a
effectively
post-ischemic
hypothesis
in
of
1987).
contributing
is
situations.
in
Ca 2 +
the
believed receptors
the
its
mechanisms,
situation
insults.
global
activity
infarct
inability
fact
and
secondary
of
occupies
die
Vascular
blocked.
cell
that
ischemia
to
that
in
if
to
is
zone
in
factor Indeed,
phase
be
deprived
fact
death
to
injury,
not
an
response
both
a
could
explain
a
factors
to
of
in
of
in
the 31 32 increase in 2[Ca +1 is ‘the final common pathway’ to cell death was presented by Schanne et al in 1979 and has generally been accepted by many researchers.
In a recent review, Dubinsky (1993b) presented a compilation of the experimental findings that could not be reconciled with the generalized 2Ca hypothesis. For example, Michaels and Rothman (1990) reported that a GLU induced rise in 2[Ca could be prevented using a combination of CNQX and APV while neuronalj loss was only attenuated. In addition, a chemical hypoxia model, using iodoacetate and rotenone to inhibit the glycolytic pathway and the mitochondrial respiratory chain reaction, was lethal to neurons and yet removal of extracellular 2Ca while preventing the rise in 2],[Ca did not alter the fate of energy depleted, neurons (Nedergaard, 1991).
Marked increases in 2+][Ca have also been observed in situations that did not end in cell loss. For example, prolonged depolarization of neurons using high extracellular K is not neurotoxic (Dubinsky and Rothman, 1991; + Tymianski et al, 1993a) even though the 2Ca responses are comparable to those induced by lethal concentrations ot GLU or NMDA. Also, in peripheral neurons deprivation of neurons of nerve growth factor (NGF) results in apoptotic cell death that appeares to be completely dissociated from any measurable increase in j21[Ca (Eichler etal, 1992). While the involvement of 2[Ca in the initiation of many cytotoxic biochemical pathways cannot bej denied, the exact relationship between the increases in 21[Ca and cell injury cannot be established with certainty. Cellular derangementj of any kind is likely to be associated with the loss of 2Ca homeostasis since the latter is tightly regulated against a large trans such Garthwaite, EAA Alzheimer’s’ induced neurodegenerative cerebral lesions VII. early and pathways still measurements death. injury cell oxygen This other exact an membrane increase limited. spatial injury metabolism. CLINICAL A as Technical is events mechanisms add sequence potential has not The sulfite neurotoxicity radicals ischemia/hypoxia, need as to been resolution unique electro-chemical 1991). most in disease leading Ideally, the much oxidase [Ca 2 +] IMPLICATIONS but, limitations to and role of discussed problem advanced Sufficient be disorders to of events as at Some to multiparameter and for severe Ca 2 + used is which neuronal the they deficiency and neuronal a excitotoxic amyotrophic of factor on present hereditary leading hypoglycemic, for above. an are intracellular of since evidence gradient. such do establishing the increase extended these injury symptoms not death. in measurement are as the to time, the Many interfere neuronal measuring techniques Huntington’s cell neurological such also same also lateral This pathogenesis in their acidosis periods the death studies [Ca 2 i epileptic believed of exists as two-way argument with sclerosis sequence this injury spatial excessive techniques of extremely of are which to injury. also disorders intracellular causing critical disease, and time to in establish fluorimetric and of relationship, the be could suggest (Meldrum of traumatic chronic could production in temporal mediated parameters events pathogenesis difficult injury, with order Parkinson’s of be a be signaling metabolic that causal made high Ca 2 + and to causative leading makes neuronal injury to resolution by discern GLU of temporal establish. of about disorderly free disease, of cell causing the origin to in the cell are 33
enhancement well
processes Schwartzkroin,
enhancing
use
function.
while
for
are,
function.
transmission
and
approach
selective
in
control
1988;
excitotoxic
VIII.
poisoning
disorders
relationship
some
the
of
as
theoretically,
consequently,
Another
The
minimally
THERAPEUTIC
VGCC
Olney
of
abnormally
this
animal
and
phospholipases
large
is
such
The
such
the
(Meidrum
mechanisms
pathogenic
that
between
may
etal,
blockers
of
approach
potent
Ca 2 +
use-dependent
1989;
number
as
interfering
models
as
FOR
EAAs
advantageous
have
the
stimulated
Guam
massive
1989).
buffering
and
excitatory
APPROACHES
removal,
Tymianski
many
Ca 2
(Deshpande
are
is
profound
of
of
process.
has
Garthwaite,
disease,
directed
C
excitotoxic
neurological
+
with
the
A
and
and
environmental
mediated
encouraged
major
EAA
open-channel
capacity
are
major
other amino
powerful
A 2 ,
in
eta!,
and
The
neurolathyrism
also
at
synapses
and
this
problem
TO
or
excitatory
reducing
synapses
unacceptable
1991).
insults
most
acid
activation
1993b).
being
diseases
Wieloch,
the
in respect
EXCITOTOXIC
the
antagonism
neurons
excitotoxins
inhibition
antagonists
direct
antagonists
with
mediating
considered
(McDonald
search
the
since
that
neurotransmitters
Also,
that
1986)
of
and
this
approach
increases
(Scharfman
side
protein
are
for
of
may
they
of
inhibition
domoate-induced
receptor
the INJURY
and that
lipid
and
required
therapeutic
effects
of
et
EAA
(Braughler
in
may
the
a!,
disease
kinases
possibly
certain
is
have
in
part
peroxidation
synaptic
the
1
and
[Ca 2 ]
PCP-site
blockade
show
of
987;
on
for
be
proven
in
down-stream use
normal
process
C
eta!
neurological
measures
mediated
normal
the
by
Gill
selectivity
and
of
by
shell
ligands
CNS
1987).
et
useful
and
A
the
a!,
as
fish
by
to 34 35
Recently, the use of neurotrophic factors for the control of excitotoxic neuronal damage has been proposed under the assumption that it is the imbalance between restorative and EAA-mediated catabolic functions that causes cell death. Thus, reduction of neuronal loss may be achieved by restoring the balance through supplementation of injured neurons with neurotrophic factors (Mattson eta!, 1993a).
IX. RECAPITULATION
Excitotoxicity is a mechanism of neuronal injury that is probably involved, to variable degrees, in the pathogenesis of a large number of acute and chronic neurological disorders. Evidence from in vitro as well as in vivo studies indicate that endogenous excitatory amino acids (e.g., glutamate and/or aspartate) may be the prime mediators of this phenomenon and that in many cases the antagonism of EAA-mediated primary, and/or more downstream effects, may be helpful in the amelioration or reversal of the disease process. Derangement of the intracellular calcium homeostasis is a central mechanism in the EAA mediated neurotoxicity and restoration of this homeostasis is the focus of many lines of therapeutic approaches including the use of intracellular calcium chelators. Other cytopathic mechanisms such as overproduction of free oxygen radicals and lipid peroxidation may also be involved in neuronal injury.
The issue of primary versus secondary effects in neurotoxicity is complicated by the extensive network of positive feedback regulatory mechanisms between the various causes of cell injury which are responsible for the perpetuation and amplification of the primary insult. The resolution of the cause-effect relationship issue must await the availability of less intrusive high spatial/temporal resolution, multi-parameter intracellular monitoring techniques.
the
metabolite,
associated
locations
distal
Wasserman
II.
parvalbumin fairly
intracellular
calcineurin
activation
proteins
in
proteins that
binding
nervous
number
I.
the
CaBP INTRODUCTION
induction
Morrissey
are
CaBP
Calbindin-D28K
large
convoluted
nervous
site
of
characterized
(Dalgarno
system
(CBPs)
HISTORY
CaBP
while
with
was
intracellular
vertebrate
and
1
and
Ca 2 +
known
(PV),
,25-dihydroxy-cholecalciferol.
of
system
and
synthesis
first
recoverin)
tissues
chicken
exist,
Taylor
the
and
tubules
buffers.
cairetinin
eta!,
AND
Wasserman
as
buffer
discovered
renal
(CaBP)
tissues
the
by
the
and
concentrations
in
performing
intestinal
DISTRIBUTION
1984).
is
and
the
1966.
EF-hand
are
cortex.
EF-hand.
proteins
they
To
totally
(CR)
is
with ic.
presence
in
known
this
a
and
(1971)
the
can
The
Ca 2
and
It
CaBP
CALBINDIN-D28K
dependent
particularly
proteins
transcellular
It
buffer
was
have
chicken
isolated
generally
Although
is
trigger
CaBP.
to
and
binding
reported
of
a
expression
subsequently
mediate
no
member
protein
a
can
are
specialized
proven
oviduct.
from
proteins
on
be
high
other
protein
potentially
the
Ca 2 +
a
the
Ca 2 +sensitive
classified
group
chick
of
close
most
and concentrations
function
families
active
a
(e.g.
found
Its
transport,
large
that
high
the
intestinal
belong
association
studied
expression
function
as
vitamin
calmodulin,
exists
enhanced
family
affinity
in
of
but
trigger
the
proteins
Ca 2 -binding
enzyme
and
they
group
epithelium
in
avian
in
of
as
D
between
Ca 2 -
a
has
in
the
or
proteins
exist
Ca 2
large
these
of
such
buffer
been
renal
CBPs
in
by
as 36
system
populations Christakos
neurons
standpoint
its
the
and
(Buchan
mammalian
and
detected
mathematically
facilitation The
Feher for
promotes
while
protein
expression
studies
(Spencer
transport
role
expression
CaBP:
central
genetically
mammalian
latter
for
CaBP
et having
is
synthesis
demonstrated
CaBP
is
and
a!,
in
et
not
a
induced
the
study
not
and
that
is
of
and
by
most
enterocytes,
(Jande
diffusional
1992)
a!,
Baimbridge,
not
no
dependent
Ca 2
in
reduction
in
universal
vitamin
Norman,
unrelated,
peripheral
it
by
1
intestinal
Ca 2 +
also
effect
Ca 2 +
978)
vertebrate
restricted
was
inhibitor,
by
a
eta!,
transport.
number
proposed
vitamin
and
originally
that
transport D
transport
Ca 2 +
on
since
of
1980,
it
on
lagged
1981;
1988).
epithelium
nervous
calbindin-D9k.
enhanced
that
has
intra-mitochondrial
in
to
cycloheximide,
vitamin
tissues
of
flux
vitamin
D.
anti-CaBP
avian
been
Varghese
an
the
Despite
authors
Roth
a
behind
tissues,
across
This
has
It
neuronal
alternative
system,
two
has
with
Ca 2 +
D
replaced Ca 2 +
(Wasserman
eta!,
D-deficient
been
led
(Baimbridge
that
the
processes
been the
(Bronner
antibodies
exceptions
expression
eta!,
to
In
transport
transport
which
1981;
protein including
presented
enhancement
intestinal
controversy
all
the
argued
and
by
Ca 2 +
1988).
vertebrates,
establishment
the
et
animals
Cello,
prevented
and
controversial
could
and
(Parmentier,
label
a!,
tissues
of
such
accumulation
the smaller (Morrissey
from
epithelium.
and
Fullmer,
1986;
CaBP
Also,
Parkes,
1990).
the
specific be
enteric
induction
of
as
modelled
an
uncoupled
and
CaBP
CaBP
Ca 2 +
idea
molecular
striated
in
its
evolutionary
Feher,
1982).
of
functional
1981;
the
eta!,
1989).
it
nervous
distribution
neuronal
that
an
However,
is
has
expression
and
transport
of
nervous
found
1983;
muscle essential
1978).
CaBP
CaBP
been
by
the
weight,
In
Unlike
system
the
role
sub-
in
in
later 37 38
III. CHEMISTRY AND MOLECULAR BIOLOGY OF CaBP
CaBP is an acidic 28 kDa protein composed of a single polypeptide with an isoelectric point of 4.2. In the purified form it is free from lipids, carbohydrates and phosphates (Bredderman and Wasserman, 1974). It is fairly heat stable up to 80°C and has an overall apparent affinity of 0.5 pM for 2Ca This high affinity for 2Ca is also associated with higher 2Ca selectivity. over other divalent cations such as (pK 2.44 versus 6.30 for 2Mg 2j,Ca 2Mn and .2Zn Human CaBP has been sequenced and cloned (Wilson et a!, 1985, Takagi eta!, 1986). Its gene has been located on chromosome 8 and is 79% identical to chick CaBP and 98% identical to bovine and mouse CaBP. CaBP is composed of 271 amino acids with six EF-hand domains, four of which are functional (numbers 1, 3, 4 and 5) resulting in a high 2Ca binding capacity with one CaBP molecule capable of binding 4 2Ca ions. Although CaBP is not as highly conserved as calmodulin, evolutionary rate analysis indicates that
CaBP is highly conserved compared to many other CBPs such as PV or calbindin-D9k. Based on the fairly high conservation of CaBP, Parmentier (1989) has suggested that, in addition to a potential 2Ca buffering function, which does not represent a strong pressure for conservation (see later), CaBP may be involved in other vital protein-protein interactions that are specific for the sites of expression of CaBP.
The molecular basis of 2Ca affinity: CaBP and many other 2Ca + binding proteins owe their 2+Ca binding properties to a conserved amino acid structural motif known as the EF-hand. This structure was first identified in 39 carp parvalbumin and it was named after the E and F a-helices in this protein (Kretsinger and Nockolds, 1973).
An EF-hand is composed of two a-helices (E and F with 9 and 8 amino acids respectively) linked to each other by a loop of 12 amino acids rich in acidic oxygen-donor groups that form the six coordination sites for 2+.Ca Five of the coordination sites come from carboxyl or hydroxyl side-chain oxygen of serine, threonine or acidic amino acids while the sixth coordination site comes from a carbonyl group on the main chain. The oxygen-donor amino acids are precisely spaced at positions 10, 12, 14 16, 18 and 21 in the EF-hand domain forming the vortices of an octahedron site for 2+Ca binding. EF-hands are almost always found in pairs as a result of the hydrophobic bonding between several hydrophobic amino acids across the two hands. Such pairing results in a positive cooperativity between the binding sites with an increase in the affinity and selectivity of the 2-hand assembly for 2Ca as well as an increased sensitivity to small changes in its concentration (Williams, 1986).
While six copies of the EF-hand exist in each CaBP molecule, other members of the EF-hand protein family have different numbers of EF-motifs per molecule of protein, ranging from 2 domains (calbindin-D9k) to 8 domains (e.g., 1LpS in Lytechinus pictus). The primary sequence of EF-hands varies in different proteins. For example, PV has a pair of ‘normal’ EF-hands and a third mutated non-functional hand that is packed against the globular structure formed by the two functional hands. Another variety exists in intestinal vitamin
D-sensitive calbindin-D9k which has two EF-hands, the first domain of which is considered atypical because the loop between the two a-helices contains 14 amino acids instead of the usual 12 amino acids. This atypical 14-amino acid
site
region
hand
According
Ca 2
and
gene
eta!,
others of
domain
members)
likely
proteins.
EF-proteins
substitution
the
required
primary
proteins.
EF-hand
proteins
exerts
others
tertiary
Beyond
proteins
mutations,
Genetics
It
similar
1990).
binding
as
are
is
proteins.
amino
for
is
well
important
All
that
to
only
a
have
that,
characteristic
structure
has
re!ative!y
Ca 2
of
to
the
Heizmann
are
Some
site,
have
as
of
underwent
acid
the
that
expressed
been
some
retained
over
genetic
in
presumed
the
Gene
binding.
without
amino
undergone
non-EF-hand
sequence
to
proteins
of
EF-hand
of
used
time,
weak
of
stress
calmodulin,
and
duplication
the
sequence
the
the
of
acids
in
multiple
for
retaining
acquired
to
Hunziker
conservation
a
EF-hand
a
are
tertiary
EF-hands
that
proteins:
of
subfamily
have
the
tremendous
species-
which
universally
sequences.
the
in
construction
coding
reiterations
since
and
evolved
the
over
quite
EF-motif
that
conformation
(1991)
allows
have
Genetic
and/or
mutation
actual of
it
pressure
is
200
for
diverse
is
diversification
the
expressed conserved
from
lost
‘maintenance
the
Undoubtedly,
retaining
the
is
proteins
tissue-specific
to
EF-proteins
primary
of
analysis
not
their
most
then
EF-hand, produce
a
expression
a
of
on
single
conserved
‘family
the
Ca 2
the
produced
conserved
(e.g.
the
via
of
amino
of
EF-hand,
both
primary the
prototypic
the
appropriate
coordination
of
known
genes
a
this
tree’
binding
calmodulin)
large
manner.
patterns
a
EF-family,
acid
multiple
in
but
calcium-binding
diversity
the
for
the
of
of
structure’.
as
number
identity.
and
rather
capacity
multitude
the these
the
gene
promoter
S100
(Moncrief
EF
Through
the
sites
the
family
EF
while
it
(most
of
is 40
conserved
scattered
gyrus
superficial
neurons
which
collateral
1992). pyramidale
the
bundle
differences,
purposes
hippocampus
view,
including
antibodies
any
specific-expression
IV.
reflects
granule
particular
CaBP
CaBP
Expression
CaBP
and
makes
do
of
The
are
functional
fibers)
interneurons
not
of
axons
mossy
throughout
pyramidal
in
EXPRESSION
in
to
of
is
cell,
CaBP
CaBP
this
albeit
other
CaBP
the
contain
a
CaBP
will
the
membranes
highly
terminate
and
is
of
work,
fibers
rat
CAl
be
staining
less
deeply
animals.
specialization
an
CBPs
show
(Baimbridge
and
fine
hippocampus:
CAl
presented
CaBP.
the
water
ideal
(Baimbridge
region
diverse
only
that
their
IN
dendrites
in
hippocampus
uniform
neurons
stained
on
or
pattern
THE
adult marker
synapse
CaBP
For
soluble
Axons
cytoskeletal
the
physiological
(Baimbridge
than
(for
CENTRAL
etal,
example,
of
brains
moderately
with
expression staining
found
are
which,
and
for
The
the
a
that
of
protein
with
comprehensive
1991).
CaBP
the
CA3
CaBP.
Miller,
individual
proper,
principal
is
in
in
pyramidal
elements.
none
and
subject
from
specific
of
NERVOUS
the
function.
the
that
pyramidal
CaBP
the
and
Their
In
1982;
Miller,
developing
while
rat
CaBP
of the
has
the
cell
whole
proteins
its
to
the
hippocampus
populations.
neuroanatomist’s
CaBP
neurons
hilar
not
distribution
species-specific
type
Neurons
Miettinen
the
review
SYSTEM
is
1982).
neurons
CAl
neurons
also
cytoplasmic
been
deep
zone
of
for
brain.
neurons
present
see
the
of
axons
Only
their
that
associated
pyramidal
in
and
of
(Schaffer
the
is fascia
in
Celio,
the
For
the
react
not the
tissue
the
Freund,
CA3
form
contains
volume
in
stratum
the
point
dentate
exactly
rat
dentata,
1990).
with
region
a
with
of 41 42
CaBP in the guinea pig (Rami et a!, 1987) while in the domestic pig all pyramidal and dentate granule cells lack CaBP (Hoim eta!, 1990).
V. DEVELOPMENTAL EXPRESSION OF CaBP AND RELATED CBPs
An extensive literature has accumulated concerning the expression of the three major neuronal CBPs with no known specific functions (CaBP, PV and CR) in the developing nervous system. In their review, Celio and colleagues (Andressen et a!, 1993) state that the ‘complex variety of [developmental] patterns does not allow us to associate any of the three CBPs with a specific phenomenon in the developing brain’. They presented, however, a few general principles related to CBP expression. CBPs can be transiently expressed at various stages of development and CR is the most precocious member of the group while CaBP expression precedes PV expression in the central nervous system and follows it in the peripheral nervous system. The pattern of expression of a particular CBP in a certain population of neurons during development bears little relation to the final expression profile of CBPs in the adult neuronal population, which in some cases (e.g., in primates) is not reached before 1-2 years postnatal. Transient expression of CaBP and PV has been observed, for example, in neocortical pyramidal neurons of layer V in both the monkey and neonatal kitten (Hendrickson eta!, 1991; Hogan and Berman, 1991). Developing non-neuronal elements, e.g. spiral limbus and crista supporting cells of the fetal mouse inner ear, can also express CBP5 transiently while they do not, under physiological conditions, do so in the adult animal (Dechesne and Thomasset, 1988).
CaBP in the developing hippocampus: CaBP immunoreactivity can be detected in the 18-day fetal rat hippocampus (Enderlin etal, 1987). During the
found Very
under
CaBP-D28k
enough
VI.
in
slowly
adult
pyramidal
CA3
CAl
observations).
(Baimbridge important
pronounced
pyramidal
closest
neurons
(Baimbridge,
granule
later which
first
pattern
vitamin
MODULATION
Expression
little,
pyramidal
few
pyramidal
developing
pattern
physiological
to
(Baimbridge
develop
to
to
seen
cells
alter
can
weeks
D
to
layer
induce
neurons
stratum
if
is
depleted
and
temporo-septal
any,
note
be
of
but
1992).
in
neuronal
not
neurons.
neurons
post-natally),
For
in
staining
the
of
seen
post-natum,
Miller,
granule
its
CaBP
is
sensitive
that,
the
oriens).
renal
develops
OF
KG,
conditions
the
adult
known
synthesis
animals.
At
in
first
CaBP
CaBP
in
purpose
expression
1982;
unpublished
the
(together
of
and
three
cells
Further,
rat.
vivo,
week
superficial
to
about
The
deepest
gradient
slowly
EXPRESSION
intestinal
expression.
CaBP the
vitamin which
Miettinen
and
Within
days
is
A
CaBP
of
adult
post-natum
delayed
expression
single
the
with
the
very
the
within
is
after
post-natum,
observations).
layers
form
(Baimbridge
is the
produced
regulation
pattern
neurons
D
CAl
calbindins
work
a
few
expressed
dose
depletion
and
few
about
until
dentate
hours
a
of
pyramidal
layer
experimental
presented
of
Freund,
do
scattered
of
of
the
the
that
CaBP
in
14
so
vitamin
while
of
staining
numerous
(D9k
KG, adjacent
CAl granule
or
large
third
only
days
only
neuronal
do
repletion
1992),
gradually
neurons
unpublished
in
express
and
pyramidal
in
interneurons)
or
in
amounts
transiently,
D
post-natum,
the
of
this
tools
cells
a
fourth
in
to
D28k)
intensely
the
small
CaBP
and
nervous
these
the
thesis
(see
develops
CaBP
(the
assumes
have
superficial
never
week
in
hilar
neurons
number
is
expression
animals
above).
the
majority
while
depressed
it
stained
been
in
and
system
zone
is
in
in
initial
the
quite
the
with
the
the
the
of
(i.e.
CAl
is
CAl
of
43 a cells factors expression expression was target of the contrast, cerebral expression concluded prolonged frequency eta!, regulation content or development seizures convulsive certain adrenalectomy septum hippocampus. found (neurotrophin-3, In Chronic Kindling 1985). of have cultured neural of cortex in CaBP adrenalectomy changes of electrical that to is indicating (Barakat the of response threshold also associated mRNA corticosterone of be CaBP However, is pathways depletion. dentate or CaBP chick a a required been was the convulsive notable As in and and stimulation of to a of expression Lindholm dorsal cerebellum [Ca 2 j 1 . in small reported CaBP role granule a the Droz, its Lowenstein with resulted kindling, using for previously-subconvulsive However, example mRNA for pathway treatment and root the in a response, 1989). subconvulsive target eta!, cells dramatic the of to only can induction in ganglion in the levels the induce where dentate the rats eta! (Miller Baimbridge be 1993), and transient. elements dentate perforant Recently, has reduction kindling modulated (lacopino and in (1991) eventually CaBP repeated neurons, but been the and granule cultured specific stimuli granule not hippocampus in Baimbridge, expression of path (1992) reported several reported the of and the the stimulus. by electrical cells a CaBP results gradually hippocampal depletion resulted regulation muscle cells stimuli maintainance Christakos, perforant reported neuronal 24 to that and were in in enhance 1983; hours but stimulations extract that Besides cultured in fully in reduce mRNA of of an path, the that not the growth the induce later. neurons 1990a). Baimbridge CaBP developed up- of main in rat, the factor the CaBP levels the the amygdala Purkinje CaBP the They high effect of and In in 44 45 in hippocampal organotypic cultures (neurotrophin-3, basic fibroblast growth factor, brain-derived neurotrophic factor, Collazo et a!, 1992)
VII. BIOLOGICAL ROLE OF CaBP
The functional role of CaBP in neurons is not known. Because of its high concentration in certain cell types (e.g., Purkinje cells and dentate granule cells) and its high affinity and 4:1 stoichiometry for Ca-binding, it has repeatedly been proposed that its primary function is to buffer2 2Ca While 2Ca buffering must be a property for a protein with the aforementioned. biophysical properties, it probably is not the sole function since the structure of CaBP is extremely conserved, a feature more consistent with a possible vital protein- protein interaction function than with a passive 2+Ca buffering role. The functional contribution of a 2+Ca buffer in neurons is not, however, a trivial one, and many researchers have reported experimental findings suggesting that CaBP, via its high 2Ca buffering capacity, is associated with regulation of 2Ca fluxes through cell membrane (Kohr eta!, 1991; Lledo eta!, 1992) as well as with neuroprotection of neurons against excitotoxic injuries (Baimbridge and Kao, 1988; Mattson eta!, 1991), cerebral ischemia (Goodman eta!, 1993) and in neurodegenerative diseases (Yamada eta!, 1990). However, excitotoxicity, ischemia and neurodegenerative diseases are very complex processes that are not well understood and their pathophysiotogy cannot be explained on the basis of a single neuronal characteristic such as CaBP immunoreactivity and the consequent higher neuronal 2+Ca buffering capacity.
The complexity of the clinical phenomena no doubt underlies the fact that many studies have failed to detect a global neuroprotective role for CaBP in
disorders
discussed
causal
condition
CaBP
systemic
midbrain
SN
neurons
comes
in
(lacopino
dorsalis,
Alzheimer’s
loss
immunoreactivity
lacking.
disorders,
are
very
resistant
neurological
mRNA
were
strongly
of
Many
sensitive
and
relationships.
from
CaBP
in
have
but
spared
While
injections
is
and
to
may
and
an
in
evidence
the
of
presented
the
disease,
human
Andressen
CaBP
not
insults.
improved
immunoreactivity
these
protein
Christakos,
reflect
also
to
SN
lchimiya
same
(Yamada
in
ischemic
in
pars
been
of
the
brains
observations
for
several
other
A
associative
levels
For
(Freund
insult
MPTP
in
or
cerebral
compacta
detailed
eta!
a
et
Heizmann
reported
example,
generalized
reduced
eta!,
1990b)
with
injury
authors
a!
of
in
neocortical
(Lavoie
(1993).
(1988)
eta!,
CaBP
spite
in
Parkinson’s
1990).
cortex.
discussion
whereas
or
and
the
in
and
and
neuronal
and
hippocampal
(lacopino
coincidental
1990).
of
monkeys
were
and
reported
striatum,
neuroprotective
associations
the
yet
the
Braun
areas
Sparing
Another
Parent,
ventral
the
detected
pigmented
fact
of
survival
disease,
Also
and
in
CBP5
rendered
(1992)
dentate
a
hippocampus
that
marked
of
brains
CAl
phenomena
1991).
in
example
Christakos,
tegmental
between
dopaminergic
in
neurodegenerative
in
both
under
where
CaBP
and
pyramidal
role
the
granule
neurodegenerative
from
parkinsonian
loss
neuronal
some
SN
of
for
a
marked
the
area
patients
neuropathological
of
rather
conflicting
and
and
1990b)
CaBP
cells
neurons
CaBP
aspects
presence
neurons
CaBP
of
nucleus
hippocampus
populations
than
are
reductions
is
the
after
with
reported
still
in
highly
results
are
any
are
the
of
raphe
46 a
the
affinity
constants,
total
time
buffer
and
where of
buffer technical
which
enhancing
Ca 2 )
binding
passive
membrane-bound
passive mechanisms,
of
active’ THEORETICAL
a
Ca 2 +
steady
reverse
passive
course
Ca 2 +
Buffers
Calcium
concentrations,
and
are
the
regardless
constant.
Ca 2 +.
is
buffers
buffers
1D.
in
reasons.
determined
difficult
total
will
state
its Ca 2 +
content
the
of
reaction
buffer
in
affinity
where
buffering
CALCIUM
the
be
non-biological
This
system
CONSIDERATIONS
Ca 2 +
satisfy
which
equilibrium
buffering
of
more
organelles)
to
buffering
can
is
general
the
the
rate
isolate
for
fast
content
on
faster
bind
the
results
be
effective
is
energy
‘buffered’
Ca 2
the
constants
a
compared
adequately
capacity BUFFERS
physico-chemical
Ca 2
will
mechanism
definition
action,
and
buffers,
basis
systems:
or
of
in
are
state
be
extruded
test
the
during
a
reversibly
of
smaller
determined known.
calcium
are
is,
particularly
to
system
experimentally,
i.e.
of
concentration
described encompasses
AND
however,
these
Under
also
by
the
the
those
(by
change
which
is
initial
Under
without
cell.
required
BIOLOGICAL
is
definition
rate
sequestered
Ca 2
equilibrium
solely
changing,
when
with
if
complicated
The
a
+..ATPase
period
constants.
both
non-equilibrium
in
change
of
active
sequestration.
in
at
larger the
on
the
biological
the
of
order the
least
the
of
free
values
conditions
a
rate
(in
available
buffering
forward buffer
change,
concentration in
SYSTEMS
or
basis
in
to
by
intracellular
‘biologically
the
For
of
exchangers),
part
describe outcome
of
many
change
total
equal
as
of
conditions,
the
ligand
rate
Only
although
for
the
the
their
factors
content
forward
total
effects
in
the
of
of
Ca 2
(i.e.,
the
the
and 47
While
concerned
capacity
Baker
shield,
Experimental
Ca 2
functional
be
localization
processes. the
receptors
function).
and/or
allowing
usually
function
with
and
Cells
applicable
end-point
effectively
+mediated
Ca 2 +
transient.
A
Microdomains
many
Buffers
are
no
and
isolating
large sequestered
achieved
of
an
attempt
outside
in
resulting
signal,
Umbach,
microdomains
steady
with
This
to
the
specialized
of
Specificity
a
intense
number
evidence
in
a
state
buffered
all
immobile
At
biophysical
a
cell
biological
microdomains
intense
the
the
Ca 2 +
by
will
particular
state
a
in
of
by and
death.
flux
1987;
activation
sub-microscopic
of
cell)
relevant
be
activation
continuous
for
sub-cellular
active
to
of
and
may
mathematical
and
immobile
of
made
Ca 2
near
avoid
the but
systems:
both
Llinas
Ca 2
reaction
aspect
Immobile
spatial
brief
function
Ca 2
buffering
integrated
machinery
the
of
to
of
buffers
the
unwanted
of
etal,
intense
Ca 2
for
present
an
buffers:
Ca 2 +
change,
any
of
homogeneity
compartments
microdomain
In
signal
mixture
influx
a
Ca 2 +
as
level,
models
the
a
short
exists
1992;
of
systems.
flux
source. biological
a
in
for
activity
Ca 2 +
an
a
messenger
An
can
effects
and
the
or
cells
buffers
large
is
a
can
period
exhaustive
have
in
Zhou
release
increase
particular
dissipated
only
overall
equilibrium
signal
the
are are
never
concept
from
In
number
During
that
such
system
been
can
and
theory,
of
be
literature
extremely
probably
channel
by
cell
time
and
the
are
maintained
in
be
as
be
Neher,
review process
published
binding
the
by
cytosolic
and
of
certain
met.
performance.
rest
the
autonomous
can
the
used
stray
(a
diffusional
Ca 2
brief
(for
the
the
(a
probability
activation
effect
of
only
heterogeneous
1993).
of
Of as
+
point
within
to
Ca 2 +
assumptions
the
example:
least
significant
life
these
by
each
mediated
Ca 2 +
an
these,
specific
be
of
cytosol.
co
span
source)
effective
dynamic
cogent.
should fluxes
(i.e.,
buffers.
of
is
an
of
can 48
case
channels)
(e.g., extrusion/sequestration
negative spreading
bound
above the
concentration
concentration
with
receptor
entry/release
Ca 2 +
high
is
is extremely
conclusions
(1990),
evaluation
models,
short-lived
never
extent
of
The
high concentration
Near
reduction
signal
by
to
Ca 2 +
Feher
feedback
reached
sites
the
the
addition
facilitating
and
temporal
the
of
high
the
of
and
are
presentation
mobile
the
source
and
transport
signal),
the
by
for
high gradient,
point
eta!
values
of
based
maintains
functional
preventing
in
Ca 2
of quickly
experimental
VEm).
on
enough
of
precision
the
(1992),
sources
buffer,
diffusion
a
and
influx
with
a
mobile
(hundreds
on
across
fluxes,
bulk
stray
fixed
within
the
handled
of
While
the
several
to
a
will
isolation
by
and
the
cytosol
some
of
and
rest steep
Ca 2 +
buffer
Ca 2
of
evoke
work
intestinal
the
and
high
results
Ca 2 +
escape
reduction
Nowycky
the
Ca 2
of
specificity.
of
by
interesting
microdomain general
concentration
probably
from
of
micromolars).
local
buffer
helps
the
increased under
an
of
extrusion
entry
presented
Roberts
from
from the
‘unintended’
and cell.
nearby
[Ca 2 ]
restrict
of and
conclusions
to
normal
microdomain
or
the
enhancing
renal
the
the
the
Because
consequences.
In
release,
Ca 2
Pinter
(1994),
mechanisms.
microdomain
can
this
sources
microdomain
driving (e.g.,
in
cell
gradient
the
tubular
conditions
This
this
be
scheme,
flux
response.
(1993).
results
spatial
of
[Ca 2 +]
inactivation will
Sala
triggered
influx
concentration
thesis.
force
structure
cannot
the
is
epithelium
between
be
desirable
and
in
spread
steep
as
The
(and
low
by
First,
thus
for useful
can
a
These
the
reach
Hernandez-Crus
removal
reduction effectively
the
affinity
existence
discussed
thus
of reach
reducing
the
more
Ca 2 +
of
(Feher
for
in
Ca 2
Ca 2
of
a
the
the
the
Ca 2 +
of
Ca 2 +,
flux
in
et
flux
the
of
the
49 a
Relative
depend
been
isolation
effect
constants
and
extremely
activation
physiological
summation
loss
the
for
presynaptic the
in
reducing
concentration
attempt
al,
the
Ca 2 +
1992),
recovery
source
nanometer
of
The
It
loaded
Third,
Second,
and
loss
is
precision
on
affinities,
and
to
outcome
important
the
may
and
fast,
of
the
the
in
of
(i.e.,
of
restore
the
with
terminals,
concentration
other
process,
functions
peak
other
a
the
a
buffer
buffer
buffer
gradient
be
dimensions).
short
dissipation
physiological
fast
a
and
the
unnecessarily
rate
Ca 2 +
of
stray
signal
processes
cellular
cell
to
mobile
affinity
concentration specificity
the
range
buffer,
properties
stress
may
constants
while
may
types
between
signal).
signal.
whole
in
of
Ca 2 +
gradient,
lead
and
the
Ca 2
be
particularly
to
other
that
function,
Under
may
the
it
interrupted
Ca 2
may
microdomain
array
non-equilibrium
as
of
to
activated
This
the
This
and
homeostasis.
concentration
all
buffer
occur.
the
Ca 2 -mediated
an
much
are
these
the
source
result
and
may
buffering
of
plays
change
enhancement
Ca 2
such
the
the
derangements
processes
results
as
increased
(e.g.,
circumstances, not
in
and
major
a
of
it
cell’s
as
signal.
while
excessive
minor
in
depends
be
Ca 2 +
capacities
unintended,
neurotransmitter
reactions
gradient
‘remote’
in
concentration
desirable
determinants
endogenous
the
processes
discussed
increasing
signal
role,
of
Consequently,
entry
dissipation
spatial
on
(loss
waste
and
in
proteases
of
(with
summation)
binding
the
since
and
any
possibly
endogenous
of
the
with
above
and
[Ca 2
cell
of
profile
buffers.
of
microdomain
bulk
microsecond
at
release
it
prolongation
energy
the of
rate
temporal
that
all.
higher
means
+1
or
cytosol
are
the
harmful
may
away
buffer
will
kinases).
has
in
in
affinity
the
result
an
from
thus
of 50
buffer
mechanisms
Indeed,
important
properties subcellular
biologically
versus
buffers
loading
soma
will
for
in
of
domain
all
relevant
vary
determining
and
or
artificial
a
practical
priori
dendrites).
from
binding
to
events.
buffers
is
another
one
purposes,
impossible.
the
sites
cell
Similarly,
response
Additionally,
will
within
that
type
ultimately
predicting
mediate
to
the
in
to
another
experimental
same
the
the
Ca 2
determine
the
addition
properties
and
cell
+.sensitive
net
(e.g.
probably
‘biological’
situations,
of
their
artificial
of
presynaptic
processes
Ca 2
effects
from
effect
the
buffers.
one
regulatory
on
precise
terminal
will
of
the
be
as 51
and
I.
designed
neuronal
CaBP
injury,
intraneuronal
experiments
This
It
e)
and ci b) a)
d)
induced
To
Ca 2 +
as
less
like
depolarization-evoked
the
‘Increasing
especially
has
the
the
the
the
the
a
Ca 2 +
characterize
to
1E.
hypothesis
vulnerability expression
molecules,
vulnerable
Ca 2
been
stores).
test
effects
dependence
types
effect survival
calcium
excitotoxicity.
HYPOTHESIS
buffering
the
will
established
buffer,
during
neuronal
and
of
of
hypothesis
of
be
will
of
to
culture
will
primary
homeostasis
bFGF
sources
of
neurons
designed
CaBP
excito excitotoxic
and
of
capacity,
be
enhance
neurons
Ca 2
CaBP
tested
treatment
Ca 2
the
with
type
or
hippocampal
toxic AND
+
of
that: under
+
availability
by
buffering
to
expression
reasonable
Ca 2
to
on
loads
their
the
is
in
injury’.
insults.
artificially
satisfy
glutamate-induced
OBJECTIVES
an
the
primary
different
work
on
ability
signals
and
important
vulnerability
neuronal
capacity,
neurons
the
of
presented
In
consequently
on
certainty
cultures
loading
tools
to
view
following
culture
(VGCC,
culture
handle
step
survival,
in
to
of
either
OF
of
them
of
vitro
that
enhance
in
the
conditions.
in
type
excitotoxicity.
LGCC
agonist-
neurons
THE
hippocampal
objectives:
this
the
render
as
potential
the
with
with
CaBP
and
thesis
a
development
STUDY
and
loss
result
artificially
respect BA
or
age.
to
them
expression
intracellular
PTA
of
glutamate
has
role
of neurons
-
been
of
to:
of 52 53
II. To determine the effect(s) of artificially increasing the neuronal 2Ca buffering capacity using 1,2-bis-(O-aminophenoxy)ethane-N,N,N’,N’-
tetraacetic acid (BAPTA) and 5,5’-dimethyl BAPTA (DMB) on short and prolonged 2Ca + responses evoked by either depolarization or exposures to EAAs (NMDA and glutamate).
Ill. To determine the potential neuroprotective role of increasing the neuronal 2Ca buffering capacity using BAPTA and DMB on glutamate-mediated neurotoxicity.
VI. To determine the potential functional association(s) between the expression of CaBP in cultured hippocampal neurons and the kinetics of depolarization- and EAA-induced 2Ca + responses.
V. To determine the potential neuroprotective role of CaBP expression on the vulnerability of cultured hippocampal neurons to glutamate-induced excitotoxicity. 54
CHAPTER 2
MA TERIALSAND METHODS 55
I. TISSUE CULTURE
All experiments were performed on primary cultures of rat fetal and/or 4- day postnatal hippocampal neurons. a. Fetal cultures
Fetal hippocampal neuronal cultures were prepared according to Banker and Cowan (1977) with modifications (SoIc etal, 1993) as detailed below.
I) Dissection
Eighteen-day pregnant female Wistar rats were anesthetized using pentobarbital (intraperitoneal, 50 mg/kg body weight) and the abdominal wall was shaved and disinfected with 70% ethanol. A lower transverse abdominal incision through the skin, abdominal muscles and peritoneum was performed and the pregnant uterus, usually containing 13 to 17 fetuses, was excised. The mother was then euthanized with an intracardiac injection of pentobarbital.
All subsequent steps were performed under sterile conditions in a laminar flow hood.
In turn, each uterine sac was incised and the fetus was decapitated. The separated head was gently stabilized with a medium size forceps grasping the snout. Using fine scissors, a mid-line slit of the scalp and thin skull bones was extended in a caudo-rostral direction to the bregma then antero-laterally on both sides. The brain was exposed by peeling apart the skull flaps and a spatula was used to free the brain from the underlying structures. The brain was picked up by the brain stem and rapidly immersed in ice cold Ca/Mg free buffered salt solution (CMF BSS, 2 ml in a 35 mm-diameter plastic-2 tissue culture plate). 0.20 longer sterile of terminated at 0.5 (DMEM). 12 culture then DNAase/trypsin dish ii) dissection. hippocampi scissors hippocampi then dissected structure pia/arachnoid side 35°C Dissociation 10% strokes mg/mI and transferred of mm) dissected fire-polished visible Hippocampi Cerebral tube, each fetal for and chopped surrounding After for away respectively) followed by were 20 to bovine Dumont allowed hemisphere, whichever a replacing meninges a out minutes similar gentle hemispheres mixture using to 1 pooled 5 into were Pasteur a using ml by serum fresh to #5 two the mixing, number smaller tissue trituration as with were settle in transferred came in forceps. and a the bisected CMF much amount the pairs combination (FBS) pipette were periodic kept added culture hippocampus first. the and pieces vicinity of BSS of as in strokes through hippocampal separated in A excess (internal of Dumont corpus Dulbecco’s with possible to (final ice Pasteur tube using agitation. ice of the of cold ice the cold or a CMF containing enzyme sharp callosum. hippocampal a tip #5 was narrower until cold of by pipette hippocampus CMF pair CMF diameter forceps. modified the tissue BSS blunt Enzymatic visible and pieces CMF of BSS concentrations enzyme BSS was iris was blunt ice The diameter dissection. was BSS as for scissors. 0.45-0.50 of pieces in Eagle’s Each cold decanted. used vascular a a triturated tissue the dissection were digestion into mixture whitish 1 CMF 5 hippocampus duration to pipette and a ml medium gently were transfer 35 On 25 Pieces mm) tissue BSS. incubated with C-shaped Six through was mm pg/mI the with (0.15- no of for ml 3 were All medial Petri the iris ml of and 10- was a 56 the attachment Coverslips 0.2-0.3 drained few grade was borate and iii) and (dH 2 O). neurons/mi. and a method were the repeated cell in diluent a Plating coated cell cleaned hours 22 suspension a followed refrigerated performed water, Calculated Sixty standard The buffer mm-diameter off ml pellet (50 The for gentle before were coverslips of cell immediately and to p1 by cell arranged coverslips (0.15 laminin by was eighty of suspension laboratory boiling were with incubated pipetting on centrifuge suspension extensive volumes cell cell re-suspended M, a were 1 solution then to glass suspension plating, sample in at pH ml before were reach 100 60°C through of of hemocytometer. 8.4) for transferred mounted was rinsing coverslips at to gently FBS of then coverslips mm-diameter (16.7 4°C. 2 the neuronal containing attain for purified the hours in in and a desired (more autoclaved 30 1 mixed 450 pg/mI) suspension 5 The in ml the centrifuging (Baxter ml to in minutes a using plating. were p1 of custom than supernatant 6 5% pipette. final final poly-D-lysine neuronal of or prepared DMEM Petri DMEM CO 2 0.4% #1, 1 an rinsed 20 and cell 2-well in plating using dishes designed FBS times) concentrated 0.17 at Viability density for then containing with suspension wlv in twice 35°C was gradient tissue a 8-10 density L-15 mm and trypan kept (PDL, in trypan 10% carefully stainless of distilled and in to minutes thickness) each (Gibco), culture for tissue 1 allow 10% FBS 15 (see by .25x10 6 blue cell were nitric blue 24 pg/mI). covered underlying was water FBS counting below). removed steel cell exclusion hours culture- at plates in which acid. plated of 150x saline) used viable using rack 18 A in with This was on G and the as 57
processes
configuration,
vitro
originally
low above
surface
neurons/cm 2 distinct
surface
cell
iv)
neurons/cm 2
Large
culture
medium
hours
addition
specified.
35°C
serum
changed
(Falcon)
Variations
density
density
with
22
Smaller,
Two
and after
and
Unless
(HS)
age,
facing
amongst
facing
with
of
for
developed
to
mm-diameter
clear
that
the
5%
different
5-fluoro-deoxyuridine
was plating.
neuronal
in
N2
a
plating
and
and
fresh
otherwise
neurons
further
down,
plastic
up.
order
tended
C0 2 195%
18
background,
supplemented
high
the
maintained
maintained
mm-diameter
In
N2
day
in
to
Nutrients
plating
closely
cultures
and
i.e.
surface
this
3
to
our
remained
supplemented
coverslips
improve
hours
was
indicated,
form
Air
neuronal
cells
configuration
laboratory
protocols
distinct
packed
counted until
in
in
(see below.
with
were
DMEM
neurite
were
tissue
tissue
coverslips
neuronal
in
(5-FDU,
were
used
also
glial
excellent morphology
2
replenished
neurons.
neuronal
sandwiched
mI/well
in
DMEM
and
This as
were
bundles,
culture
culture
Brewer
multiplication
7
plated
order
neurons
day
attachment.
to
the
20
were
‘face-down’
used
10
condition
twice
zero
pg/mI)
of
to
morphology plates
plates
neuronal
at
and
particularly
and days
DMEM
by
plated
enhance
survived
a
with
between
in
density
weekly.
replacing
Cotman,
neurites
to
with
with
vitro
except
was
fetal
for
Finally,
the
at
cultures
containing
culture
the
neuronal
the
for
a
(0
up
inhibited
and
the
of
in
culture
neuronal
low
In
where
Dlv).
were
1989).
half
to
cell-coated 3-4
older
cell-coated
2-3x10 5
the
extensive
glass
specifying
method
incubated
density
6
the
weeks,
weeks
5%
not medium
medium
survival
cultures.
otherwise
by
coverslip
cultures.
In
culture
visually
a
horse
this was
of
single
in
final
at
1O 5
was
48
in 58
A
FBS/HS)
cell
centrifugation
After
replaced supplemented
(25
initiated
centrifuge
chopped
Petri
individual
out
atmosphere.
technical
however,
Baughman,
neuronal
These
b.
procedures
calculated
pg/mI)
pellet
Postnatal
and
dish
incubation
At
Four-day-old
The
‘face-down’
by collected
was
with
into
the
cultures.
was
differences
filled
brains
required.
tube
medium.
stages
replacing
were
1
volume
end
added
986)
slices
cultures
The
serum-free
on
with
re-suspended
with
filled
for
on
a
of
performed.
in
of
neonatal
were
FBS
papain
A
Wistar
less cultures
filter
to
30
the
ice ice
of
Mechanical
preparation
with
the
between
short
adjust
EMEM
minutes
gradient
cold
cold
dissection
then
essentially
ice
paper
Eagle’s
ice
(1
rat
rats
description
were
in
cold
L-15. L-15
mg/mI),
the
1
cold
pups
supplemented
our
EMEM
soaked
mm
were
at
were
trituration
of
minimal
cell
L-15
turned
medium.
35°C L-15
handling
procedure
With the
were
postnatal
in
density
decapitated
BSA
and
performed thickness
medium
in
of
same
medium.
the
fine
right
anesthetized
essential
ice
this
viable
(0.2
and
of
Hippocampi
papain-BSA-DNAase
with
cold
scissors,
to as
enzymatic
neuronal
side
procedure
fetal
with
cell
mg/mI)
4x10 5
for
and
cell
as
and
5%
L-15
medium
up
purification
and
the
previously
5
collected
counting
the
FBS
before
ml
by
hippocampi
cultures
neuron/mI.
and
and
preparation
postnatal
dissociation
were
highlighting
pro-warmed
exposure
brains
and
(EMEM)-DNAase
DNAase
transferred
any
dissected
was
in
described.
5%
using
(Huettner
were
1
mixture experimental
cultures
5
were
performed.
Cell
HS
to
of
ml
(25
was
the
L-15
high
fetal
dissected
(EMEM
to
from
pg/mI).
and
was
a
is,
The
CO 2 59 a. were II. days with of was schemes. determined concentrations was (CaBP) the c. glial then (EMEM-N2). plates, suspensions previously culture Principle INTRACELLULAR bFGF-treated medium supplemented used cell 1 later, the treated The For Basic For ml in face proliferation. medium the of medium the to fluorescent the On 2Opl described, for of fibroblast with up, EMEM-N2 increase with were Arabinoside-C postnatal first the first Fura-2 the (1-10 in of cultures fresh was 20 basis in 24 then an with use 24 bFGF CALCIUM each UI ng/mI the calcium hours incubator and Ca 2 + growth hours, replaced Replenishing of containing EMEM-N2 cultures of of plated bFGF stock percentage bFGF well. the 0.5% these in after (Ara-C, sensitive cells factor (2.5 0.5% measurement coverslips on MEASUREMENT solution by in at (Collazo In trials, BSA plating twice 18 our 10pM were an 35°C ng/ml). all nutrients 10pM) BSA) (human mm-diameter of equal dye (vehicle experiments cultures. the weekly. (125 maintained neurons eta!, in the were Ara-C was fura-2 following The an volume was culture recombinant ng/ml) was solution). atmosphere then 1992). tested medium and expressing was added achieved using coverslips in of incubated 2.5 medium were treatment the as 1 N2-supplemented A 48 ml was ng/ml well bFGF, range probe bFGF, added of hours of by calbindin-D28k (EMEM-FBS/HS) totally as 5% prepared EMEM-FBS/HS in of replacing protocol of control used several R&D 6 to later bFGF. CO 2 or each replaced for 12 Systems) to in as cultures was dosage EMEM Two half well all 1 inhibit air. ml 60 61
experiments involving intraneuronal 2+Ca measurements. Fura-2 is a 2+Ca chelator with a BAPTA-like 2Ca binding site coupled to a fluorophore in a way that makes its fluorescence sensitive to 2+Ca binding. In the absence of free 2Ca fluorescent emission at 510 nm wavelength 510(EM will have a peak intensity, when fura-2 molecules are excited at 362 )nm while +bound 2Ca fura 2 molecules will have a peak emission intensity when excited at 335 nm wavelength. This 2Ca dependent shift in peak excitation wavelength is associated with a negligible shift in the peak emission wavelength from 510 nm in the 2Ca-free fura-2 to 505 nm in Ca-bound fura-2. These properties make fura-2 a very useful probe for free2 2+Ca measurement using a dual- excitation/single-emission wavelength protocol (Fig 2. 1).
As the free Ca concentration 2+ 2+])([Ca around fura-2 increases, the EM510 at 380 nm excitation wavelength )(Fl380 decreases while fluorescence intensity at 335 nm excitation wavelength )(Fl334 increases. The ratio between Fl334 and Fl380 /F1=Fl3343803341380 which also increases with increased [Ca has a wider dynamic range than the individual 2+], ),(R fluorescence intensity values (Fig 2.2). Assuming that Ca:fura-2 complex has a stoichiometry of 1:1, 3341380 is related to 2 according to the following equation R 2[Ca (Grynkievicz eta!, 1985): j
* * ]21[Ca = ( Kd (R3341380 - Rmin) / (Rmax - )R3341380 In this equation the proportionality coefficient 1, is the ratio of Fl380 in zero 2[Ca to Fl380 in saturating 2],[Ca Rmin and Rmax are R3341380 ] +1 fluorescence ratios in zero and saturating 2[Ca respectively, and Kd is the 62 affinity constant of fura-2 for 2+Ca under specific experimental conditions of pH, temperature, viscosity and ionic strength.
Rmin, Rmax and f are influenced by the optical properties of the experimental equipment (e.g., transmittance and reflectance of filters, mirrors, prisms and lenses) as well as by the spectral sensitivity of the photo-sensitive device and the electronic gain level in the acquisition and digitization equipment.
Intraneuronal free 2+Ca level can be probed using fura-2 if the dye is introduced into the cytoplasmic compartment. However, fura-2 is a highly charged polyanionic tetra-carboxyl molecule and is thus plasma membrane impermeant. A variety of loading techniques have been devised to accommodate particular experimental paradigms and cell types. These methods include direct intracellular injection of fura-2 penta-potassium salt, cell permeabilization and the use of the acetoxymethyl ester of fura-2, fura-2 AM (Tsien, 1981). The latter is the preferred method for loading a large number of cells, especially small cells, simultaneously and is the method of choice for neuronal cultures. The membrane permeant fura-2 AM diffuses easily into the cytoplasmic compartment where it is quickly de-esterified by cytosolic esterases to yield the anionic membrane impermeant fura-2, that becomes trapped inside the cell, and formaldehyde that quickly diffuses out. The use of R3341380 as the experimental measure of free 2+Ca (ratiometry) has significant advantages over the use of raw fluorescent intensity values. In addition to the wider dynamic range mentioned above, ratiometry corrects for a variety of confounding factors encountered with the use of all ion-sensitive fluorescent probes. Cell-to-cell variability in fura-2 fura-2 fura-2 measure active of wide remedies, characterized free probe the bleaching. index single-excitation/single-emission be is dependent correction are uniformities intensity result path loading a fura-2 performed. excitation also minor Ca 2 + variety through for of Although extrusion. AM If in in (e.g., fura-2 corrected [Ca 2 +] 1 choice cultured artifactual requires that the are problem efficiently concentration as processes in of detection in long welt a are different path. the is spectral single other for the changes excited The hippocampal only automatically established as without optical for many differences literature This with they such Ca 2 + affinity cell standard ratiometry of cell characteristics (Fig at is reasons. over intra-experiment minimal system (edge-to-center affect the as esterase the 362 sensitive 2.1) so of photo-bleachin:g, (Table a neurons need and corrected isosbestic UV fura-2 that nm, ion wide Fl 334 while (e.g. and subcellutar Our apparent optical activity sensitive for EM 510 2.1). potential fluorescent range is and and for and it laboratory mathematically non-homogeneous a is by increase point Ca 2 + function dye elements, Fl 380 affinity Neurons a the and becomes of ratiometry. gradients sequestration major probes problems, loss neuronal leakage dye and cell makes equally. probes has in coefficients, of due confounding has it are size) where and intracellular independent the can long in intensive or it to known been activity. are as Moderate raw routines and sensitive amount extrusion be leakage illumination Uncontrollable experience and well ratiometry available a extensively fluorescence variable very to fura-2 practically as factor shading light Also, de-esterify for of of or enough non- their of useful fura-2 ambient with photo- its optical was path) cannot patterns) the with for the use time a dye no our to use in can 63 distance microscope fluorescence c. de-esterification warmed before compartmentalization temperature, in hydrophobic mixing test pipette (30 b. more necessary IND0-1 imaging are BSS Image Fura-2 pg implemented tube expensive The Fura-2 Five any (1.5 on in tip offers BSS 40x system. and Acquisition 25 a imaging AM to acquisition Ca 2 + ml equipped ml/coverslip) vortex p1 fura-2 imaging objective fluorescence and neurons produce stirred of all Loading anhydrous of than BSS measurements in allowed the The hardware fura-2 stirrer AM. almost vigorously system with advantages that supplemented of were a hardware single-excitation/dual-emission (Zeiss final fura-2. at Neuronal AM. were to for a was DMSO) every well 33°C 100 cool was solution using LD fura-2 captured required using for loaded were After UV-Achroplan watt commercially down of built for was cultures Attofluor dual-emission with ratiometry dual-excitation of 60-70 a attempted mercury loading, around added with vortex to to 6 on 0.04% pM room achieve were a minutes. no imaging ZeissAttofluorTM slowly, fura-2 a stirrer. cultures arc available 0.6, but visible Zeiss temperature in BSA then microscopy lamp implementation dispersion order systems. Ph2). AM. software. Ca 2 while were Axiovert-lO incubated At The subcellular were and digital to this + BSA Excitation fura-2 placed sensitive stirring, ensure a for transferred concentration of is long fluorescence and digital significantly 60-90 with the AM fluorescent in of complete working vigorous using probe a light the the solution 15 minutes to fura-2 a was ml pre fine and 64 65 filtered through 10 nm band pass 334 nm or 380 nm excitation filters which were positioned into the light path using a computer-controlled solenoid filter changer. A 395 nm dichroic mirror and a 510 nm long pass filter completed the optical requirements for fura-2 excitation and emission. The photosensitive detector was a high sensitivity, Attofluor intensified charge-coupled device
(CCD) camera and images were digitized on a Matrox-AT image processor. The imaging software controlled the excitation filter changer, camera gain, image processor in addition to data processing, display and storage. The software was operating on a dual-monitor IBM-compatible 80486-33MHz. A schematic illustration of the imaging hardware is shown in Fig 2.3.
Using the imaging software, suitable gain and exclusion values were selected. Up to 99 regions of interest (ROls) could be positioned over the cell images in the selected experimental field. ROls were generally placed within the bounds of cell somata and average fluorescence intensities of all the pixels within the ROl were obtained at 334 nm and 380 nm wavelength. The frequency of ratio image acquisition was varied dynamically between 1 ratio every 1.5 seconds to a ratio every 900 seconds depending on the experimental protocol. Less frequent acquisitions were used whenever possible to reduce the risk of UV-induced photo-bleaching and photo-toxicity. For the same reasons, neutral density filters in the excitation path were used whenever fluorescence intensity was sufficiently high. On-line background correction was never necessary since cellular autofluorescence was below detection.
Background (BG) readings for both excitation wavelengths were, however, recorded from several cell-free regions in the field and used later for BG correction during off-line data analysis. The major source of these BG values was electronic noise in the acquisition hardware, although other factors such 66 as autofluorescence in the perfusion media and glass coverslips and light scatter in the optical path made some contribution. Although frame averaging could be performed using Attofluor software it was not used since illumination intensity and signal-to-noise ratio were very stable. d. Test drug exposure protocols and perfusion
Several perfusion chambers were used in experiments involving intracellular 2+Ca measurements. The most elaborate chamber was used for experiments involving transient (less than 30 seconds) exposure to test drugs. The chamber was manufactured in the workshop of the Department of Physiology (UBC) according to our design specification. The chamber design permitted excellent laminar flow, relatively rapid medium exchanges, regulated temperature and control over CO2 around the perfusate (Fig 2.4). An alternative chamber with a fixed glass bottom (0.17 mm thick) was also used which was more secure against leakage, especially during 24-hour experiments, and allowed for a much faster and more efficient exchange of medium as well as a simpler coverslip replacement. However, it lacked temperature and atmospheric control.
A peristaltic pump was used to superfuse cells with BSS at a rate of 1.8- 2 mI/mm at 21-22°C. Unless otherwise indicated, BSS for perfusion was 2Mg free and contained 1 pM tetrodotoxin (TTX) to block voltage-gated sodium channels. Drugs that required transient application were prepared in
BSS and applied into the chamber through a 1 ml syringe connected to a 1 Y2” 30G blunt needle pointing to the inlet of the chamber. The drug application assembly was fixed to the microscope stage using a small C-clamp. The peristaltic pump was stopped during drug application and the exposure to test 67
drugs was terminated by resuming perfusion together with a flush of 1 ml of BSS through the drug application syringe.
In some experiments where long exposures to test drugs were needed, an alternative perfusion method was employed. The glass coverslip was glued onto the bottom of a well in a 12-well tissue culture plate using a trace of silicon rubber (Dow Corning). A small perfusion-head assembly was attached to the wall of the well with one perfusion inflow line and two suction lines. One of the suction lines was positioned near the bottom of the well and could be used to empty the well completely while the other suction line was well above the bottom and allowed about 1 ml of BSS to remain in the well at all times during perfusion. With the use of a 1 ml pipette and properly timed control of which line was open, a total exchange of the medium in the well could be achieved in less than one second (Fig 2.5). This configuration was particularly useful in experiments where relocation of the cells under examination was required following subsequent immunohistochemical staining (see below). e. Fura-2 calibration
An in situ calibration method was used to convert fluorescence ratios to absolute 21[Ca Fura-2 loaded cells were treated with 5-10 pM of the non fluorescent] ionophore, 4-Bromo-A23187 in BSS while recording fura-2 . 2Ca fluorescence ratio. In the presence of standard BSS 2[Ca +1 of 1.8 mM, the acquired ratios represented Rmax. The medium was then replaced by 2Ca +4ree BSS, supplemented with 200 pM EGTA, and Rmin was obtained. The proportionality coefficient, f, was calculated as the ratio of Fl380 Zero Calciumto 68
Fl380,MaximumCalcium We used the published fura-2 Kd values of 135 nm at 20°C and 224 nm at 37°C (Grynkievicz eta!, 1985).
Calibration was not performed routinely at the end of every experiment but generally when there was any change in the optical properties of the system (e.g., re-alignment or installation of a new lamp). Although our 2+Ca imaging results are presented as absolute 2+Ca concentration in nM, it is important to stress that, in most experiments, control results were obtained from sister cultures under identical experimental conditions and thus trends of change have more relevance than the absolute values of change in 2+.Ca A variety of calibration techniques are employed by different laboratories (e.g., in situ, in vitro and spectrofluorimetric). Each of these methods relies on a different aspect of the experimental setup, i.e. cells, dye and hardware properties, and they tend to produce different calibration parameters even when performed in the same laboratory. Indeed, problems with different calibration methods have led some authors, e.g. Dubinsky (1991a), to report data as fluorescence ratios rather than absolute 21+][Ca together with a sample of the calibration values (Rmin, Rmax and ,f values) to facilitate ‘coarse’ comparisons of results across different laboratories. In our imaging system
Rmin, Rmax and I averaged 0.2, 10 and 8 respectively. f. Relocation Experiments
In order to relocate a particular field on a coverslip following 2+Ca imaging and subsequent immunohistochemistry (IHC), the coverslip was attached with a minimal amount of silicon rubber to another coverslip with hand-made diamond pen grid scratches. Care had to be taken to avoid having the silicon rubber in the way of the test fields as it impaired microscopic 69 visualization. Also while the rubber was setting, two exchanges of the medium were performed to remove the traces of acetic acid. With the two coverslips attached back-to-back, the grid was visible and the assembly was sufficiently sturdy for handling during imaging and IHC.
An alternative marked field setup was used for experiments performed using the multi-well culture plates. In this case the grid was drawn with a fine tip permanent marker pen on the undersurface of the tissue culture plate and the test coverslip was attached, with the same precautions as above, to the plate using traces of silicon rubber. The use of the culture plate as an attachment platform had the advantage of making relocation extremely easy as orientation in the horizontal plane was maintained by the mechanical stage of the microscope, i.e., no field rotation errors existed.
Ill. FURA-2 DATA ANALYSIS a. Background correction and calibration
Experimental data of 2Ca imaging experiments were initially stored in binary Attofluor data files (ADF files) that could be converted into standard comma delimited ASCII file formats. The magnitude of the data generated made it practically impossible to use commercial spreadsheets and statistical packages to perform data processing and analysis. To automate and speed up this process several programs were written in Microsoft QuickBASICTM v4.5 to perform off-line BG correction, selected ROls separation, ratio calibration, descriptive statistical calculation, and preliminary graphical display of results. Final graphs and tables were generated using Borland Quattro ProTM v5.O or Poly Software International PsiPlotTM v2.O. temperature, AM prepared acetoxymethyl capacity buffers control used IV. were Ca 2 + measured the respectively, Ca 2 + (change/mid-span) rate responses [Ca 2 +] Ca 2 BAPTA b. Kinetic stock increase LOADING of to performed Two BAPTA, response response buffering For molecule, change were study loading) over of as solution the experiments in analysis artificial cultured 20 (slope-up) prepared unless the the of the time NEURONS dimethyl-BAPTA esters of mM to was the properties using several response neurons N-(o-methoxyphenyl)-iminodiacetic consequences [Ca 2 in its was course Ca 2 solutions peak otherwise neurons. described BSS. duration. and a by studying and used parameters Quick WITH change vigorously studied stock buffers, between during (e.g. duration, Neurons recovery as in specified. BASIC using and BAPTA The anhydrous solutions a Ca 2 different of in which (Fig descriptor BAPTA 25% artificially Ca 2 . half-BAPTA were were calculations slope mixing mid-span phases program 2.6). transients OR [Ca 2 +] 1 and After of used CaBP incubated and of DMSO. ITS Finally, a These these of (slope-down). [Ca 2 +] 75% calculated increasing and 5,5’-dimethyl-BAPTA, written to ANALOGUES loading, the immuno-reactivity were remained and in describe buffer-AM of low-span the included relation Working populations in acid statistical changes the all for the aspect cultures amount the obtained peak (half-BAPTA), that above mixture and of The basal Ca 2 solutions parameters compounds the measured ratio purpose. change compare comparisons with duration were of 50% magnitude as and levels buffering at the different and room rinsed or of peak during buffer were that Ca 2 + as or these 25%, of one were the the of 70 71 twice in BSS and left for 20-30 minutes to ensure complete hydrolysis of the AM ester before testing. When loading was conducted at 33°C, at least 60 minutes were allowed for complete de-esterification of the buffer ester.
V. INDUCTION OF EXCITOTOXICITY a. Exposure protocols
Two paradigms were used for exposing hippocampal cultures to excitotoxic amounts of glutamate or NMDA. ii 30-minute exposure protocol
Test coverslips were transferred from culture medium into 2 ml of a pre warmed BSS and allowed to cool down to room temperature. BSS was then replaced with an equal volume of BSS containing the excitotoxic drug. After
30 minutes a triple exchange of the BSS was performed to remove all traces of the excitotoxic drug and cultures were incubated for 20-22 hours at room temperature prior to neuronal viability assessment.
II) Extended exposure protocol
This alternate exposure protocol was devised to avoid some problems encountered with survival of control cultures using the above method. A lOx concentrated solution of the excitotoxic drugs in BSS was filter-sterilized (Costar Syrifil-MF 0.22 pm) and appropriate volumes were added directly to the test cultures without replacing the culture medium. The cultures were incubated for the test period (20-24 hours) in 5% 2CO at 35°C prior to viability assessment. satisfactory plating well) extremely culture Wroblewski LDH when using technique negligible. glial ii and neurons. b. injury where exposure temperature), neurons no protocols. Lactate Viability alteration sensitive contribution release used 0.1 compared site. Several This increases released to and % the dehydrogenase method LDH high assumes in In protocol It Triton-X. results and assessment method some glia of index other. our required techniques and release neuronal endogenous environmental LaDue to the to may hands, also no of the using that of LDH in has LDH These the loss Total have neuronal minimal in simulated our total (1955), LDH plating many release the LDH an previously releasable were (LDH) of hands accumulated differences releasable releasable LDH excitotoxins culture is any release advantages manipulation conditions tested was released density injury and in more release may potential both also fraction cited medium Trypan in in for be LDH, closely LDH in (up control control culture only method. extremely in are understandable use (pH, the publications over growth the to pool blue of assayed and has from not usefulness in in 106 bicarbonate cultures: medium and cultures total (Koh viability measured vivo exclusion probably removed been injured High factors neurons/35 variable treatment using medium-exchange and excitotoxic that reported density during no was assessment and in Choi, neurons which diminishes quickly the after content reported medium from view unacceptably efficiency groups mm incubation. method 1987). as neuronal culture the situations one of from and a diameter and exchanges, the reliable cultured of culture sister is that of the This of cultured lysis the This high the to 72 73 culture LDH content variability that may have been caused by the variable glial number in our cultures.
While the rationale behind the use of the LDH release method is the ability of healthy cells to retain macromolecules, viability assessment using the trypan blue exclusion method relies on the ability of healthy neurons to exclude extracellular trypan blue (0.04% in BSS) from their cytoplasm unless their membrane integrity is breached. We used trypan blue exclusion in some preliminary experiments but live/dead cell contrast was too low for photography and trypan blue itself was toxic, making it impossible to use for more than a single time point assessment of viability in the same sample. ii) Fluorescent viablilty probes
Fluorescent live/dead cell markers offer a significantly improved performance over traditional viability assessment methods. They have very high contrast and sensitivity while maintaining extremely low cell toxicity levels. Combining live and dead cell markers in the same procedures gives simultaneous indications of viability index, i.e. percentage survival, that can be easily followed as a function in time. The probes we selected for use were calcein AM and ethidium homodimer (EthD).
Calcein is a fluorescein analogue with high fluorescence yield, slow fading and long retention half-life inside the cytoplasm. As a lipophilic acetoxymethyl ester, calcein AM has access to the cytoplasmic compartment where it is hydrolyzed by cell esterases to produce the polyanionic membrane impermeant calcein molecule. In healthy cells with an intact cell membrane, calcein is retained and fills the entire cytoplasmic compartment including the finest neuritic processes. When excited at 460-490 nm, calcein emits a bright
Criteria
fixation.
Nikon
iii)
conditions,
‘viability’
dead
yellow/green
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Contax
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Morphological
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number
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temperature,
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and
when
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a 74 75 fairly homogeneous cytoplasm with minimal vacuolation, one or more neuritic extensions at least twice as long as the neuronal soma, and a general outline of the soma not indicative of excessive cell swelling. Dead or dying neurons showed distended spherical somata, dark granular cytoplasm and loss of neurite integrity. The results of morphological assessment correlated very closely with those of fluorescent viability assessment methods.
VI. STAINING AND PHOTOGRAPHY a. Immunohistochemistry
Cultured neurons intended for histological processing were fixed by replacing the bathing medium or BSS with 2% calcium acetate in 4% paraformaldehyde at room temperature for 1-2 hours. The cultures were then rinsed twice with phosphate buffered saline-azide (PBS-Azide, 0.04% sodium azide in 0.1 M PBS, pH 7.4) and stored at 4°C until processed.
Three-layer IHC was performed at room temperature except where specified (Sloviter and Nilaver, 1987). The staining procedure was initiated by replacing PBS-Azide with Tris buffer (0.1 M, pH 7.6) for 15 minutes. The endogenous peroxidase activity was removed by incubating cultures with freshly prepared 20H (1 % in Tris) for 30 minutes. Residual 20H was removed and cultures were incubated with Tris-A followed by Tris-B for 15 minutes each on a 60 RPM rotating shaker. Primary antibody in Tris-B was then added and allowed to react with its target protein for 48 to 72 hours at
4°C. After the cultures were rinsed again in Tris-A and Tris-B, the second layer of the reactants (biotinylated Protein-A, 1:400 in Tris-B) was added for 1 hour. This was followed by washing in Tris-A and Tris-B (15 minutes each) and then incubation for 1 hour in avidin/biotin-peroxidase (1:1000 Vectastain 76
Elite ABC Kit, Vector Laboratories, CA) in Tris-B. A double rinse in Tris (15
minutes each) was then followed by colour development using a DAB/glucose
oxidase mixture for 30-90 minutes. The colour reaction was terminated by the
removal of DAB reaction mixture and rinsing twice in 0.1 M Tris buffer.
Cultures, on coverslips, were then dehydrated in alcohol, cleared in xylene and mounted on standard microscope glass slides.
No attempt was made to quantify the intensity of neuronal staining and the neurons were classifies only into immunopositive or immunonegative sub- populations for the antigen under study (e.g., CaBP and CaBPN).
Primary antibody sources and dilutions
Primary antibodies for CaBP and parvalbumin (PV) were raised and characterized in our laboratory by Dr. KG Baimbridge. They were used at dilutions of 1:1500 and 1:800 respectively. Neuron-specific enolase (NSE) was obtained from Dako, Copenhagen and was used at 1:2000 dilution. Monoclonal antibodies against glial fibrillary acidic protein (GFAP, Sigma) were used at a dilution of 1:100. b. Cresyl violet (CV) staining
CV staining was performed according to standard procedures (Culling,
1974) alone or as a counter stain after other immunohistochemical methods. A schematic of the CV staining procedures is shown in Table 2.2. c. Photography
For black and white photographs, Kodak TekPan ASA 50 was exposed at the specified sensitivity rating and developed using HC-1 10 developer, dilution 77
D. Final prints were made on glossy Ilford Multigrade Ill paper (RC Deluxe). Colour slides were taken using Fujichrome-1600d exposed at 800 ASA
(fluorescent microscopy) or Kodak Tungsten 160 exposed at 160 ASA (light microscopy). Both were processed commercially.
VI. STATISTICAL METHODS
Statistical comparisons between different treatment groups (e.g., CaBP versus CaBPN neurons) were performed using unpaired t-tests. Multiple comparisons (e.g., in some excitotoxicity experiments) were performed using Newman-Keuls multiple comparisons test. In order to test the effects of buffer-loading on the kinetic parameters of 2+Ca responses, comparisons were made between the parameter values in the same group of neurons before and after the buffer-loading procedure. Estimates of 50EC values were obtained by fitting the experimental data using non-linear regression to Hill’s equation. Two statistical programs were used for data analysis: KwikStat v3.3 (for t-tests and multiple comparisons) and NonLin v2.1 (for non-linear regression fitting). 78 Sources for Materials
Gibco: CMF BSS, DNAase, DMEM, EMEM, Fetal bovine serum, HBSS, Horse serum, Laminin, L-15 medium, Trypsin. Molecular Probes: BAPTA-AM, DMB-AM, BAPTA-AM, Calcein-AM, Ethidium Homodimer, Fura-2 AM. MTC Pharmaceuticals: Pentobarbital. R&D Systems: bFGF.
Sigma: 5-FDU, Ara-C, DNAase, N2 supplement, PDL , TRIS base, and other unlisted laboratory chemicals.
Preparation of media and BSS
Media sterilization: All liquid media were filter sterilized (Costar, 0.22pm filters) and stored at 4°C for up to 4 weeks.
Dulbecco’s Modified Eagle’s Medium (DMEM) Diluted as per instructions and supplemented with: HEPES 10mM NaHCO 22 mM Serum3 (HS 5% and FBS 5%) or N2 supplement pH adjusted to 7.35 and stored at 4°C.
Minimal Essential Medium with Earl’s salts (MEM) Diluted as per instructions and supplemented with: Glutamine 0.4 mM D-Glucose 10 mM HEPES 10mM NaHCO 22 mM Serum3 (HS 5% and FBS 5%) or N2 supplement pH adjusted to 7.35 and stored at 4°C.
N2 Supplement (concentrations in working media)
Insulin 5 pg/mI Transferrin 100 pg/mI Progesterone 20 nM Putrescine 100 pM Sodium Selenite 30 nM BSS for perfusion in 2+Ca measurement and excitotoxicity experiments NaCI 139mM KCI 3.5mM 79 NaHPO 3mM 24NaHCO 2 mM HEPES3 acid 6.7 mM HEPES-Na 3.3 mM D-Glucose 11 mM 2CaCI 1.8 mM 2MgCI 0.8 mM Glycine 2pM TTX 1pM pH is adjusted to 7.35 with 0.1 N NaOH Notes: - For Ca-free BSS, 2CaCI was omitted and 200 g1M2 EGTA was added. - Mg’ was omitted from BSS to obtain Mg free BSS. -2 - High extracellular potassium medium (HK) was prepared by equimolar replacement of 46.5 mM NaCI with KCI to reach a final concentration of 50mM K. Other solutions Calcein-AM and Ethidium homodimer solutions Stock solutions: Calcein-AM 1 mg/mI DMSO Ethedium homodimer 1 mg/mI 2OdH store at -80°C. Working stock solution: Calcein AM Stock solution 100 p1 Ethedium homodimer stock 50 p1 0.9% Saline 850 p1 store at 4°C.
Note: - Final cell labelling solution is prepared as lOOx dilution of the working stock in culture medium or BSS. Borate buffer
Sodium tetraborate 150 mM pH is adjusted to 8.4 using HCI
DAB Glucose oxidase mixture (per 100 ml of Tris-B)
DAB 5 mg/dl Glucose oxidase 0.3 mg/dl Ammonium chloride 40 mg/dI I-D(+) Glucose 200 mg/dl 80 Phosphate buffered saline (PBS, pH 7.4) NaCI 150mM NaHPO 8mM 24POKH 2mM Tris Buffers
Tris (Tris Base) 0.1 M adjusted to pH 7.6 Tris-A Tris with 0.1 % Triton-X Tris-B Tris-A with 0.005% BSA 81
Figure 2.1: The excitation spectrum of fura-2.
The emission intensity at 510 nm is plotted (ir arbitrary units) as a function of the excitation wavelength for fura-2 under Ca + free and saturating’ high 2Ca + conditions (reconstructed from Molecular Probes Catalog 1992-94 by Richard P Haugland, figure 20.1, page 114). The major feature of this excitation spectrum is the shift in peak intnsity from 362 nm to a shorter wavelength of 335 nm, as the ambient Ca’ changes from 0 to a saturating concentration of several millimolars. Another feature is the presence of an isosbestic wavelength of 362 nm where fluorescence is independent of 2+Ca (dotted line and arrow head). Dashed line A and B (at 334, and 380 nm respectively) represent the wavelengths used for fura-2 excitation in our imaging system. These wavelengths are chosen because they offer a wider dynamic range of the fluorescence ratio than the range offered by the peak emission lines (362 and 335 nm)
A B
> (I) C U) C C 0 1-’ (‘3 C.)x w
250
Excitation Wavelength (nm) 82
Figure 2.2: Changes in fura-2 fluorescence in a typical 2+Ca response. An idealized 2C response in a fura-2 loaded cell is illustrated showing that an increase in [Ca j1 is detected by an increase in the ratio (thick line) of the fluorescence intensity at 334 nm to the fluorescence intenity at 380 nm (F1IF1334380 thin lines). Note that with the increase in [Ca’], the Fl34 is increased while Fl380 is decreased. The change in the fluorescence ratio has a wider, dynamic range than the change in the individual fluorescence intensities.
240 2.4
200 2.0
Cl) C D Cl) 160 1tI.’J 0) C.) C C D a) C.) 0 U) 0) 120 (‘3 0 1.2 U-
80 0.8
40 0.4 0 50 100 150 200
Time (s) filter) epifluorescence filter and controls The operation. processing Figure I right 3 changer and c. b. a. functional 2.3: the components Optical Computer Excitation an This unit Illustrated filter intensified mechanisms. (CPU, microscope unit blocks unit changer, unit light also that diagram 80486-33MHz), of CCD are: including source block includes includes shutter optics camera. of A). that the the a activation (dichroic the data includes imaging image Input/output neutral monitor mirror, processor and hardware. the density and the Hg UV controller Arc intensified an filters (ImPrc), objective image Bulb, (NDF), (I/O) the monitor central CCD and CCD which shutter emission camera Camera (top and left 83 84
Figure 2.4: Schematic diagram of the perfusion chamber.
Side View Gas Mixture Heating
Glass CoverSlip
OutFlow InFlow Rubber 0-ring
Top View
well
and This
perfusion
respectively.
plate.
Figure
complete
and
C-clamp
2.5:
Arrow
a
and
1
Schematic
ml
perfusion
orientation
Thin
exchange
suction
pipette
dashed
Objective
at
diagram
(top
and
lines:
a
(in
rate
arrows
suction
left)
or
suction
out
of
of
allowing
a
1-2
indicate
of
head
perfusion-head
the
mI/mm.
line
is
well)
for
1’
reaching
the
fitted
fast
lines
Thick
Tissue
4
indicates
to
exchange
assembly.
close
used
a
solid
side
culture
inflow
to
for
arrows
C-clamp
well
the
of
continuous
well
all
or
of
bottom
the
indicate
suction
a
with
12-well
medium.
of
cells
gentle
fast
the 85 86
Figure 2.6: Kinetic parameters used in the description of 2+Ca responses. Note that the percentage values specified represent the level of the percentage of change from basal to peak values (and not from zero to peak) in the relevant units used, i.e., ratio or nM calcium. The solid horizontal bar represents the duration of exposure to the agonist (e.g., NMDA).
C E :3 C)
C-)
(Es :3 0G) (Es C
ci) a) U-
100 150 200 250 300 350 400 450 Time (s) 87
Table 2.1: Problems associated with fura-2 fluorimetry, and their remedies Compiled from Roe etal(1990).
Problems Solution(s) Interference with he 2Ca signal Minimize loading, use high (i.e., buffering Ca ) sensitivity detectors (e.g., a intensified CCD).
Sequestration of fura-2 in Reduce loading concentration of subcellular organelles. Fura2-AM and loading temperature.
Phototoxicity of the cells and Reduced UV exposure using neutral photobleaching of intracellular fura-2 density filter, use shorter (UV light effects) experimental protocols, reduce frequency of acquisition and use high sensitivity photo-detectors. Active extrusion of the fura-2 by Use anion exchange blockers (e.g., anion exchangers (uncommon in SITS and DIDS) neurons) Incomplete hydrolysis of the fura-2 Use other loading methods (e.g., AM ester. microinjection and ATP permeabilization). Quenching of fura-2 fluorescence by Use the heavy metal chelator, TPEN. heavy metals. 88
Table 2.2: Outline of the cresyl violet staining method.
1. The tissue is dehydrated by passing through a series of graded alcohol solutions (70%, 90%, 100% and 100%).
2. Tissue is de-fatted in xylene (twice).
3. Tissue is rehydrated through graded alcohol solutions (100%, 100%, 90%, 70% and 35%). 4. After rinsing in 2O,dH stain in 0.1% (w/v) CV in 2OdH (30 seconds to 2 minutes. 5. Rinse in 2OdH to remove excess CV. 6. De-stain in CV differentiator with frequent inspection until the required de staining level is reached.
7. Dip a few times in 95% alcohol, 100% alcohol and then in xylene. 8. Mount on glass slide using Permount. 89
CHAPTER 3
CHARACTERIZATION OF HIPPO CAMPAL NEURONAL CULTURES 90
The objectives of these experiments are to characterize relevant aspects of the cultured hippocampal neurons prepared as described previously. This characterization will cover the survival of neuronal cultures, neuronal 2+Ca binding proteins as well as 2+Ca responses induced by exposure to excitatory amino acids (EAA) and high extracellular K (HK).
I. SURVIVAL OF HIPPOCAMPAL NEURONS IN CULTURE
Different types of primary hippocampal neuronal cultures were used in the course of the present experiments. Culture conditions were varied to suit the particular need of different experimental paradigms. Lower density cultures
(10 neurons/cm were plated on 18 mm diameter number 1 glass coverslips and maintained)2 in a face-down position (sandwich method) in 6-well tissue culture (TC) plates with 1.8-2 ml of TC medium until used. Higher density fetal 5(2x10 neurons/cm and postnatal 5(4x10 neurons/cm cultures were routinely)2 maintained in a face-up configuration)2 on 18 or 22 mm diameter glass coverslips in a volume of TC medium adjusted to the total number of cells plated (0.8-2 ml). a. Identification of neurons in cultures
Using phase contrast microscopy together with light microscopy of cresyl violet stained cultures, neurons and glia could be distinguished on a morphological basis. The use of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) immunohistochemistry confirmed that the distinction between neurons and glia on a morphological basis was accurate and it was thus used routinely for experiments requiring neuronal counting. At
7 Dlv, neurons constituted approximately 90%, 80% and 80% of the cells in face-down fetal cultures, face-up fetal cultures and 4-d postnatal cultures, 91 respectively. Different neuronal types were also identified on the basis of their morphology. Pyramidal and bipolar neurons represented the vast majority of neurons and were near equally prevalent (50% and 45%, respectively) while multipolar neurons represented only a small fraction (< 5%) of the identified neurons. These proportions were closely maintained in different culture types and over different culture ages (Fig 3.1). b. Age dependence of neuronal density in culture
As the cultures grew older more neuritic arborization was evident but the total viable neuronal density, as estimated morphologically, was greatly reduced. Fetal neurons cultured using the ‘sandwich’ method showed the best survival rates. After an initial rapid loss of neurons with only 75% and 52% of the initially plated neurons remaining morphologically normal after 1 and 3 DIV respectively, the rate of cell loss slowed down with 48% and 37% of the neurons remaining alive after 7 DIV and 14 Dlv, respectively.
Fetal neurons cultured in the face-up configuration suffered a much higher initial loss and only 28% and 11 % were alive after 1 and 3 Dlv, respectively. One week after plating 8.7% of the neurons were alive but these were almost completely lost during their second week in vitro with only a 1.4% survival rate after 14 DIV. Although face-up cultures initially had twice as many cells as the face-down cultures, the latter had 12 times more surviving neurons after 2 weeks in culture.
In four-day postnatal cultures only 20% of the originally plated neurons survived after 1 DIV and only 5.4% were viable on day 3 in vitro. After 7 and 14 DIV 3% and 2.5% of neurons were still alive. 92
Fig 3.2 shows the progress of survival of neurons in the three culture types tested. c. Effects of basic fibroblast growth factor (bFGF)
Addition of bFGF to the culture medium resulted in a major change in the survival pattern and the glia/neuron ratio of postnatal cultures. Several concentrations of bFGF were tested by adding small volumes of a concentrated stock solution of bFGF containing 5% BSA to the culture medium in the wells on days 0, 1 and 3 after plating. Final bFGF concentrations were 0.1, 1, 2.5 and 5 ng/ml of the culture medium.
Aside from the lowest concentration of 0.1 ng/ml, which had no noticeable effect on the cultured cells, bFGF produced a concentration dependent increase in neuronal density and glial/neuronal ratio compared to time-matched controls. Glial overgrowth was so excessive that antimitotic treatment with Ara-C had to be instituted after 24 hours rather than the usual
48 hours. Glial morphology was also different from that in untreated cultures.
Glia were larger, more phase bright and many had a spindle-shaped appearance. At a concentration of 2.5 ng/mI, neuronal density after 3 DIV was 125% of the BSA treated control cultures (p < 0.05).
With continued examination after the cessation of bFGF treatment these cultures displayed a faster loss in neuronal viability compared to their BSA treated counterparts so that on day 6 in vitro, (i.e., 3 days after the last bFGF treatment) both cultures had an approximately equal density of viable neurons.
This trend of accelerated neuronal loss in bFGF-treated cultures continued and after 12 Dlv, neuronal survival of the bFGF-treated cultures was only 25.5% of the BSA-treated control cultures with p < 0.001 (Fig 3.3). 93 d. Sensitivity to glutamate-induced excitotoxicity
All types of cultured hippocampal neurons were sensitive to glutamate- induced neurotoxicity when tested after 7 DIV. Toxicity could be demonstrated either by brief exposures to high concentrations of glutamate or by continuous exposure for 24 hours to lower concentrations of glutamate. Details of this process will be discussed in a later section.
II. Ca1 BINDING PROTEINS INCULTURED HIPPOCAMPAL NEURONS
In vivo, immunoreactivity to a variety of 2+Ca binding proteins has been demonstrated in different hippocampal neuronal populations with varying developmental patterns. The most relevant of these 2+Ca binding proteins to the present work are calbindin-D28k and parvalbumin. a. Calbindin-D28k (CaBP)
CaBP was expressed in at least a small percentage of neurons in all the culture types tested. The frequency of its expression was a function of culture type and culture age (Fig 3.4).
In face-up fetal cultures, the number of CaBP neurons declined progressively from 59 ± 11 neurons/mm (4.9 ± 0.7% of total) after 1 DIV to 8 ± 4.1 neurons/mm (2.1 ±2 1.1% of total) after 7 DIV. After 14 DIV no CaBP neurons2 were detected in the cultures. Thus, the reduction in the number of CaBP neurons in the face-up fetal cultures continued throughout all culture ages tested (0-14 DIV) and was always larger than the reduction in the number of CaBPN neurons, i.e., the percentage of neurons expressing CaBP decreased with time in vitro. 94
In fetal cultures maintained face-down, the change in the number of
CaBP neurons had a very different pattern. From an initial plating density of 75 ± 18.8 2cells/mm (3.9 ± 1.3%), the number of CaBP neurons fell to 16 ± 12.5 2cells/mm (1.8 ± 0.8%) at 1 Dlv, and to 8 ± 7.3 neurons/mm (0.8 ± 0.7%) at 3 DIV. By the end of the first week in vitro, this2 trend of decline has given way to increased CaBP expression and the number of CaBP neurons markedly increased to 60 ± 14.3 neurons/mm (6.4 ± 1.8%) and 50 ± 16.9 neurons/mm (6.7 ± 2.0%) at 7 and2 14 Dlv, respectively (Fig 3.4A,B). 2
In postnatal neurons (face-up), the temporal pattern of CaBP expression was more like the fetal neurons in the face-down cultures than like the fetal neurons in the face-up cultures. After an initial loss of CaBP expression that peaked after 3 DIV (6 ± 5.6 neurons/mm on day 3, down from 75 ± 14.4 2cells/mm at plating time), there2 was a progressive increase in the number of neurons expressing CaBP reaching a peak after 12 DIV when 235 ± 30.2 neurons/mm were CaBP, representing 100% of all the surviving neurons. At2 19 DIV the postnatal neuronal density fell to 38.5 ± 10.0 neurons/mm but all of these neurons were still CaBP (Fig 3.4A,B). 2 b. Parvalbumin
Unlike CaBP, parvalbumin immunoreactivity, which is a generally accepted marker for subpopulations of GABAergic neurons (Celio, 1990), could not be demonstrated in any of our cultures at any culture age. c. Effects of bFGF on 2Ca binding proteins At discussed above, bFGF enhanced neuronal survival in postnatal cultures but its withdrawal caused a more rapid neuronal loss. Following 95
treatment on days 0, 1 and 3 in vitro with 2.5-5 ng/ml bFGF, the number of CaBP neurons increased dramatically such that after 6 and 12 Dlv, 69.6 ± 8.8% and 100% of the neurons were CaBP, respectively. These numbers were significantly higher (p < 0.001 at both ages) than those obtained from the BSA-treated cultures at the same ages in vitro (7.3 ± 4.9% and 59.8 ± 10.8%, respectively) (Fig 3.5).
The effects of bFGF on fetal cultures were much less dramatic and more variable from one culture to the other. However, we tested the effect of culture media conditioned with postnatal cultures treated with either bFGF or BSA, by collecting half the culture medium from these cultures twice weekly (as part of the routine of culture feeding) and freezing the harvested medium at -80°C. The conditioned media were then used to supplement the feeding medium for fetal cultures (face-down) treated with additional bFGF or BSA. While bFGF and conditioned media from BSA- and bFGF-treated postnatal cultures were capable of enhancing CaBP expression independently, combining bFGF with either of the postnatal culture-conditioned media was far more effective in increasing the number of fetal neurons expressing CaBP (Fig 3.6).
The effect of bFGF on glia, neuronal survival and CaBP expression in
postnatal cultures contrasted sharply with its total lack of effect on PV expression in the same cultures. Even in the presence of 5 ng/ml bFGF, no PV immunoreactivity could be detected.
III. Ca RESPONSES IN CULTURED HIPPOCAMPAL NEURONS a. 2+][Ca in non-stimulated neurons In all types of hippocampal cultures tested, the average resting ]2[Ca varied within a narrow range (50-150 nM). Occasionally, some cells showed 96
significantly higher fluorescence ratios which were associated with a swollen appearance and cytoplasmic vacuolation of these cells. Such cells were
excluded from subsequent data analysis. Fig 3.7B illustrates the degree of 1 variability in 2[Ca ] at rest and during an NMDA-induced 2Ca response within a set of neurons being recorded simultaneously from a single coverslip.
The tight regulation of 2+][Ca observed in our neurons appears to be dynamically controlled in the face of on-going tonic 2Ca fluxes. Thus in many cultures, changing 2[Ca +1 of the perfusion medium was accompanied by a corresponding change in the recorded 21[Ca Also the addition of EAA antagonists (e.g., MK-801 or APV) or VGCC.] blockers (e.g., nifedipine) was sometimes associated with some reduction in ]2[Ca (Fig 3.8). b. Effects of excitatory amino acids on 21[Ca In the presence of extracellular 2+,Ca agonists of all the major classes of glutamate receptor subtypes were capable of increasing neuronal 2+]j[Ca in a concentration dependent fashion. Unless otherwise indicated, all 2+Ca responses were measured in the absence of extracellular 2+Mg and in the
presence of ‘1pM TTX.
1) N-methyl-D-aspartate (NMDA): Activation of NMDA sensitive glutamate receptors increased 2[Ca in almost all morphologically identifiable neurons in culture. The 50EC forj NMDA-induced 2Ca + responses was 25 ± 1.6 pM in the absence of extracellular 2Mg (Fig 3.7A). In the presence of 0.8 mM extracellular 2Mg 200 to 400pM NMDA were required to produce an effect comparable to, that of 20 pM NMDA in the absence of extracellular 2Mg • 97 NMDA-evoked changes in 21[Ca were abolished by the classical non- competitive NMDA antagonists, iMK-801 (2 pM) and dextromethorphan (100 pM) in an agonist-dependent manner, as well as by the competitive antagonist, APV (40 pM) (Fig 3.8). Blockade of voltage-gated 2Ca channels (VGCC) using 2 pM nifedipine, resulted in a 40% reduction in the peak 2Ca responses to NMDA indicating the involvement of voltage-gated 2Ca fluxes in the NMDA-induced responses. Changes in extracellular pH )0(pH had dramatic effects on NMDA-induced changes in 21[Ca At 0pH 6.9 in 7C0HC0-buffered BSS without 2Mg 2[Ca responses.] to 2OpM NMDA32were on the average 52 ± 5% lower, than controlj responses at 0pH 7.3 while raising 0pH to 7.9 increased NMDA responses by 32 ± 7% more than the control responses (Figs 3.9, 3.15).
2) 2+Ca responses to other EAA: Transient exposure of hippocampal neurons to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid (KA) caused a reversible increase in 2+].[Ca In the absence of extracellular 2Mg at 0pH 7.3, 8OpM KA and 4OpM AMPA evoked 2Ca responses comparable in magnitude to 20 pM NMDA. AMPA- and KA-evoked responses were sensitive to CNQX, a competitive AMPA/KA antagonist, and disappeared in the absence of extracellular 2+.Ca
Synthetic (+)quisqualate (QA) was the most potent of all the EAA tested in evoking 21[Ca responses with a Kd of 0.5 ± 0.09 pM. However, in all experiments j performed it was obviously less efficacious than NMDA, AMPA or KA (Fig 3.10). QA evoked responses were dependent on the presence of extracellular 2+Ca and sensitive to 0pH in a manner similar to other EAA. Beyond higher potency and lower efficacy, QA displayed an extreme lack of 98 homogeneity in agonist concentration-dependence of 21+][Ca responses in different cells within the same experiment. While some cells showed the familiar graded peak increases, a large fraction of cells showed ‘step function’- and ‘all or none’- type of concentration dependence (Fig 3.11). It should also be noted that, regardless of the pattern of the cell response to QA, QA-induced 2+Ca responses were abolished in the absence of extracellular 2+.Ca
Glutamate (GLUT) is the primary endogenous agonist for GLUT receptors and in cultured neurons it induced 2+Ca changes that were a composite of the pattern of activation evoked by the excitation of the GLUT receptor subtypes. Its use was generally avoided whenever possible to minimize uncertainty regarding the receptor subtypes involved. However, it was used to study excitotoxicity-associated 2+Ca changes and in 2+Ca buffering experiments (see later) where an examination of neuronal 2+Ca handling, rather than the route of 2+Ca influx, was the main research objective. c. Depolarization-induced 2+Ca fluxes
Increasing extracellular K+ to 50 mM for 20 seconds depolarized neurons and resulted in the activation of several types of voltage-gated 2+Ca fluxes and a detectable rise in intraneuronal 2[Ca +1. A variety of selective 2+Ca channel blockers were used to identify subtypes of VGCC in our cultured neurons.
1) Contribution of endogenous GLUT: In the presence of glutamate receptor blockade using AP5 (40 pM) and CNQX (20 pM), the depolarization induced neuronal 2+Ca signal was reduced by 5 ± 2% of the control value indicating only a minor contribution of depolarization-induced release of endogenous GLUT to the overall 2+Ca change recorded during exposure to 0high-K (HK) medium. Nonetheless, all voltage-gated 2Ca channel (VGCC) 99
blockage studies were conducted in the presence of a combination of APV and CNQX.
2) Subtypes of VGCC: Nifedipine, a highly selective L-type 2Ca channel blocker, caused a concentration-dependent reduction in HK-induced rises in 21[Ca with an IC50 of 11 ± 2 nM. The maximum effect of nifedipine was attained at a concentration of 1-5 pM which resulted in more than 80% reduction in HK-induced 2Ca responses. At these high nifedipine concentrations, reversal of the blockade was very slow (Fig 3.13). The residual nifedipine-resistant 2Ca changes were further reduced by 50% in the presence of 10 pM Conus geographicus snail neurotoxin, omega-Conotoxin
GVIA (CgTx). The remaining nifedipine- and CgTx-resistant component of the HK-induced 2Ca signal were abolished by the application of 1:1000 crude funnel web spider (FWS) venom (Fig 3.14).
In summary, depolarization-induced changes in 2[Ca are predominantly (>80%) due to activation of the nifedipine-sensitive L-type 2Ca channels while the rest is equally contributed by fluxes through CgTx-sensitive N- channels and channels insensitive to both DHP and CgTx but sensitive to the FWS venom. Any involvement of the low-threshold rapidly inactivating T-type 2+Ca channels can be excluded since the extended 20 seconds depolarizing stimuli and the low temporal resolution of the imaging system makes the detection of the rapidly inactivating T-channel contribution to the signal very unlikely.
3) The Effect of extracellular pH on VGCC fluxes: Changes in depolarization-induced 2+Ca responses at different pH values were very similar to those observed with NMDA-induced changes in 2].[Ca In HEPES-buffered 100
BSS, at 0pH 6.9, HK-induced 2Ca responses were 39 ± 1% lower than the control value at pH 7.4 while raising 0pH to 7.9 was associated with increased 2Ca response by 49 ± 3%. In 7C0HC0-buffered BSS corresponding values were a reduction of 37 ± 321% at 0pH of 6.9 and an increase of 78 ± 3% in 0pH of 7.9. The 50EC for 0pH of HK-induced responses in 7C0HC0 buffered BSS was 7.02 ± 0.05 (Fig 3.15). -32 Sensitivity to extracellular pH was also evident if the dominant nifedipine sensitive component of the 2Ca + flux was inhibited. In the presence of 10 pM nifedipine, residual nifedipine-resistant HK-induced 2+Ca responses showed a 40% reduction and a 25% increase compared to control values as 0pH was changed from 7.3 to 6.9 and 7.5, respectively. d. Effects of the absence of extracellular 2+Ca In addition to 2+Ca fluxes through plasma membrane voltage- and neurotransmitter-gated channels, hippocampal neurons have been reported to possess EAA- and caffeine-releasable intracellular 2+Ca stores (Murphy and Miller, 1989; Harada eta!, 1992, Uneyama eta!, 1993). Several approaches were employed to test the existence of such stores in our preparations of cultured hippocampal neurons. In nominally 2Ca-free BSS, supplemented with 0.8 mM 2Mg and 200 pM EGTA, exposure of hippocampal neurons to HK, NMDA or AMPA was not associated with any detectable increase in 21[Ca (Fig 3.16). In other experiments NMDA and EAA] quisqualate, two reported to induce release of
2Ca + from intracellular 2Ca + stores (Murphy and Miller, 1989; Harada et a!, 1992), applied at concentrations as high as 400pM could not elicit any intracellular 2+Ca release in the absence of extracellular 2+.Ca This 101
refractoriness persisted even when applications of these agonists were performed very shortly after removal of extracellular 2+Ca in an attempt to avoid or minimize the potential depletion of intracellular 2Ca stores (Fig 3.17). Also, the application of the same agonists immediately upon re-introduction of
extracellular Ca evoked increases in 2+ 2[Ca that were not different from those recorded immediately before exposurej to 2Ca +4ree medium (Fig 3.17).
A direct and presumably more specific way of activating metabotropic GLUT receptors is to use isomers of ACPD (1-amino-i ,3-cyclopentane- dicarboxylic acid) as agonists (Manzoni eta!, 1991; Manzoni eta!, 1992). In our hands, three out of the four optical ACPD isomers were capable of evoking rises in 2j:[Ca 1R,3R-, 1S3R- and 1S,3S-ACPD, but only in the presence of extracellular 2Ca Except for 3 cells out of 200 cells tested, these 2Ca responses were. completely abolished by 2pM MK-801. ACPD-induced 2Ca responses were also significantly reduced in the presence of extracellular 2+Mg (Fig 3.18-3.20). In the presence of MK-801 or extracellular 2Mg changes in ACPD-induced 2+Ca responses were similar to changes in NMDA-induced, responses suggesting that active ACPD isomers were activating NMDA channels rather than the metabotropic subtype of GLUT receptors. e. Effects of other 21+Ca release agonists
Caffeine treatment (3-25 mM) of cultured neurons did not evoke any significant change in 2+].[Ca In some cultures less than 10% of the neurons demonstrated a small transient rise in their 2+][Ca on application of caffeine (Fig 3.21A). The more consistent finding was that in the presence of caffeine both NMDA- and depolarization-induced 2Ca responses showed a 25%
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DISCUSSION
SURVIVAL OF HIPPOCAMPAL NEURONS IN CULTURE
Three types of neuronal cultures were used in our experiments: fetal face down (lowest plating density), fetal face-up (medium density) and postnatal face-up (highest density). In all culture types, a remarkable decline in the number of viable neurons was observed as the cultures got older. Face-up cultures, both fetal and postnatal, lost more than 90% of the initially plated neurons after 7 DIV. Maintaining fetal cultures face-down (‘sandwich’ technique), has been in use in our laboratory since 1985, and cultured neurons maintained this way displayed a significantly lower incidence of spontaneous neuronal loss after plating with an approximately 50% survival rate after 7 DIV.
Brewer and Cotman (1989) reported that the use of sandwich culture technique could improve the survival of fetal hippocampal neurons grown in serum-free media, and they attributed the improvement to a lower oxygen tension in the limited diffusion space around the neurons. The authors also argued against a contribution of glial paracrine factors in this limited diffusion space as the cause of enhanced survival. While oxidative stress in face-up cultures may harm neurons, it is our opinion that glial factors may play a significant role in the improved survival under these conditions. In our experiments, survival of face up cultures was critically dependent on higher plating density, including glial plating density, delayed application of antimitotic drugs and the limiting of the culture medium volume/well. All these techniques help reduce volume/density ratio suggesting that some secreted factors are the major determinants of neuronal survival and that these factors may well be glial in origin. 104
In order to define more precisely the potential effect of growth factors we examined the effect of adding bFGF to cultures grown in serum-free conditions.
The inclusion of bFGF in the culture media resulted in a significant enhancement of survival of postnatal neuronal cultures. This effect may be mediated via an action on glia since the presence of bFGF results in a remarkable increase of glial number and size, as well as a dramatic change in morphology.
After 3 DIV bFGF treatment was discontinued and as a result neurons, conditioned with bFGF treatment, displayed an accelerated neuronal loss far exceeding that observed in sister cultures treated with vehicle only. This phenomenon may reflect an apoptotic neuronal death similar to that observed in response to withdrawal of NGF in cultured rat sympathetic neurons (Edwards and Tolkovsky, 1994), DRG neurons (Eichler and Rich, 1989), and bFGF withdrawal in rat ventral mesencephalic dopaminergic neurons (Mayer et a!, 1993)
A very important offshoot of the significant time-dependent neuronal loss in vitro is that the results obtained from cultured neurons represent, at best, the characteristics of a highly selected neuronal population. The sensitivity to growth factors in vitro also indicates that the development of cultured neurons can deviate significantly from their in vivo program in a manner dependent upon the culture conditions. A direct extrapolation of the results obtained with the in vitro preparations to the intact animal should thus be done with extreme caution.
CaBP EXPRESSION IN CULTURED NEURONS
The expression of CaBP in cultured neurons was found to be a function 105 of the culture method and the type of neurons, i.e. fetal or postnatal. All culture types displayed an initial loss of CaBP neurons for the first 3 days in culture. In fetal face-up cultures this trend continued until no CaBP neurons could be detected after 14 DIV. In face-down fetal cultures the number of neurons expressing CaBP then increased and the ratio of CaBP/CaBPN neurons stabilized after 7 DIV. In postnatal cultures, the number of neurons expressing CaBP increased steadily after 3 DIV, in spite of the ongoing time- dependent loss in the total number of viable neurons and by 12 DIV all surviving neurons were CaBP. It would seem, therefore, that all of the postnatal neurons have the capacity to express CaBP, although the factors controlling this expression are not clear. It may be a part of the developmental process in vitro or it may be a function associated with the selected fraction of neurons that survive in vitro over time.
bFGF treatment of postnatal cultures enhanced neuronal survival and increased the expression of CaBP even after the bFGF treatment was stopped. Although the percentage of neurons expressing CaBP varied between batches of cultures, it was always significantly higher in bFGF-treated cultures than in controls, and reached 100% expression in the majority of cultures tested.
While it is possible that the expression of CaBP was a direct effect of bFGF, our data support the possibility that bFGF may be necessary but not sufficient for CaBP expression, and that other secreted factors, possibly of glial origin, may be required. This is particularly evident in fetal neuron cultures in which bFGF-treatment alone produced only a small increase in neuronal CaBP expression whereas adding bFGF in the presence of conditioned medium from postnatal cultures produced a very large increase in the number of neurons expressing CaBP. than observed was although subtype had was capable Ca 2 scattered Miller, the is differentiated cultured et cultured expression state synthesis between turn the of expressed a! glial pattern a effect also completely one Almost release (1 This of step RESPONSES 1982; 993) cells of agonists, differentiation neurons, rat single type capable with a interneurons of result, of function-like responding specific of of hippocampal that all factors in only cultured Miettinen bFGF neuronal of CaBP. expression other postnatal neuron neurons dependent Quis concluded together of in NMDA, which IN is, induction evoking that a EAA. However, neurons. CULTURED receptor sub-population at to and of responses population. and appearance tested, can leads least cultures glutamate neurons seen the AMPA with This on not Freund, that an be of neurons the in to with in at the regardless phenomenon increase On it CaBP achieved the part, and enhanced neonatal to (data all HIPPOCAMPAL is from presence use the Additionally, as rather different 1992). of increasing growth in of kainate, synthesis due with well interest CA3 basis an of not CAl in by antimitotic than to of undifferentiated or [Ca 2 a as shown), survival of factors pyramidal bFGF of subsequent affinities. an their may adult pyramidal with to extracellular to concentrations the our +11 rather action the the note NEURONS reflect culture treatment, rats an in graded data can and leads expression prototypic agents all increase than neurons the on in neurons Quis-induced cause an cultures we the constitutive conditions, vivo, us glial recent to response Ca 2 +. increase an cannot to to presence a does of a effect cells in (Baimbridge limit highly in ionophore conclude of and transition tested Quis [Ca 2 j 1 report which CaBP Quisqualate not distinguish which the in pattern a upon responses were typically few of CaBP reflect number of CaBP in that more that of in the Ray all and 106 107
were also dependent on the presence of extracellular 2+Ca and a ‘pure’ metabotropic response could not be elicited even when selective metabotropic agonists, such as 1S,3R-ACPD and 1R,3S-ACPD, were used. Interestingly 2+Ca responses induced by these ACPD isomers, which are commonly used to test for the presence of metabotropic glutamate receptors in neurons, were in our hands mediated solely by activation of the NMDA receptor-associated ionophore as indicated by the sensitivity of these responses to the non- competitive NMDA antagonists, 2Mg and MK-801.
Depolarization of our cultured neurons was also associated with an increase in ]21[Ca via VGCC. Using blockers of different types of VGCC we determined that the largest fraction of the 2Ca signal was mediated via 2Ca influx through L-type channels, while less than 20% of the signal was equally contributed by channels sensitive to CgTx or to funnel-web spider venom.
Both NMDA- and depolarization-induced 2Ca signals were sensitive to changes in extracellular pH. While increased extracellular acidity resulted in the reduction of NMDA- and depolarization-evoked 2+Ca responses, these responses were progressively potentiated as extracellular alkalinity was increased. These results are in agreements with the reported neuroprotective effect of reducing 0pH and the detrimental effects of high extracellular alkalinity on cultured neurons (Giffard eta!, 1992; Kaku eta!, 1993).
INTRANEURONAL 2Ca STORES AND INTRACELLULAR RELEASE
We conducted several experiments to test for the presence of intraneuronal releasable 2+Ca stores in our cultured neurons. In the absence of extracellular 2+Ca no intracellular release could be demonstrated using NMDA, Quis or tACPD. The latter two agents in particular have been demonstrated by to extracellular small induced neurons between continued by and lack Ca 2 + [Ca 2 j nor established presence such Manzoni many fill the ryanodine filling any On intracellular caffeine-releasable as release, authors recent in [Ca 2 ] 1 (DRG) that the detectable central etal, of of presence a agents small K+induced other basis those within observation could to and prepared 1991; signal (neocortical, release Ca 2 number of types caffeine stores were EAA-releasable of the induce these Manzoni extracellular could stores. endoplasmic from of Ca 2 + Ca 2 + also Ca 2 + must by of results intracellular was any be neurons. Shmigol unsuccessful. hippocampal 5-8 eta!, stores influx from be obtained, [Ca 2 +] 1 capable day we extremely Ca 2 +. intracellular an 1992). reticulum which, whereas eta! conclude postnatal Ca 2 + lP 3 response of particularly and (1994) This sensitive inducing these Attempts For labile stores in (Murphy brain that Ca 2 conclusion rats. example, DRG indicative of authors and our using only stem) intracellular substantial when The stores to neurons dependent and cultured demonstrate a neither central other concluded, and small has preceded of Miller, or intracellular a peripheral that been well differences Ca 2 + large increase neurons thapsigargin neurons upon 1989; emptying supported by the served caffeine store the a either in had high 108 109
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and
glial
color. 3.1:
After
the
Figure (A)
marker brown bipolar
for represents staining
A
B I- 0
U
0 c3 U,
microscope
represent
fetal
highest squares),
rapid
used.
In
Figure
all
2000
1000
1500
500
face-down
the
initial
0
A
3.2:
percentage
hippocampal
fetal mean
I
loss
Neuronal
field
face-up
of cultures
±
(0.245
standard
neurons
survival
survival
neuronal
(solid
mm had 5
I
that
ieviation rates
). circles)
the
in
culture
Age
slowed
vitro
lowest
of
the
and
varies
(days
of
types
down
3
initial
at postnatal
10
culture
least
with
in
tested,
after
plating
vitro)
1 the
types
2
(solid
4
fields,
different
fetal
DIV
density,
(B). squares),
15
(A).
face-down
HPF
Data
culture
they
Although
points
high
there
had
conditions
(open
power
the
was
the
20
110 a
LI-
(1)
‘ 4 -J
ci)
C) ci) C
(‘3 C) ci)
> (‘3 >
20
40
60
Figure
0 L
0
3.2
B
5
Age
(days
10
in
vitro)
15
20 111
microscope
density
cultures
BSA-treated
unpaired
and
Postnatal Figure
C,)
3
20
10
30
40
0
in
3.3:
after showed
- vitro
—
t-test)
cultures
bFGF
field
cultures
12
demonstrated
2DIV
neuronal
DIV
(0.245
an
enhances
treated
accelerated
(p
(solid
<
mm 2 ),
survival
with
0.001
circles).
neuronal
a
significantly
n
2.5
neuronal
Days
using
during
ng/ml
12
After
6D!V
survival
unpaired
fields
in
bFGF
loss
of
cessation
vitro
higher
bFGF
in
or
and
administration
vitro.
more.
t-test).
(p
were
(open
of
<
bFGF
0.001
significantly
HPF
circles)
12DIV
treatment, compared
=
after
high
on
2
days
lower
power
DIV
to
these
0,
using
the
in
1 112 ()
02 0
z o_
LL a, Cr)
D
symbols).
more.
neurons synthesis
detectable squares)
number
immunoreactive
culture
In
Figure
40
30
20
50 60
10 70
0
fetal/face-down A
0 3.4:
I
types.
of
detected
after
resulted
CaBP
HPF
but
Survival
an
CaBP’
cells.
initial
at
high
in
cultures
nçurons
and
different
an neurons
5 However,
I
power
drop
increase
number
(open
decreased
in Age
in
microscope
the persisted
vitro
of
in
in
squares)
(days
number
the
CaBP
fetal/face-up ages
10
steadily
absolute
(A).
in
field
is
and
of
cultured
vitro)
presented
CaBP’
The
postnatal
(0.245
until
number
cultures
percentage
no
neurons
mm 2 ),
15 neurons,
in
CaBP
of
cultures
(solid
Fig
CaBP
3.4B
n
varies
of circles)
=
active
neurons
CaBP(H
(solid
12
(same
in
fields
20
different
the
were
or 113 Percentage of CaBP Neurons
0 0 0 0 0 0 0 0 ‘.1 0 CD
-I CD c) w
0,
CD CD
0
C/) 0
0
(1
N) C
Th
+
C-)
0
z
0
C-) CU 0) C)
C) C CU
o C U)
I.
respectively,
and
neurons
(solid
Postnatal
Figure
100
40
20
60
80
0-
3
circles)
in
3.5:
at
vitro
cultures
all
bFGF
using
(p
demonstrated
the
2DV
<
increases
culture
treated
0.05,
unpaired
p
ages
with
neuronal
<
a
t-test).
0.001
significantly
tested
2.5
Days
ngfml
n
CaBP
and
6DIV compared
=
in
p
12
of
vitro
higher expression
<
bFGF
fields
0.001
to
percentage
(open
or
the
after
more.
in
BSA-treated
circles)
vitro.
2,
12DIV
6
of
and
CaBP
on
12 cultures
days
DIV.
0,
1 115
was
more)
However,
conditioned
compared
regardless
neurons
but
enhanced Figure In
12
C)
0 z
4-,
fetal a)
(‘3
C-) ci) 0)
C 0 (0
0) D 0 C
Cl)
significant
highly
produced
3.6:
40
30
10
50 60
70
80
face-down
0
and
combining
in
to
of
significant
In
medium,
the
glia
BSA
whether
fetal
(p<0.05),
a
presence
also
treated
more
neurons,
face-down
bFGF
BCM)
produced
(p<0.001
postnatal
than
increase
controls.
of
treatment
or
bFGF
additive
media
bFGF
cultures,
Treatment
a
neurons
in
modest
treatment
in
both
conditioned
Addition
(bFGF-conditioned
the
with
enhancement
bFGF
cases).
had
percentage
increase
BCM
Group
alone
of
been
effect
media
or
by
produced
in
with
treated
postnatal
in
on
of
CaBP
CaBP
conditioned
medium,
CaBPIH CaBP
FCM
with
expression
expression
9nly
neurons.
(n
expression BSA
=
FCM).
neurons
a
by
10
moderate,
(BSA
postnatal
fields
that
is
when
or 116 117
Figre 3.7: NMDA induces a concentration-dependent increase in intraneuronal [Ca ] In 69 neurons, 2[Ca +1 increased in response to exposure to NMDA (solid triangles) for 25 seconds at different concentratjons (2.5, 5, 10, 20, 40, 80 and 160 pM) in the absence of extracellular Mg + 50(EC 26.3 ± 1.6 pM). The magnitude of the peak response was concentration-dependent and approached a maximum between 80 and 160 pM of NMDA. In B, a 20 pM NMDA-induced response (n=45 neurons) is illustrated to show the extent of normal variability in cell responses by plotting the mean (solid line), mean ± SEM (dotted line) and mean ± standard deviation for the set of neurons tested.
A
1500
1200
C + 900 CD (-) 1. CD
a) 600 C-) CD .4-I
300
0 I I I . I. III 0 500 1000 1500 2000 2500 3000 3500 4000 Time (s)
eq
+
C-)
G) 1
CD C.) CD
400 200 600
800
Figure
0
0
3.7
B
100
Time
(s)
200
300 118 c’J
+ (.) (‘3 C-) 0 C C (‘3 I.
(‘3
antagonist-free
NMDA
inhibition induced
respectively,
In
Figure
31
200
400
600
800
0
neurons,
0
3.8:
antagonists
by
was
25
APV
solid
rapidly
seconds
extracellular
medium.
500
and
bars)
is
Mg 2
quite
and
application
resulted
The
completely
1000
noticeable
abolish
application
reduction
in
Time
of
complete
completely
1500
reversible
NMDA
in
of
of
this
(s)
APV
resting
experiment.
inhibition
(20
NMDA-induced
and
on
2000
pM,
[Ca 2 ]
perfusing
Mg 2
solid
of
the
on
2500
(40
triangles).
the
[Ca
application
Ca 2
neurons
ii
and
responses.
3000
increases
This
1.5
of
with
mM,
the 119 120
Figure 3.9: NMDA-induced 2Ca responses are modulated by changes in the extracellular pH. The mean of 20 pM NMDA-induced 2Ca + responses (solid triangle) in this record (n = 20 neurons) shows that an increase in 0pH to 7.9 (solid horizontal bar) was associated with a 30% increase in the NMDA-induced 2+Ca response compared to responses in a 0pH of 7.3. Lowering 0pH from 7.3 to 6.9 (open horizontal bar) was associated with a 37% decrease of the 2+Ca response.
1000
800
C + 600 C)
Co D 400 C) Co I... C 200 -
A A A
I I I I I 0 I I 0 1000 2000 3000 4000 5000 Time (s) 121
Figure 3.10: Quisqualate is highly potent in inducing intraneuronal 2Ca responses. Exposure of 49 neurons to increasing concentrations of quisqalate (Quis, open squares) resulted in concentration dependent increases in [Ca+] with 50arEC below 1 pM of Quis. In spite of this maximum Quis-induced1 Ca + potency, responses were much lower than those induced by a 20 pM of NMDA (25 seconds exposure, solid triangle) which induced a submaximal response to NMDA (compare to figure 3.7a).
800
600 C +
(‘3 C-) 400 (‘3
ci) ci (‘3 4-, C 200
0 0 500 1000 1500 2000 2500 3000
Time (s) 122
Figure 3.11: Quisqualate-induced 2Ca responses show that QUIS does not have a homogeneous potency on hippocampal neurons.
A characteristic feature of the Quis concentration response curve was the extreme heterogeneity of its potency on different neurons. This is illustrated in this graph where 3 neurons, from the same experiment, were exposed to increasing concentrations of Quis (solid triangles; concentrations from left to right: 0.1, 0.2, 0.5, 2, 5 and 20 pM). While the overall 50EC of Quis in this experiment was 0.65 ± 0.17 pM, individual cells showed wide patterns of response including a steep ‘all-or-none’ response pattern (e.g., middle panel).
400 -
A 200
0
400
B 200 -
0 A A A A A A
400 -
C 200
0 0 500 1000 1500 2000 2500
Time (s)
C
with
cells.
extracellular
concentration:
Unlike
homogeneous
Figure
B
A
parallel
A
Quis
3.12:
representative
(Fig
Mg 2 i
(or
NMDA-induced
1000
1
1000
1500
than
1000
500
1
500
500
2.5,
500
500
proportional),
0
3.11),
0
0
0
Quis-induced
5,
displayed
10,
NMDA-induced
sample
20
750
Ca 2 +
and
a
but
of
fairly
responses.
3
not
4OpM
responses
neurons
Time
homogeneous
equal,
Ca 2
1500
from
(s)
show
increases
responses
in
left
neurons
a
to
2250
potency
graded
right
in
(solid
are
[Ca
in
rsponse
more
on
the
triangle;
+11.
3000
all
absence
responding
pattern
NMDA
of 123 124
Figure 3.13: Nifedipine inhibits depolarization-induced 2Ca responses In 41 neurons, exposure to high 0K+ (HK; 50 mM) for 20 seconds resulted in an increase in the mean t][Ca which was completely reversible upon restoration of low mM)2 K+ Exposure of (3.5 0 neurons to nifedipine (solid horizontal bars) at concentration. of 0.05, 1 and 5 pM caused an increasing inhibition of the HK-induced responses (80% reduction at 5 pM nifedipine compared to control responses). Reversibility of inhibition at this concentration was slow and incomplete 1 hour after cessation of exposure to nifedipine.
800
600
+
C-) 400 0.05gM 1.0gM 5.0gM 0 C)
200
0 I I I I 0 2000 4000 6000 8000 10000
Time (s) ______
125
Figure 3.14: Multiple types of VGCC contribute to HK-induced 2Ca responses in cultured hippocampal neurons.
In the presence of APV and CNQX (40 and 20 pM, respectively, solid bar) HK induced 2+Ca responses were not significantly changed. Under these conditions, nifedipine (10pM, top open bar) caused more than 80% reduction in the HK-induced responses. The nifedipine resistant component of the response was further reduced by 50% in the presence of CgTx (middle open bar). The remaining nifedipine/CgTx resistant response was nearly abolished by 1:1000 crude funnel web spider (FWS) venom. Graph represents the mean response in 9 neurons.
500 APV 4OpM + CNQX 42OuM
400 I Nifedipine 10pM
C -
300 - omega-CgTx 5pM C-)
D FWS Venom j5 200 - 1:1000
100-
0 I I I 0 2250 4500 6750 9000 Time (s) 126
Figure 3.15: HK- and NMDA-induced 2Ca responses are modulated by changes in extracellular pH. Changing 0pH from 6.7 to 7.9 resulted in a 72% and 99% enhancement in the magnitude of HK-induced response (solid triangles) and NMDA-induced responses (open triangles), respectively (n=59). The two pH ramps indicate the change in 0pH which occurred in steps of 0.2 pH units, and the horizontal segments indicate the control 0pH of 7.3. For both HK and NMDA-induced responses, extreme 0pH effects were rapidly and completely reversible.
800
600
7.3 400
ci) C.) CD 200 I AAAAAAAAA A I I 2zD 0 0 2000 4000 6000 8000 10000 12000
Time (s) 127
Figre 3.16: HK-induced and EAA-induced Ca responses are abolished in + 2 Ca free media.
In 55 neurons, exposure to HK (open triangles), 200 pM NMDA (solid triangles) and 40 pM AMPA (solid diamonds) resulted in 2Ca + responses that were completely abolished if the exposure to HK or EAA was performed in the absence of extracellular 2+Ca (solid bars).
750
500 +
CD C)
CD
ci) C) 250
0 0 3000 6000 9000
Time (s) 128
Figure 3.17: The effects of Ca-free medium on NMDA-induced Ca responses. 2 2 Exposure of 61 neurons to 200 pM NMD for 25 seconds (open triangles in the presence of 0.8 mM extracellular Mg + resulted in an increase in [Ca +], that was totally abolished in nominally Ca-free perfusion (solid horizontal bar) even when NMDA was applied within2 5-7 seconds of perfusion with 2Ca free medium. Within 5 seconds of resumption of perfusion with standard perfusion medium, ANM (200 pM, 25 seconds) induced a response that was identical to the pre-’Ca9 +.4ree perfusion.
400
300 C +
(‘3 C) 200 (13 D
G) C-) (‘3 .4-’ C 100
0 0 300 600 900 1200 Time (s) 129
Figure 3.18: The effect of extracellular 2+Mg on the calcium responses to NMDA and ACPD isomers. In 47 neurons, switching from Mg-free to 0.8 mFMg-containing perfusion medium resulted in a 2depression of the Ca + responses induced by the transient application of NMDA (200pM, solid triangles), 1S,3R-ACPD (1 mM, open squares), 1S,3S-ACPD (1 mM, rosses) and 1R,3R-ACPD (100pM, solid squares). Removing extracellular Mg + was2 associated with fast recovery of the responses. The rank order of responses to different agonists wa retained under both perfusion conditions. Percentages of depression by Mg’ (±SEM) were 68.1 ± 3.05%, 75.6 ± 2.90%, 71.9 ± 2.66 and 66.7 ± 3.26, respectively.
800
700
600 C + 500 C’4 CD Ci 400 CD D a) C.) 300 CD 4-I C 200
100
0 0 1000 2000 3000 4000 5000 6000 Time (s) 130
Figure 3.19: MK8O1 inhibited ACPD induced rises in neuronal free calcium. In 45 neurons, the addition o2pM MK-801 to Mg-free perfusion medium abolished the increase of [Ca induced by NMDA2 (100 pM, solid triangles), 1R,3R-ACPD (100pM, open squares), 1S,3S-ACPD (1 mM, crosses) as well as 1S,3R-ACPD (1 mM, solid squares). The figure represents the mean [Ca of 38 cells. The effect of MK8O1 was not reversible even after prolonged]2 (>1 hr) perfusion with MK-801 free mdium. Notice the use-dependent effect of MK 801 on the NMDA-induced Ca responses.
600
500
C 400 + c..J CD ci 300 CD
0a) CD 200 1-’c
100
0 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Time (s) 131
Figure 3.20: An example of a single cell where MK8O1 did not abolish the calcium response to 5 mM 1S,3R-ACPD.
In 191 of 194 neurons, MK8O1 abolished completely both NMDA- and ACPD induced Ca + responses (Fig 3.19). In the remaining 3 cells (only 1 cell is shown), [Ca increased in response to the application of 1S,3R-ACPD despite the2 presence of 2 pM MK8O1. These responses were, however, much reduced comparedj to the control responses (Numbers below the symbols represent the agonist concentration in millimolars).
400
A NMDA 350 D 1S,3R-ACPD
• 1R,3S-ACPD 300 0 1S,3S-ACPD c • 1R,3R-ACPD + 250 C”
C-) 200 D 2pM MK8O1
AL1O • L1 AALI .2 1 1 1 1 5 5 .2 .2 5 1 1 I 0 I I 0 20 40 60 80 100 Time (mm) 132
Figure 3.21: Caffeine induces only a small increase in intraneuronal 2+].[Ca In 56 neurons, exposure to 10 mM of caffeine (solid horizontal bar) resulted in a transient small increase in +][Ca that was not potentiated when the exposure to caffeine 21 highK+ was repeated after a depolarizing stimulus using (open triangle)(A). In (B), 38 cells were exposed o 20 pM NMDA for 025 seconds (open triangles). The NMDA-induced Ca + response was reduced, in a reversible manner, in the presence of 10 mM caffeine (solid horizontal bar).
A
600
500
C 400 + C.,’
C-) 300 D
U) C-) ( 200 C
100
0 0 500 1000 1500 2000
Time (s) 133
Figure 3.21 B
600
C 400 + c’4 CU C-)
(U D a) C) CU 200 C
0 0 500 1000 1500 2000 2500
Time (s) 134
CHAPTER 4
THE EFFECTS OF ARTIFICIAL BUFFERS ON NEURONAL 2Ca + RESPONSES adjusted DMSO-treated before The duration, 33°C employed minutes vigorously ester used neurons derivative, THE neuronal experiments the VGCC Statistical paired derivative, intraneuronal different first LOADING in (buffer-AM), (20 Since Loading This any t-test or order at with to protocol cells cultures. NMDA or a significance mixed room measurements dimethyl-BAPTA the on chapter final the 60 to presented treatment to the Ca 2 were control the neurons PROCEDURE highest compare pM, 20 achieve temperature buffer-AM receptors in required used kinetics prepared mM will balanced washed buffering 60 group of a concentration in groups with buffer-AM examine minutes, different the final the this were concentration of and concentration from (DM8) in was the & pre- salt Ca 2 (20 effects concentration capacity, chapter CONTROLS: were BSS measured made. artificial the pM, 33°C). a included and + solution levels stock was 20 without responses performed consequences of present post-loading 15 were mM using achieved the of of solution Ca 2 minutes, After using of (BSS). with enhanced stock the buffers the performed of 20 in BAPTA induced 20 corresponding buffer the loading using fura-2 Ca 2 the or solution by was Two pM values. RT). 60 DMSO was of buffer-treated incubating neuronal an and BAPTA pM of buffer prepared increasing microfluorimetry. on by loading for The estimated unpaired the in its for fetal activation concentration the Comparisons DMSO second for buffer-AM dimethyl 60 or acetoxy-methyl Ca 2 + required protocols face-down cultured in 20-40 its minutes the t-test. DMSO, group using and dimethyl of buffering. protocol minutes either for across a were (e.g., All at a 1 5 the 135 136
0.1 % DMSO if the highest buffer-AM concentration was 20 pM). A second control group was treated with half-BAPTA-AM (hBAPTA-AM), a control compound with a BAPTA nucleus modified to eliminate its 2Ca binding capacity. Neither DMSO nor hBAPTA treatment resulted in any significant change in the kinetics of the 2+Ca responses when compared to the controls. For the rest of this chapter, all the comparisons reported use the hBAPTA group for reference unless comparing the BAPTA-treated group to the DMB treated group.
As a result of the increased intracellular 2+Ca buffering capacity after loading neurons with BAPTA or DMB, in situ calibration using the 2Ca ionophore, Br-A23187, becomes increasingly difficult. For the purpose of this section, the results are presented as background-corrected but uncalibrated ratios. Statistical comparisons were done on experiments performed under identical optical and gain settings. Under such conditions a larger ratio value represents a larger absolute concentration of 2+Ca in spite of the non-linear relationship between the fluorescence ratio and the corresponding 2+Ca concentration. Reference to 2+Ca ‘concentration’ in this section implicitly means 2+Ca concentration as estimated by the background-corrected fluorescence ratio’.
I. BAPTA EFFECTS ON SHORT 2Ca TRANSIENTS
a) Changes in Kinetic parameters: Loading neurons with BAPTA-AM (20 pM, 15 minutes, RT) resulted in significant changes in the magnitude and time course of the 21[Ca responses induced by brief exposure of neurons to NMDA (20 pM,j 25 seconds) or to high extracellular K (HK; 50 mM, 20 seconds). Both treatments resulted in nearly equal 2+Ca responses and the 137 changes recorded in these responses after buffer loading were similar in both the depolarization-induced and NMDA-induced 2Ca transients. The kinetic parameters used in the analysis are illustrated graphically in Fig 2.6.
In 14 experiments using NMDA, baseline 2+Ca levels were similar in the control and BAPTA-loaded cultures. Peak 2+Ca levels were, however, significantly lower in the BAPTA-loaded neurons (74.3 ±8.7% of the control values, p < .001). Changes in 21[Ca were slower in the BAPTA-loaded group ] with in compared to the control group the rate of the increase 21[Ca being of ] 52.6±13.1% control while the rate of recovery was only 23.7±9.4% of the control value (p < 0.001 in both cases).
BAPTA-loading resulted in 2Ca responses that not only had smaller peaks and slower rates of rise and recovery, but were also of longer duration than the control responses. The mid-span width was 160.1 ± 28.6% of control while the low-span was 222.4±69.2% of the control values (p < 0.001). With smaller peaks and more prolonged responses, the aspect ratio of the 2+Ca transient was also significantly reduced in the BAPTA-treated neurons (43.4±13.6%, p < 0.001). An illustration of the effects of BAPTA-loading on transient 2+Ca responses is shown in Fig 4.1.
b) Kinetic differences between the effects of loading with BAPTA and
DMB: In 10 experiments, fetal neurons were loaded with DMB, which has higher affinity for 2Ca than that of BAPTA. Using the same loading protocol as above (20 pM, 15 minutes, RT), these DMB-loaded neurons displayed changes in the kinetics of 2+Ca responses that were similar to those observed in BAPTA-loaded neurons both in the percentage of change from control values and in the statistical significance levels (see Table 4.1) with two exceptions.
show
BAPTA-AM.
medium.
those
recovered
experiments
loading
medium
experiments,
[Ca 2 +]
the
using
after
order
from
Although
were
were
control
II.
significantly
neurons
Both
AM
BASELINE
loading
those
any
induced
then
fura-2
to
attempted. the
a)
In
with
in
treatment
esters
at
compare
[Ca 2 ],
(p
initial
Simultaneous
mid-span
change
the
quickly
the
room
compared
in
<
respectively).
fluorimetry.
DMB-AM
In
with
shorter
exposure
responses
the
neurons
by
Ca 2
0.05
was
experiments,
addition,
temperature
increased
depolarization
to
in
Ca 2 +
control
The
the
with
performed and
and
the
pre-exposure
CHANGES
in
between
Ca 2
and
effects
(Fig
of
hBAPTA
DMB-loacled
recording
low-span
background
responses
in p
raw
cultures,
neurons
hBAPTA-AM
<
the
during
4.2).
In
buffers.
the
fluorescence
0.005
was
all
of
at
coverslips
buffer-loaded
DURING
or
was
cases,
loading
buffer
room
from
Similar
parameters
exposure
levels
associated
to
by
within
additional
respectively)
neurons
fluorescence
20
performed
For
NMDA
temperature
cell-free
loading
Ca 2 +
(10
pM
on
of
THE
changes
that
these
the
data
neurons
perfusion
to
out
BAPTA-AM
compared
neurons
receptor
experiments
with
of
same
BAPTA
responses
received
buffer-AM
on
areas
experiments,
from
of
before
the
(Table
during
were
an
neuronal
10
set
while
with
Ca 2 +
cells
LOADING
were
on
with
increase
activation,
and
different
any
of
to
also
4.1).
in
were
the
the
BAPTA,
ester:
monitoring
neurons
that were BAPTA-loaded
responses
4
the
the
kinetically
Ca 2 +
Ca 2 +
observed
out
coverslip
application
incubation
as
in
ester-free
responded
perfusion
performed
PROCEDURE
In
treatments.
of
the
and steep
DMB
measurements
responses
13
before
4
mean [Ca 2 +] 1
were
[Ca 2 j 1
distinct
out
during
did
or
as
of with
and
of the
to
not
in
15 138 blocker Ca 2 presence to Ca 2 , the required. than induce response Since to Cells responses widely perfusion [Ca 2 i cultures associated the altered (Fig responding exposure examine the presence beginning 4.2). a that the levels ci hi buffer-AM BAPTA-AM result Ca 2 + but (Church background during that The Cell-to-cell of to association showed Second, did to to the with the cells loperamide, specific indicating BAPTA-AM of mechanism of influx NMDA responded not or magnitude exposure mechanism(s) a the et an (Fig after application non-specific respond a we (2 al, via increase lipophilic fluorescence close variability: channel and 4.3A) experiments) between 1994), the that used the BAPTA-AM to to by HK of of association to termination activation Ca 2 BAPTA-AM the did BAPTA-AM loperamide, an in treatments the activation (lower either AM to involved. perturbation such [Ca 2 increase Within AM not test individual flux (Fig ester, did stimuli, indicate compounds, +1 panel, did cell-to-cell the of 4.3B). through to of cultures not in exposure suggested loading-induced additional specific in not a (top First, route the exposure a potent Fig [Ca 2 +] cause neuronal of possibly set any cause magnitude and The the in 4.3A,B). the of that of variability fluorescence Ca 2 + the individual with the middle broad any cell use 3 that experiments plasma an as to showed experiments, glial response absence Ca 2 AM change well increase membrane of an conductances the spectrum of increases panels, DMSO cells, increase and ester membrane as peak buffer-AM cell flux. an of data in intensity to peak in never responses were elevation suggestive the alone [Ca 2 +] 1 extracellular Fig buffer-AM permeability [Ca 2 In even Ca 2 + in in from Ca 2 + the baseline 4.3A,B). performed [Ca 2 ] 1 [Ca 2 ] 1 : responded was rather might was ‘1. shifts in non- channel of varied Fig not of 139 at in
which
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than
loperamide
observed
under
to
reflect
required
were
in
with
Ca 2 +
4.4,
the
in
of
by
140 in 141
loading was also increased in order to test the effects of a much higher buffering capacity on the plateau 2Ca levels under high 2Ca flux conditions.
Several cultures were tested for the consequences of loading neurons with BAPTA-AM or DMB-AM (20 or 6OjiM, 60 minutes, 33°C) on 2Ca responses induced by short exposures to NMDA (20 pM, 25 seconds) compared to short (25 seconds) and long (3-4 minute) exposure to glutamate (250pM). The resulting effects on 2Ca responses are shown in Fig 4.6(A-E). Control cultures were prepared by treating them with DMSO (0.3%) or hBAPTA-AM (60 pM) under conditions identical to those used for loading the buffer-AM (60 minutes, 33°C).
In the hBAPTA-AM loaded neurons (Fig 4.6A), responses to NMDA (20 pM, 25 seconds), glutamate (250 pM, 25 seconds) and to glutamate (250 pM, 4 minutes) were identical to the responses obtained from the 0.03% DMSO treated group and from ‘naive cultures (data not shown). Except in neurons loaded using 2OpM of DMB-AMt (Fig NMDA-induced increases in 4.6D), 2[Ca were severely reduced in both the BAPTA-loaded groups (20 and 60 pM)i (Fig 4.6CE) and in the 60 pM DMB-loaded group (60 minutes) (Fig 4.6B) compared to the hBAPTA-loaded group (Fig 4.6A). This buffering effect on the
NMDA-induced responses was more evident than that observed in neurons loaded using the ‘20 pM, 15 minutes, RT’ protocol, and the larger effect confirmed that the buffering capacity was much larger in neurons loaded using the ‘20 or 60 pM, 60 minutes, 33°C’ protocol. Like their effect on NMDA induced 2Ca responses, BAPTA and DMB loading (20 pM, 60 minutes, 33°C) induced a marked attenuation of 2+Ca responses that were induced by a short (25 seconds) exposure of neurons to glutamate (Figs 4.6D, 4.6E) glutamate continued recovery minutes evident DMSO glutamate 40 cultures acids, DMB-AM exposure are EXPOSURE IV. slower recovery during Ca 2 + exposure cells transient pM, exposed BAPTA compared In for Under peak In (0.3%), the in with at after were the began contrast phases to (60 responses, the extended to presence resulted exposure. 8-minute increase responses EFFECTS 250 control to 250 complete excitotoxic pM, the buffer-loaded initially graded glutamate, immediately, to pM being pM termination to 60 control in of intervals. and intervals cultures, the glutamate the glutamate a minutes, challenged ON during glutamate. recovery concentration-dependent particularly rapid recovery situations markedly magnitude Ca 2 groups. and groups rise for and of extended i.e. We 33°C). probably on was to exposure LEVELS 25 in with it phases cultures therefore diminished On baseline neurons in prolonged compared In characteristically [Ca 2 seconds, of relatively vivo, spite termination 3 the In Ca 2 + concentrations other 11 DURING of the to peak treated such of loaded Ca 2 + which the tested NMDA. followed (Fig flux to absence NMDA- this unchanged endogenous extended Ca 2 + responses the as EXCITOTOXIC levels 4.6). situations, of relative declined with with the cerebral control exposure displayed Exposure and response of by effects of hBAPTA-AM either usually extracellular responses a in lack glutamate-induced NMDA: to excitatory only 30-minute groups, ischemia, the the NMDA BA of to of a to within to a buffer GLUTAMATE fast Ca 2 + PTA-AM a glutamate, effect 250 little 2.5, a 30-minute with were 3-4 (60 were initial amino neurons Mg 2 , pM 3 loaded in changes 10 on minute pM) the the or much and the 142 or 143
component that achieved more than 80% of the total recovery within 10 minutes followed by a slower component that completed the restoration of j2[Ca to pre-exposure levels within an additional 20-30 minutes (Fig 4.7A,B). In the BAPTA-loaded neurons (Fig 4.7C), NMDA-induced 2Ca responses were significantly diminished to the extent that exposure to 2.5 pM NMDA was not accompanied by any detectable rise in 2+][Ca at all. However, exposure to 10 and 4OpM NMDA were associated with an increase in 21[Ca but responses to both ] concentrations were blunted and prolonged compared to those in the control cultures. The incomplete recovery of 2[Ca between consecutive NMDA challenges, separated by 8 minute intervals,i resulted in a staircase appearance of the 2+],[Ca presumably due to the accumulation of the residual 2+.Ca Responses induced by a 30-minute exposure to glutamate were, however, not reduced in peak magnitude in spite of the considerably slower rate of increase in 21+][Ca compared to control cultures. The small recovery component normally occurring during exposure to glutamate was characteristically lost in the BAPTA-loaded neurons and actually replaced by a slow and steady increase in 21+][Ca After resuming perfusion with glutamate- free BSS, recovery of 21Ca .was so delayed that 40 minutes later barely 40% of recovery was achieved. In the DM8-treated cultures, 2+Ca responses followed a pattern similar to that observed in BAPTA-loaded cultures with two differences. First, NMDA-induced responses (at 10 and 40 pM) were less diminished than in the BAPTA-loaded cultures although recovery was still incomplete at the 8-minute response spacing that was used. Second, the rate of increase in 2[Ca + ] on exposure to 250 pM glutamate was faster than in the BAPTA-loaded neurons (Fig 4.7D). 144 DISCUSSION
The present experiments were designed to gain some insight into the potential effects of artificial buffers, with well established physico-chemical properties, upon stimulated increases in 21+][Ca in neurons. The experiments were facilitated by the use of cultured neurons (making direct measurements of 2[Ca with fura2 technically straightforward) and the use of membrane permeantj derivatives of artificial 2+Ca buffers. Effects on 2[Ca +11during the loading procedure: Exposure of neurons to the acetoxymethyl esters of BAPTA, DMB or hBAPTA (but not to DMSO) was, unexpectedly, associated with a rapid and largely persistent increase in baseline 2[Ca +1 that recovered completely when the exposure to AM compounds was terminated. This effect was not observed in the absence of 20+][Ca nor in cells that did not respond to NMDA or HK before the buffer loading. The addition of loperamide, which is a voltage-gated 2Ca channel blocker, in the continued presence of BAPTA-AM, resulted in complete reversal of the increase in 21[Ca induced by BAPTA-AM. We concluded from these findings that 2Ca j influx through specific 2Ca channels, most likely VGCC, was the primary event responsible for the increase in 2[Ca that was induced by the AM ] three derivatives tested. Such an effect on 21[Ca during buffer loading is interesting since several studies have reported ani increase in the amplitude and duration of EPSP in rat hippocampal slices and in acetyl choline release in frog neuromuscular junction observed during exposure to BAPTA-AM and DMB-AM respectively (Niesen et a!, 1991; Robitaille and Charlton, 1992). The authors of these studies suggested that the buffer-AM effect was mediated via inhibition of -2Ca dependent potassium conductance secondary to enhanced 2+Ca buffering. To 145 the contrary, our results suggest that the increase in 2[Ca during loading may be independent of 2+Ca buffering for several reasons.i First, the effect on 2[Ca appeared very rapidly reaching a plateau that lasted for the duration of exposurej to the AM compounds with a similarly rapid recovery following their removal. Unlike this square wave response, the increase in buffering capacity should assume a ramp-like waveform that reaches a peak when the buffer-AM is completely hydrolyzed, probably several minutes after termination of exposure to the buffer-AM. Second, exposure to hBAPTA-AM resulted in an increased 2[Ca despite the fact that the hydrolysis products of hBAPTA-AM have no Caj buffering ability. Thus, the observed increase in may be 2 2[Ca a direct effect of the BAPTA nucleus AM ] and/or the esters with the activation of VGCC, directly or secondary to perturbation of the membrane lipids by the lipophilic esters, being a possible mechanism. Effect on resting 21[Ca Cytoplasmic BAPTA or DMB had minimal effect on the resting ]2[Ca :]suggesting that enhancing 2Ca buffering capacity did not affect the cellular mechanisms responsible for setting 2+][Ca at rest (e.g., 2Ca +..ATPase and basal ‘leak’ influx). The only exception to this was observed with the maximum loading regimen (60 pM BAPTA-AM at 33°C) where a very small increase in resting i21[Ca was observed (0.1-0.2 ratio units). Effects on transient 2+Ca responses: When neurons were exposed to NMDA (20 pM for 25 seconds) or high extracellular K (HK, 50mM for 20 seconds), 21+][Ca increased rapidly to a plateau and then recovered completely and rapidly following wash out. Loading neurons with BAPTA or DMB resulted in changes consistent with the effects of increased neuronal 2+Ca buffering capacity, such as lower peak 2+Ca responses and a damping of onset and recovery kinetics. However, these changes did not result in a ‘miniature 146
response’ as the recovery was so slow and prolonged that the duration of the
recovery phases and the responses in general were considerably longer-lasting in the buffer-loaded compared to control neurons. The prolongation of the recovery phase may have major functional consequences on the net biological effect(s) of the 2Ca buffer. Examining the 2[Ca responses shown in Fig. 4.1 raises the possibility that in buffer-loaded neurons, while Ca-mediated processes with low affinity may be reduced, or even not evoked2 at all, other ‘high-affinity’ processes may be stimulated for longer period of time when compared to control neurons exposed to the same stimulus (e.g., 20 pM NMDA for 25 seconds).
The duration of 2+Ca responses was assessed using the mid-span and low-span kinetic parameters, both of which were significantly prolonged after neurons were loaded with BAPTA or DMB. Moreover, mid- and low-span intervals were the only parameters that displayed significant difference between the two buffer-loaded groups used (Table 4.1) with BAPTA loading being associated with longer 2Ca responses than the DMB loading. A possible explanation for this difference may be that DMB has a higher affinity for 2Ca than that of BAPTA, and at resting 2j,[Ca as well as any other 2],[Ca the fractional saturation of DMB will be larger than for BAPTA. Thus the ‘effective’ buffering capacity available for the stimulated 2Ca influx may be more for BAPTA-loaded neurons than for DMB-loaded neurons when the total buffer concentration is the same (as might be reasonably assumed when neurons are loaded under identical conditions). As soon as the available buffering capacity is saturated by 2Ca influx, the kinetics of 2Ca change should be similar to those of control value, i.e., faster. This possibility is supported by the conclusions of Augustine eta! (1991) concerning the differences in effect of 147
dibromoBAPTA, BAPTA and DMB in squid giant presynaptic terminals on neurotransmitter release.
Effect of 2Ca buffers on prolonged 2Ca responses: The ability of artificial 2+Ca buffers to reduce the amplitude of 2+Ca responses during situations of excessive stimulation is an essential requirement for neuroprotection under the excitotoxicity/calcium hypothesis of neuronal death.
Our results indicate that while both BAPTA and DMB were capable of reducing peak responses induced by transient stimulation, peak 2+Ca levels during prolonged exposure to NMDA or glutamate were hardly changed, even though the rate of rise of 2[Ca to those peaks was markedly reduced in buffer- loaded neurons, andj more so in BAPTA-loaded than in DMB-loaded neurons. An equal peak 2[Ca in the presence of 2Ca buffers also indicates that the net 2Ca influxi was larger in the presence of intracellular BAPTA or DMB. A larger net influx of 2+Ca could be a result of reduced 2Ca +.mediated inactivation of either VGCC (Kohr and Mody, 1991) and/or NMDA channels (Legendre eta!, 1993). Recovery of ]2[Ca to pre-stimulation levels should, understandably, be extremely slow in the presence of additional intracellular buffers as a result of the larger net 2+Ca influx during stimulation, and the competition between the recovery mechanisms (e.g., 2+Ca pumps and exchangers) and the high affinity 2Ca binding sites of the added buffer. Larger 2+Ca influxes and slower recoveries are probably the basis for the ‘staircase’ appearance of the 2+Ca profile observed in buffer-loaded neurons during repeated stimulation. The buffer in this situation may be acting as an integrator of the calcium signal (i.e., a capacitance effect). The repeated stimuli that normally leave no residual effect on j21[Ca can thus temporally 148
summate in the presence of the buffer giving rise to a high and prolonged 2Ca signal with potentially harmful consequences.
EFFECTS OF BAPTA UNRELATED TO BUFFERING ACTIVITY
A very important but largely ignored aspect of the effects of artificial 2+Ca buffers on cells are the non-buffering properties of these buffers. A large number of studies have utilized BAPTA and its analogues to analyse the role of 2Ca in cell function and a sensitivity of a biological process to buffer loading has generally been accepted as evidence that this function is 2Ca +.dependent. While this conclusion may be true in a large number of cases, since 2+Ca buffering is undoubtedly an effect of many BAPTA-like compounds, an increasing body of evidence, including our own observation that BAPTA-AM evokes a direct increase in 21[Ca suggests that some of the BAPTA effects are directly mediated by BAPTA,j rather than secondary to 2Ca chelation.
Several studies have implicated BAPTA and many of its analogues, including Quin2, in enhancing the synthesis and release of different prostaglandins and arachidonic acid (Raspe et a!, 1989; Van der Zee et a!, 1989; Boeynaems et a!, 1993). BAPTA loading has also been associated with severe depletion of cellular ATP (Tojyo and Matsumoto, 1990), and inhibition both of protein kinase-C (Dieter et a!, 1993) and phospholipase-D activation (Coorssen and Haslam, 1993). Other non-buffering effects of BAPTA have been reported including direct blocking of muscarinic inhibition of 2Ca currents (Beech eta!, 1991) and competitive inhibition of 31P binding to its receptor (Richardson and Taylor, 1993). For a highly charged molecule such as
BAPTA, diverse direct interactions should not come as a surprise and these
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long
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the 149 150
Figure 4.1: The effects of BAPTA loading on short-duration 2Ca transients. The mean 2Ca + response of 53 neurons exposed to 20 pM NMDA for 25 seconds (solid horizontal bar) is illustrated (solid line) compared to an NMDA induced 2+Ca response after the same neurons were loaded with BAPTA by exposure to BAPTA-AM (20 pM, 15 minutes, RT)(dashed line). The mot noticeable changes were the reduction in th peak magnitude of the Ca + response, the slower rate of increase in [Ca] and the slow and extended recovery phase. The time axis of the second response has been shifted to facilitate the comparison of the two responses.
2.0
0 4-, CD
ci) C.) 1.5 C ci) C-) Cl) ci) 0 U 0 OD 1.0 (Y) (1 C)
0.5 0 100 200 300 Time (s) 151
Figure 4.2: Changes in 21[Ca during exposure to BAPTA-AM. Fifty-four neurons were jexposed to 0.1% DMSO (open horizontal bar) and later to 20 pM BAPTA-AM (solid horizontal bar) at room temperature. Exposure to BAPTA-AM, but not to DMSO, resulted in a marked increase in the mean [Ca that was reversed at the end of exposure to BAPTA-AM. The peak magnitude2j, f both NMDA- (open triangles) and HK depolarization-induced (solid triangle) Ca responses was reduced after loading the neurons with the buffer, BAPTA.
3.0
0 2.5
CD
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C) C) 0.5
0.0 0 1000 2000 3000 4000 5000 6000 7000 Time (s) 152
Figure 4.3: Heterogeneity of the responses to BAPTA-AM exposure. A) Intracellular 2Ca changes in cells during exposure to BAPTA-AM (solid bar) varied from one cell to another as shown in representative cells in 3A. B) Raw fluorescence values at 334 nm and 380 nm (dotted and solid lines respectively) of cells shown in 4.1A indicate that neither addition nor cessation of exposure to BAPTA-AM (20 pM) was associated with detectable shifts in the raw fluorescence intensity suggestive of altered background fluorescence in the presence of BAPTA-AM, regardless of the individual cell response to the AM- ester. The response to BAPTA-AM expsure in individual cells was apparently correlated with the magnitude of the3Ca + response in these cells to HK (open triangles).
3.0
A 2.0 0 4J (‘3
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Time (s) 154
Figure 4.4: Loperamide reverses BAPTA-AM induced increases in 21[Ca In 50 neurons exposed to 2OpM BAPTA-AM (solid horizontal bar), the [Ca in . was abruptly increased and remained high the presence of BAPTA-AM.J21 Addition of loperamide (25 pM, open horizontal bar) to the bathing medium in the presence of BAPTA-AM resulted in an immediate terminatin of the BAPTA AM induced 2+Ca change. A subsequent, NMDA-induced Ca + response (open triangle) had a smaller peak after BAPTA exposure compared to the pre exposure response.
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LL 0 1.0 OD C,,
C,, C,, 0.5
0.0 0 500 1000 1500 2000 2500 Time (s) 157
Figre 4.6(A-E): The effect of buffering on short- and extended-duration + 2Ca Ca responses.
Neurons were treated for 60 minutes at 33°C with 60 pM of the AM ester of hBAPTA-AM (78 neurons, Fig 6A), DMB (72 neurons, Fig 6B) or BAPTA (40 neurons, Fig 6C). Figures 6D and 6E display results of neurons loaded for 60 minutes at 33°C with 20 4uM of the AM ester of DMB (49 neurons) or BAPTA (66 neurons) respectively. 2+Ca responses were evoked by NMDA (20 jiM, 25 seconds: open triangles) or 250 pM glutamate for 3-4 minutes (solid bar) or for 25 seconds (solid triangles). In comparison to control response in hBAPTA-Ioaded neurons, transient 2+Ca respones in buffer-loaded neurons were much more reduced than the prolonged Ca+ responses to a 3-4 minute glutamate exposure. BAPTA was more effective than DMB at the same loading concentration in reducing the peak response to extended glutamate application and in slowing the rates of 2+Ca increase and recovery. The higher loading concentration (60 pM) of each buffer was more effective than the lower (20 pM) concentration of the same buffer under the same loading conditions of time and temperature.
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2500 2500 Figure 4.6 D,E 159 3.0
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0.0 0 500 1000 1500 2000 2500
Time (s) 160
Figure 4.7(A-D): The effect of 2+Ca buffers on ‘excitotoxic’ exposure to glutamate.
Neurons were treated for 60 minutes at 33°C with 60 pM of the AM ester of hBAPTA (48 neurons, Fig 7A), BAPTA (62 neurons, Fig 7C) or DMB (69 neurons, Fig 7D). Figure 7B displays results of neurons treated for 60 minutes at 33°C with 0.03% DMSO (47 neurons). 2Ca responses were evoked by NMDA (2.5, 10 and 40 pM, 25 seconds: open triangles) or 250pM glutamate for 30 minutes (solid bars). In the control groups (A, B) 8-minute intervals between consecutive NMDA responses were adequate for complete recovery of [Ca+] to its resting pie-exposure level. After the 30-minute glutamate exposure, recovery was relatively rapid and almost complete within the experiment time. In the buffer-loaded neurons (C, 0) short NMDA-induced 2+Ca changes were extensively reduced (in the BAPTA-loaded more han in the DMB-loaded neurons). However, recovery of intracellular Ca+ w,s incomplete between stimuli and residual Ca accumulated giving th Cat+ tracings a step-ladder 21 + appearance. A 3O-minue glutamate-induced Ca response showed a slower +]j rise and recovery in [Ca in the buffer-loaded groups than in the control groups, and in the BAPTA-loaded neurons than in the DMB-loaded neurons. However, the peak of the response was not lower in magnitude in the buffer- loaded neurons compared to the control neurons.
A
3.0
0 2.5
(V
a) U 2.0 C a) C-) C,, a) 0 1.5 D LJ 0 10 C)
C’) C’) 0.5
00 0 1000 2000 3000 4000 5000 6000 Time (s)
Figure
D
C
B
4.7
B,C,D
C’) C,)
C’) 0
U C,,
co C,,
C,) C,) C,,
Q3
0 C,,
U 0
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D 0, C-) 0)
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0)
D
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0)
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C
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05
2.5
1.0
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15
2.0
1.5
2.5
0
0
1000
1000
1000
2000
2000
2000
Time
Time
Time
3000
3000
3000
(s)
(s)
s)
4000
4000
4000
5000
5000
5000
6000
6000
6000 161
Aspect
Low-Span
Mid-Span
SlopeDn
SlopeUp
Peak
Parameter
buffer-loading
both
any This
in of
in
capacity Table
NMDA-induced
the
different
the
of
Ratio
table
buffer
4.1:
individual
values
the
(Ratio/s)
(Ratio/s)
(s)
by
(s)
lists
listed
The
experiments
groups
loading
of
(p
and
perentage
Ca
that
experiment.
parameters
<
were compares
+
0.001).
neurons
parameter
BAPTA-loaded
responses
222.4
160.1
Mean
43.4
23.7
52.6
74.3
(%
Notes:
after
significantly
of
changes
was with * * * *
*
control)
All
loading
the
NS:
p-Values
All
Means
before
‘n’
in
the
cultures
after
values
pre-loading
calculated
neurons
effects
BAPTA
measurements represents
69.19
13.61
28.58
neurons
not
13.07
9.43
8.74
S.D.
(n=14)
in
changes
different with
loading
are
the
the
statistically
of
compare
using
of
ranging
or
calculated
the
the
kinetic
neurons
increased
loaded
value. from
DMB.
an
with
corresponding
in
0MB-loaded
buffer,
unpaired from
150.8
133.4
experiment
Mean
39.9
27.5
47.9
66.9
the
(%
are
BAPTA-
from
significant
the
buffer,
parameters
of with
were The
from
the
based
listed
values
control)
40
expressed
neuronal
t-test.
value
control
compared
the
BAPTA
and
to
loaded
parameters
on with
90.
parameter
(n=1O)
18.28
23.14
20.52
11.49
19.26
10.88
percentage
S.D.
of
DMB-loaded
ratios.
of
of
a
that
buffering
values
or
with
transient
number
the
as
to
with
percentages
parameter
its
‘mean’
the
<0.005 and
<
before
NS
of
NS
NS
NS DMB.
0.02
p
buffer
in
of 162 163
CHAPTER 5
CALBINDIN-D28K EXPRESSION AND NEURONA L 2Ca + RESPONSES 164
The objective of the experiments described in this chapter is to compare EAA- and depolarization-induced 2Ca responses in CaBP(H and CaBPN hippocampal neurons using fetal face-down and postnatal cultures. Our working hypothesis was that CaBP neurons, much like BAPTA-loaded neurons, would display 2+Ca responses with lower peaks and slower recovery rates when compared to responses in CaBPN neurons. All statistical comparisons were performed using unpaired t-test.
I. THE ASSOCIATION BETWEENCaBP EXPRESSION AND RESTING INTRANEURONAL j2[Ca Basal ‘resting’ levels of intracellular 2+Ca were found to be significantly higher in CaBP neurons (96.9 ± 6.63 nM, n=82 neurons) when compared to CaBPN neurons (75.3 ± 1.14 nM, n=1004) with a difference of 21.6 nM (p <0.002).
II. THE ASSOCIATION BETWEEN CaBP EXPRESSION AND TRANSIENT INCREASES IN j21[Ca IN CULTURED FETAL HIPPOCAMPAL NEURONS a. NMDA-induced 2Ca transients:
In 25 different experiments, exposure to NMDA (20 pM, 25 seconds) resulted in an increase in 2[Ca to 383.8 ± 20.8 nM in CaBP neurons (n=82) which was significantlyj larger (p < 0.001) than the peak 2Ca concentration in CaBPN neurons (303.7 ± 4.84 nM, n=1004).
The absolute increase (Basal-to-Peak) was also significantly larger in
CaBP than in CaBPN neurons (286.9 ± 20.69 nM, and 228.4 ± 4.79 nM respectively, p < 0.01). The duration of the 2Ca transient was longer as seen by measurement of the mid-span and low-span parameters which were 16
neurons were
neurons
resting
duration
the
peak
fetal
resting
CaBPN
closely
parameter
studied
individual
and
different
well
Similarly,
the
in
seconds
respectively
CaBP
application
basal-to-peak
their
Interesting
It
as
culture
A
still Ca 2 +
is
‘basal’
typical
Ca 2 +
reflected
neurons
during
parameters,
displayed
while
of
also
and
in
larger
statistics).
the
experiments.
was
the
response
and
the
were
(both
concentration
29.1
of
rates
the
Ca 2
experiment
the
of
transient
found
two
for
variants
CaBPN
observed
significance
the
the
exposed
changes
peak,
a
recovery
p
seconds
the
of
lower
populations
though
level
frequency and
<
It
agonist
to
change
CaBP
must
0.001
neurons
Table
basal-to-peak
are
was
be
the
was
in
is
peak
to so
larger
in
longer
phase
measured
shown
the
illustrated
to
be
basal-to-peak
longer
than
20
that
in
the
).
5.3
not
with
Ca 2 +
note
population
stressed
(See
same
(5.14
[Ca 2 j
pM
Widening
in
CaBP
lists
of
higher in
the
that
in
in
the
which
that
NMDA the
the
Table
change
Fig
and
experiment.
from
aspect the
±
in
the
of
CaBP(+)
during
CaBP
that
Ca 2 +
0.48
Fig
in
in
5.3A
the
CaBP
the
frequency
(Fig
neurons the
of
change
5.1
the
the
4
for
5.1
the
on the
ratio
CaBPN
out
width
and
differences
transients,
and
5.2).
for
whole
NMDA
CaBP 25
exposure
where
population
significant
transients
than
of
full of
4.97
were
seconds.
neurons.
3B.
(n=
the
of
with
the
neurons
pool
listing
receptor
in
the
85
3)
25
In
than larger,
±
CaBPN
transient
were
which
were
to
Fig
was of
0.14,
Ca 2
neurons
experiments
was
as
differences
The of
NMDA
in
neurons
(n=82).
5.3A
compared
and
activation,
all
higher
not
the
observed
proportional
neurons
a
p=0.7).
transient
mean
was
parameters
particular
of
the significantly
CaBPN
CaBP
than
tested,
an
before
similar
The
of
in
the
to
8-DIV
in
the
the
as
the
165
the to 166
CaBPN neurons yet the recovery was slower in spite of the reduced peak
height. Fig 5.3B illustrates how a moderately larger peak response in the CaBP neurons was associated with much longer recovery where mid- and low-span parameters were almost double those of the CaBPN neurons in the same field.
Another illustration of the difference in the recovery process is evident in the extent of recovery of intracellular 2+Ca after NMDA receptor stimulation of the CaBP neurons. Over the observation period (6-8 mm), CaBPN neurons tended to have rapid and complete recovery to pre-stimulation levels of 2+]i[Ca whereas CaBP neurons displayed a residual increase in intracellular 2+Ca over pre-NMDA exposure levels (Fig 5.1, 2 and 3).
CaBP(H neurons were also over-represented in the few neurons that
‘collapsed’ on challenging them with NMDA (15 out of 23 neurons in the 25 experiments conducted using NMDA). CaBP immunoreactivity was also more frequently found in neurons that had extremely high 2Ca levels at the beginning of the experiment (10 out of 19 neurons). Such neurons were excluded from the statistical analysis since kinetic parameters could not be measured. b. Depolarization-induced 2+Ca transients:
In another set of 18 experiments with a total of 742 neurons, of which 59 were CaBP(+), depolarization-induced 2+Ca responses were evoked by exposure of neurons to high extracellular concentrations of K+ (50 mM). Similar statistical analyses were conducted and the results were very similar to those obtained from NMDA-induced 2+Ca responses. Only the parameters that were significantly different with NMDA stimulation were also significantly the when exposure were although that ‘narrower’ tested exposed is experiments, cultured whether NMDA to expected EXPOSURE two Ill. values CaBP(H different almost that postnatal THE were stimulation In On The much compared was spite or as of exposure neurons the to ASSOCIATION the and double in [Ca 2 ] results if HK qualitatively BAPTA, well responses faster 613 the CaBP the OF of kinetic magnitudes CaBP 8-day CaBPN exposure as these NMDA CULTURED case of to procedures that reported at (compare functioned the in to which which postnatal old trends a the NMDA, of neuronal observed with kinetic statistical more neurons similar (20 cultures (see depolarization-induced postnatal BETWEEN of reduced 80 in high pM, observed tables POSTNATAL advanced the (Fig chapter the differences CaBP were postnatal intracellularly to populations displayed in 25 aspect transients significance of previous those 5.4, fetal the 5.1 cultures. later seconds). 4 CaBP 4). neurons, in day-postnatal Tables stage magnitude and recorded cultures ratios neurons fetal found between However, many HIPPOCAMPAL EXPRESSION section were 5.4). were as of This cultured of 5.2 and The a that to development. Ca 2 of the of displayed Ca 2 + greater from very and be of resulted were faster the postnatal the total were differences it hippocampal the CaBP, transients. was 5.3). neurons comparable cultured characteristic same buffer AND not number and peak recovery observed NEURONS increases in of what and in the taller TRANSIENT interest in Ca 2 a would In fetal vitro between a frequency of rates fetal a neurons would Absolute manner to among and series in neurons response neurons in age. trends, TO pre exist the to neurons, of [Ca 2 ] 1 test be NMDA the change of the were similar in that 14 167 to that was recovery aspect neurons 2.61 of neurons. same significantly versus 37.72 proportion rate neurons, had statistics were population larger CaBPN p difference comparison =0.479). the in significantly complete of The and Also also population. recovery in 13 ratio ± 15.06 increase neurons (p was the net during 2.82 for 1.17 With out in larger to was < (695.94 of larger between However, contrast CaBP postnatal result the incomplete 0.01). of to ± the seconds nM/s larger that process, was the 0.56 (70.65 in pre-exposure higher 14 increase The Ca 2 (p CaBP was experiments, increase the ± in 53.25 Another peak with < fetal population nM/s) rate peak cultures. the 17.46 respectively) rates that 0.01), resting ± were transients over in fetal CaBP CaBPN [Ca 2 i 1 2.50 of ± neurons [Ca 2 ] 1 from the mid-span and of nM, recovery prominent the only 3.00 levels was CaBP [Ca 2 ] 1 change, nM peak (939.95 recovery that recovery same p neurons and slightly was and nM/s not and in < while (Fig than and in and response 0.001) only of was markedly time fetal significantly 72.74 neurons, compared finding the 5.5). in ± the [Ca 2 ], basal-to-peak phases in low-span of longer (p CaBP slightly 43.05 almost CaBPN course. CaBPN 1 [Ca 2 j 1 same < and 5 Table ± in to out 0.001), different in postnatal 1 of the the though to NMDA nM) .08 identical parameters larger the neurons. of parameters, larger neurons. neurons the 5.4 in the postnatal basal-to-peak 25 nM compared the CaBP parameters Ca 2 + an lists postnatal mid-span, was statistically fetal (18.25 in respectively CaBP increase CaBP in the compared parameter A significantly transients. CaBP(H in cultures cultures as neurons notable CaBP the to ± CaBPN indicators of the changes neurons the that neurons 1.08 CaBPN the to with and tested, mean CaBP was is (by The in 168 169
IV. THE ASSOCIATION BETWEEN CaBP EXPRESSION AND PROLONGED 2Ca RESPONSES
In a smaller series of experiments, extended exposure of cultured fetal hippocampal neurons to NMDA (20 pM, 2 or 5 mm) or glutamate (500 pM, 5 mm) resulted in a prolongation in the 2+Ca response. Under these conditions, CaBP neurons demonstrated larger peak responses and slower rates of recovery than their CaBP counterparts (Fig 5.6A,B).
DISCUSSION FETAL NEURONS: In fetal cultures, resting pre-stimulation 2[Ca and peak 21[Ca during NMDA-induced responses were significantly higherj in CaBP jneurons than in CaBP neurons. Larger peak responses were not simply a reflection of the higher pre-stimulation ]2[Ca since the basal-to-peak change in 21[Ca was also significantly larger than in the in ] CaBP neurons. Also, some experiments where pre-stimulation 2+][Ca was similar in both populations, the response amplitudes were still larger in the CaBP neurons.
Larger response peaks in CaBP neurons were associated, in fetal cultures, with a proportional prolongation in the recovery of 2[Ca +j, compared to CaBPN neurons at both the mid-span and low-span levels. While longer span values are to be expected in association with larger 2+Ca responses, prolonged recovery may not be entirely due to larger peak responses. In some experiments where the peak of the 2+Ca response was lower (or marginally larger) in CaBP neurons, the duration of 2+Ca responses was still more prolonged than that of the CaBPN neurons. Slower recoveries of 2+][Ca to pre-stimulation values were also reflected in an incomplete recovery of the 170 mean 21[Ca of CaBP(H neurons that was observed in 15 out of 25 experiments,j while incomplete recovery was observed in the mean 2[Ca +1 of the CaBPN neurons in only 3 out of 25 experiments. The differences observed between 2+Ca responses to transient NMDA exposure in CaBP and CaBPN neurons were also observed when 2+Ca responses were induced by depolarization using high extracellular K+ and when exposure to NMDA or glutamate was extended for several minutes.
These results lead us to conclude that the development of CaBP immunoreactivity in fetal hippocampal neurons is associated with a change in EAA- and depolarization-induced 2+Ca responses characterized by larger magnitudes and longer durations. It is also associated with a tendency for an elevated basal 2+][Ca and incomplete recovery following a 2+Ca load.
In general, larger peaks and longer-lasting responses could result from larger 2+Ca influx, greater intracellular 2+Ca release, less efficient recovery mechanisms or a combination of these factors. The larger 2+Ca influx could be a result of an increased 2+Ca channel density and/or altered channel kinetics (e.g., larger unitary conductance levels, slower inactivation or desensitization, enhanced sensitivity to agonist or to depolarization). Similar factors could contribute to a larger intracellular release of 2+.Ca Impaired recovery may result from impaired extrusion/sequestration mechanisms or as a result of competition between recovery mechanisms and a high capacity intracellular calcium buffer.
While our experiments cannot precisely identify which of these mechanisms are responsible for the changes observed in 2+Ca responses in CaBP fetal neurons, it is unlikely that a single mechanism underlies all the 171 changes observed (i.e., elevated basal 2i,[Ca larger peak responses and slower recovery) since they were not all present in every experiment. For example, slow and prolonged recovery was pronounced even when mean response amplitudes were lower in CaBP neurons than in CaBPN neurons. Very similar changes in both NMDA- and depolarization-induced 2Ca responses also suggest that the difference between the two neuronal populations may be more downstream to 2+Ca influxes, although a parallel change in agonist- and voltage-induced fluxes in CaBP neurons cannot be excluded.
Changes in 2+Ca responses associated with the onset of expression of CaBP in fetal neurons may be required for developmental purposes (e.g., synaptogenesis), but the same changes may also heighten the vulnerability of CaBP neurons to 2Ca + mediated injury. This issue is addressed in more detail in chapter 6 but a coincidental finding in the present set of experiments indicates that this may actually be the case since CaBP neurons were significantly over represented in neurons that had ‘de-regulated’ 2+Ca levels before exposure to NMDA or depolarization, and in those that collapsed, i.e. showed no sign of recovery, after stimulation and for the duration of post- stimulation period.
It is important to stress that statistical comparison of two populations can be complicated and biased when one of these populations has a much lower frequency of incidence than the other. In our case, CaBP neurons constitute approximately 8% of the total neurons in fetal cultures. A few extreme responses can cause a significant bias in the calculated parameters, and subsequently skew the results of significance tests. In order to avoid this situation the results of our experiments were obtained by pooling the results of 172
all the CaBP neurons before performing statistical comparisons to the pooled results of the CaBPN neurons. However, we contend that the consistent and
reproducible trends observed in the large number of experiments performed lend more support to the validity of the observations than does the actual numerical results of the analysis although in our results complete agreement exists between the two approaches (Table 5.3).
POSTNATAL NEURONS:
Fetal neurons are in a phase of extensive developmental change and may not be representative of neurons in adult animals. For this reason, postnatal cultures were also studied to test whether the trends observed in fetal cultures would persist during later stages of development. Compared to fetal neurons, postnatal neurons had larger peak responses regardless of their CaBP immunoreactivity. Despite these larger 2+Ca responses, recovery in postnatal neurons was surprisingly fast with mid- and low-span values that were 40 to
50% shorter in postnatal neurons for responses that were more than double the magnitude of fetal response, indicating a significant increase in the efficiency of recovery mechanisms relative to the NMDA-induced 2+Ca load. Also, it is interesting to speculate that larger 2+Ca responses are associated with larger 2+Ca influxes that may have saturated cellular 2+Ca buffers and resulted in the faster initial recovery of the 2+Ca responses observed in postnatal neurons.
Within the postnatal cultures, CaBP neurons displayed significantly larger peak 2+Ca responses compared to CaBPN neurons although basal 2+][Ca was similar in the two groups. Mid-span and low-span parameters were marginally longer in CaBP neurons but not in proportion to the increase 173
in peak levels since the rates of increase and recovery were significantly faster in the CaBP neurons. Larger peaks and faster rates of increase and recovery caused 2+Ca responses in CaBP(H neurons to look more ‘pointed’, a feature that was numerically expressed as a significantly larger aspect ratio.
It is clear from our data that the presence of CaBP is associated with greater differences in resting and stimulated 2+][Ca responses in fetal neurons compared to postnatal neurons. However, we cannot conclude that CaBP is the cause of these differences. Further, we have made no attempt to quantify the amounts of CaBP expressed in neurons derived from these two sources, mostly due to the capricious nature of immunohistochemistry as a detection method for this protein. The determination of neurons as being simply “positive” or “negative” on the basis of immunohistochemistry may well mask the possibility of large absolute differences in concentrations of CaBP in fetal and postnatal neurons which could account for the differences observed.
Although CaBP has been the focus of a large number of studies, only a few of these have been aimed at identifying the functional correlates of the expression of this protein. In 1991, Kohr eta/studied the high voltage activating (HVA) 2Ca current in acutely dissociated adult hippocampal dentate granule (DG) neurons prepared from normal and kindled rats using the whole cell recording technique. Kindling, while inducing seizure activity in rats, causes a selective loss of CaBP from DG cells (Miller and Baimbridge, 1983). Comparing CaBP-deficient DG neurons from kindled rats to normal CaBP DG neurons from non-kindled animals, indicated that HVA (L-type) ‘Ca displayed marked inactivation in CaBP-depleted cells when compared to normal, CaBP
DG cells, which showed very little inactivation. This difference in inactivation was significantly diminished by the inclusion of the fast 2Ca buffer BAPTA in 174
the recording pipette indicating that the difference in inactivation was the result of a reduced 2+Ca buffering capacity, presumed to be due to the loss of CaBP. Peak ‘Ca was similar in both cell types and their respective I-V relationships were identical. The authors concluded that CaBP may have a physiological role as an intracellular 2+Ca buffer in the immediate submembraneous zone with important functional consequences (Kohr eta!, 1991; Kohr and Mody, 1991).
Mattson eta! (1991) used primary cultures of E18 fetal hippocampal rat neurons, plated at very low density (50 neurons/mm and maintained in media containing 55 mM d-glucose, 20 mM KCI2 and 10% fetal bovine serum, to study neuronal ) vulnerability to excitotoxic injury and calcium responses in relation to CaBP expression. Their results showed that CaBP neurons are better capable of handling 2Ca loads induced by exposure to glutamate or to the 2Ca ionophore Br-A23187 (4 neurons). Pre-stimulation }2[Ca and peak 2+Ca responses were similar in CaBP and CaBPN neurons but the extent and rate of recovery was significantly faster in CaBP neurons. The use of 2+Ca ionophore, presumably inducing equal 2+Ca loads in different neurons, led the authors to conclude that the difference between the two populations was not a result of different glutamate receptor density. However the assumption of an equal 2Ca load induced by A23187 is based on the assumption of a uniform neuronal surface/volume ratio which may not be valid for cultured neurons with different CaBP immunoreactivities since, in our own experience, Ca8P neurons were generally larger than CaBPN neurons (data not shown).
A novel and potentially very useful approach to the study of the functional role of CaBP was employed by Liedo eta! (1992) where they used a retrovirus to transfect 3GH pituitary cell line with the cDNA of CaBP. The recombinant 175
cell line displayed reduced magnitudes of both L- and T-type ‘Ca upon current- evoked depolarization. Inactivation of both L- and T-currents was also
enhanced in the cell line expressing CaBP (a result entirely opposite to that of Kohr and Mody, 1991) but peak 2Ca responses to depolarization were decreased. However, in the recombinant cells resting 2[Ca was increased, which in itself could provide an explanation for the enhanced inactivation observed in these cells. Analysis of the decay time of 2[Ca transients indicated a ] the loss of fast decay component evident in the wild type cell line, while the slow component of decay was not significantly altered. These authors also increased intracellular CaBP content by including it in the patch pipette. Using this loading method, increased CaBP content of the cell was not associated with reduced 2+Ca current amplitudes and resting intracellular 2+Ca was not significantly changed. Peak 2[Ca responses to depolarization were, however, reduced and the fast decay component was lost while the slow component was slightly prolonged.
A more recent study also utilized patch pipettes for loading cultured postnatal dorsal root ganglion (DRG) neurons with CaBP (Chard eta!, 1993). Introduction of CaBP had no effect on the inactivation or rundown of calcium currents. Basal 2+][Ca was unaltered while depolarization-induced changes in 2+][Ca were reduced in magnitude and the rates of increase and decay of ]21[Ca were markedly diminished in the CaBP-Ioaded cells. These results are therefore consistent with a fast 2Ca buffering action of CaBP in DRG neurons.
Direct comparisons between the results of the above-mentioned studies and our own are extremely difficult since diverse preparations and experimental paradigms were employed. A particularly important experimental difference is the time scale of the stimuli used to induce 2+Ca responses. While
their
of
conditions
The
(1991) capacity,
recovery
results while
loss
with
following
neurons.
‘acute’
results
currents
and
taken
saturation
milliseconds
using
electrophysiological
CaBP
common
conceivably
of
individual
However,
the
In
not
20
into
were
but a
showing
loading
spite
will
effects
fast in
of
fast
second
stimulation
producing
The
were
of
account.
their
CaBP
[Ca 2 +]j
depend
obtained
in
of
feature
recovery
cytoplasmic
Ca 2
elevation
kinetic
this
of
duration,
this
very
sample
of
larger
extended
stimuli
wild
transfecting
conclusion
after
buffering
cautionary
identical
on DG
measurements
different
of
was
by
properties,
Ca 2
component
type
the
size
our
or
cells,
or
our
Chard
transmembrane
Ca 2
quite
recovery
resting
longer.
full
results
GH 3
was
responses
fluorescent
effects
action
resulting
from
cannot
profile
remark,
et GH 3
evident
binding
cell
quite
in
a!
[Ca 2 +]
and
and
Thus
ours.
were
any
of
cells
of
to
(1993)
with
of
be
small
[Ca 2 +]i,
a
in
CaBP.
these
transfection,
the
in
in
particular capacity
Ca 2
slightly
[Ca 2 +]
total
conducted
Ca 2 +
reconciled
with
larger
CaBP-containing
our both
It
and
lack
is
in
and
studies
fetal
Ca 2
understandable
acutely-loaded
extended
CaBP
regulatory
fetal
flux,
is
Ca 2
of
extended
measurements
their
during
consistent
cell
inactivation
neurons
with
and
using
loads
consistent
was
(Liedo
seems
type.
neuronal
influx
longer
recovery
postnatal
that
associated
mechanisms,
stimuli
and
recovery.
patch
et
and
to
(Kohr
with
DRG
of
a!,
that
the
stimuli
of
be
culture
with
were
in
Mattson
pipettes,
1992).
of
Ca 2
the
phase
a
eta!,
CaBP
agreement
possibility
the
neurons.
delayed
tens
a
with
Similar
conducted
present
should
high
effects
and
1991),
of
The
et
the
a!
176
of be 177
Another difficult observation to explain is the larger amplitude of 2Ca responses in association with a 2+Ca buffer such as CaBP. While expression of CaBP is probably not the only distinguishing feature between CaBP and CaBPN neurons, and thus other mechanisms might be responsible for larger 2Ca responses, it is possible that the relief of Ca-dependent inactivation of Ca channels in immediate 2+ the submembraneous2 zone (Kohr eta!, 1991) per se can cause an increase of 2+Ca influx that outweighs the enhanced buffering capacity associated with CaBP expression. Another possible explanation is that a 2Ca buffering action of CaBP causes ‘uncoupling’ of physiologically paired messages. For example, altering the time relationship between changes in 21[Ca and the production of 31P or AA might result in a change in the temporalj and/or spatial relationship between inhibitory and stimulatory effects of these second messenger pairs with the subsequent enhancement (or inhibition) of other 2+Ca process such as release, uptake or extrusion.
It should be made clear that none of the studies discussed here, including our own, can establish any causal, rather than associative or coincidental, relationship between altered 2+Ca responses on one hand, and CaBP expression on the other. The only exception could have been the transfection experiments (Lledo et a!, 1992) although the use of wild type cell line as a control, rather than a retrovirus-infected cell line (without CaBP-cDNA or with a
‘dummy’ cDNA) precludes establishing a cause-effect relationship. This lack of an appropriate control group makes it difficult to evaluate the conclusions of that study.
While our results are not consistent with the hypothesis that CaBP is exclusively functioning as a simple 2Ca buffer, similar to BAPTA, prolonged 2+Ca responses and delayed recovery in fetal neurons indicate that buffering 178
2+Ca may be one function of CaBP in neurons. This conclusion is supported by the results of Kohr et al (1991), where BAPTA could reverse the enhanced inactivation of 2Ca currents observed in DG cells that lost their CaBP, and by the results of Lledo etal(1992) and Chard etal(1993), where peak 2Ca responses and rates of change in 2+][Ca were reduced whenever cellular CaBP content was increased. Larger 2+Ca responses in both fetal and postnatal CaBP neurons most probably reflect other functional consequences of CaBP expression, although they may as well reflect a different aspect of the phenotype development in which CaBP expression may be coincidental or a secondary phenomenon. 179
Figurç 5.1: NMDA-induced 2+Ca responses are larger and longer-lasting in CaBPIH fetal hippocampal neurons. Eighty-seven cultured fetal hippocampal neurons wer simultaneously exposed to NMDA (20 pM) for 25 seconds. Three were CaBP’ and displayed a higher initial base line +][Ca a larger peak response and a slower recovery (solid line) when compared21 the mean of 84 CaBPN neurons line). The (dotted recovery of the ,former was also incomplete during the interval studied.
600
CaBP, n=3 500 J
:3 400 C) 0(‘5 1 (‘5 300 :3 0G) (‘5 -4-, C 200 u) 1ci) U-
CaBP n=84
0 100 200 300 400 500 600 Time (s)
transient
recovery intracellular
Of
neurons Figure
U-
0 a) 1) (ES C) ci) I
C) E
C
79
neurons
5.2:
even
was
and
0
Larger
Ca 2 +
extended
complete.
in
exposed
the
levels
and
100
absence
longer-lasting
recovery
to
were
NMDA,
of
200
similar
initial
(solid
5
CaBP
Ca 2 +
to
Time
high
line,
CaRPN
300
resting
responses
mean
(s)
neurons
neurons
of
Ca 2 +
400
5
are
responded
neurons),
(dotted
levels.
observed
500
line)
although
with
in
and
CaBP
a
600
larger
initial 180 181
Figure 5.3: Comparison of the mean 2Ca response in CaBPU and CaBP neurons following exposure to 20 pM of NMDA for 25 seconds. A) Although the peak of the mean 2Ca response is smaller in CaBP neurons (n=5, solid line), recovery parameters, i.e. mid- and low-span, are longer than those in CaBPN neurons (n=32, dotted line).
B) In this experiment, a small increase in the peak response in CaBP neurons (n=2) was associated with an increase in the mid- and low-span parameters (37.3 to 57.5 seconds and 71 .2 to 99.0 sends respectively) and a slower and incomplete recovery compared to CaBP neurons (n=32).
A
250-
C 200
- 150 :3 0a) 100 LCaBP+,n=5 ci) ci) U- 50
NMDA
I 0 I 500 550 600 650 700 750 600 Time (s) CaBP ______
182
Figure 5.3 B
700•
600-
—500C E D 400 CaBP+,n= - I -j300o - C 200 ci)
U— 1•
100 . .. ,n=32 NMDA I 0 I 0 100 150 200 250 300 Time (s)
CaBP the
B) A)
responses
(dotted is
Figure LL 0 -4-,
1 I 0
a) ci) D ctS 0 C 1 E
C
slower
In
Five
aence a
-,
A
5.4:
different
neurons.
100 CaBP
line)
in
and
Depolprization-induced
CaBPIH
of
in
150
slow
the
raised
experiment,
neurons
same
200
incomplete
compared
initial
field.
(solid
250
[Ca]
larger
line)
to
recovery
300
CaBP
increases
and
in
had
Time
5
longer-lating
350
CaBP’
higher
when
fetal
(s)
in
400
neurons.
compared
[Ca 2 +1 1
resting
neurons
450
responses
[Ca 2 i 1 ,
are
to
compared
500
larger
79
larger
CaBP’
were
550
and
to
evident
recovery
Qeak
69
600
neurons
183 in 184
Figure 5.4 B
350
300 CaBP+n=5
-250C E 200
150 1 0 4-, .... C ; 100 - - 1) - - .. - IL LCaBP , nt=69 50
0 I I 500 550 600 650 700 750 800 850 900 Time (s) 185
Figure 5.5: Mean [Ca in cultured 4-day postnatal hippocampal neurons (8- DIV) exposed to 202j, pM NMDA for 25 seconds is larger in CaBP neurons than in CaBP neurons.
Within the postnatal cultures, CaBP neurqns (solid line) showed larger pea responses and faster rates of change in [Ca] compared to postnatal CaBP neurons (dotted line). Recovery was marginally delayed but was complete in almost all cases.
C E D 0 0
C”
0 C” 4-,
a) U
0 100 200 300 400 500 600 Time (s) The CaBPN for the shared The similarities NMDA glutamate Figure U- 0 C 1 ctS U) 0 U) U) 1 0 D E C 2 mean mean prolonged mm. A 5.6: for a neurons, larger of peak results shorter to In Extended 54 the (B) Ca 2 + peak [Ca 2 J 1 CaBPN and 74 pattern in times larger neurons [Ca responses recovery exposure neurons in Figs frequently +] 3 Ca CaBP’ and were 5.1-5.4). was + of in (dotted responses a fetal CaBP exposed slower much observed neurons Time hippocampal line) In slower. incomplete (A), neurons in (s) to when in was CaBP 500 3 C 8 BP(H CaBP larger pM exposed (solid neurons recovery neurons. glutamate than neurons line) neurons to to that retained 20 of NMDA for exposed pM [Ca 2 +] of (solid 5 71 NMDA or mm. many line) than to 186 187
Figure 5.6 B
500
450
400
350
300 C-) 250- Z3 200 CaBP, flt3 I
50 CaBP n=7rJ
0 0 500 1000 1500 2000 2500 3000 3500 4000 Time (s)
Low-Span
SlopeDown
Mid-Span SlopeUp(nM/s)
Basal-to-Peak
Peak
Parameter
Basal
Table
25 responses
seconds
Ca2+
Ca2+
5.1:
(s)
(s)
(nM)
(nM/s) (nM)
-
in
Comparison
(nM)
(NS fetal
=
CaBP
111.7
228.4
303.6
Mean
55.3
11.2
75.3
2.8
not
CaBP(-)
statistically uing
+
151.93
153.37
57.09
21.51
36.21
3.18
9.32
S.D.
and
n=1004
unpair
CaBP
S.E.M.
0.68
1.80 0.10
0.29
4.79
4.84
significant). 1.14
-
neurons
t-test)
142.5
287.0
383.8
Mean
71.3
12.8
96.9
2.4
of
CaBP(+)
exposed
the
187.37
188.02
76.26
36.02
10.33
60.06
2.21
S.D.
kinetics
n=82
to
S.E.M.
20.69
20.76
20
8.42
3.98
0.24
1.14
6.63
of
pM
the
NMDA
Ca 2
Mean
30.86
16.00
58.59
21.58
80.18
-0.41
value
1.55
Difference
for
<0.001
<0.001
<0.01
188
<0.01
<0.01
(NS)
(NS) p 189
Table 5.2: Comparison (uing unpairec t-test) of the kinetics of the CaBP+1 2Ca responses in fetal and CaBP’ neurons depolarized by exposure to 50 mM 0K for 20 seconds (NS = not statistically significant).
CaBP(-) n=683 CaBP(+) n=59 Mean Difference
Parameter Mean S.D. S.E.M. Mean S.D. S.E.M. value p
Basal Ca2+ (nM) 78.6 38.96 1.49 99.8 65.81 8.57 21.23 <0.05
Peak Ca2+ (nM) 301.6 119.58 4.58 358.6 146.16 19.03 56.98 <0.01
Basal-to-Peak (nM) 223.0 121.25 4.64 258.7 140.27 18.26 35.74 <0.05
SlopeUp (nM/s) 12.9 9.28 0.36 15.0 11.04 1.44 2.14 (NS)
SlopeDown (nM/s) 2.8 2.43 0.09 2.3 2.15 0.28 -0.58 (NS)
Mid-Span (s) 49.2 29.65 1.13 65.1 36.80 4.79 15.83 <0.01
Low-Span (s) 97.0 50.61 1.94 140.6 82.97 10.80 43.66 <0.001
Low-Span SlopeDown
Mid-Span
SlopeUp
Basal-to-Peak
Peak
Basal
Parameter
that
mean
CaBP’
occurrence the
pooled
This
Table
NMDA-
(e.g.,
Ca2+
Ca2+
CaBP’
significant
table
values
larger
5.3:
(nM/s)
(s)
çlata
(s)
or
(nM)
(nM/s)
neurons
(nM)
lists,
HK-induced
(nM)
neurons
is
from
peak
of
listed
p-values
the
for
in
all
response
each
parameter
the
next
CaBP(+)
of
the
NMDA
(25
Ca
the
NMDA
neurons
reflected
experiments)
kjnetic
to
same
22
19
17
21
19
19
exposure
or
8 the
>
response
CaBP(-)
longer
under
group).
p-value
parameter,
experiment.
(e.g.,
the
study
mid-span)
trend
76%
88%
32%
68%
76%
76%
84% was
1004
obtained
The
%
in
I
the
in
associated
purpose
<0.001
<0.001
<0.01
<0.01
<0.01
CaBP’’
the
(NS)
(NS)
The
the
p
number
in
frpm
CaBP
the
relationship
percentage
CaBP(+)
High
of
neurons
(18
CaBP
t-test
with
of
this
experiments)
K+
experiments
and
14
13
12
13
13
14
5
a
listing comparisons
>
exposure
versus
larger
between
CaBP(-)
of
CaBP
neurons
such
is
parameter
to 82
neurons.
72%
78%
28%
67%
72%
72%
78%
where
the
than
show
%
of
f
the
190
<0.001
in
<0.01
<0.05
<0.01
<0.05
(NS)
(NS) p 191
Table 5.4: Comparison (using unpaired t-test) of the kinetics of the 2Ca responses in cultured 4-day postnatal CaBP and CaBP’ neurons exposed to 20 pM NMDA for 25 seconds (NS = not statistically significant).
CaBP(-) n=533 CaBP(+) n=80 Mean Difference
Parameter Mean S.D. S.E.M. Mean S.D. S.E.M. value p
Basal Ca2+ (nM) 72.7 24.93 1.08 70.7 22.37 2.50 -2.09 (NS)
Peak Ca2+ (nM) 695.9 403.21 17.46 940.0 385.02 43.05 244.01 <0.001
Basal-to-Peak (nM) 623.2 402.30 17.43 869.3 384.05 42.94 246.10 <0.001
SlopeUp (nM/s) 37.7 27.01 1.17 53.2 26.82 3.00 15.53 <0.001
SlopeDown (nM/s) 15.1 12.94 0.56 18.3 9.69 1.08 3.20 <0.01
Mid-Span (a) 36.7 8.84 0.38 39.3 6.31 0.71 2.61 <0.02
Low-Span (s) 56.6 18.11 0.78 59.4 11.89 1.33 2.82 (NS)
Aspect Ratio 18.9 16.37 0.71 23.1 11.81 1.32 4.16 <0.01 192
CHAPTER 6
EXCITOTOXICITY IN HIPPOCAMPAL NEURONAL CULTURES temperature. identifiable glutamate resulted resulted assessed (± their protocol a. from I. vacuolation minimize assessment injury neuronal The results on exposure EXCITOTOXICITY SE) Fetal glutamate-induced influence sensitivity cultures included Fetal Although The of that in in at Cultures: variability injury, after 68.6 of EC 50 a neuronal neurons objective room cultures were of only. reduced cultured tested In of a the swelling to ± of all viability the further temperature different comparable 23.1 excitotoxins cytoplasm Criteria 192 the as were injury absence which after of IN sensitivity hippocampal a neuronal results this pM ± and ‘NAIVE’ 24 result assessment killed 33 7 culture which used had hour and chapter to loss of pM. and to (RT) reported of 10 by at been injury for the extracellular to those incubation NEURONAL was of the conditions a loss DIV. using in glutamate neurons In morphological phase glutamate addition is maintained using the culture both concentration-dependent will to of obtained below a presence present neuritic also 30-minute brightness groups, fluorescent to in of and Mg 2 +, age CULTURES toxicity are HEPES-buffered high concentration be Mg 2 by face-down in treatment the processes. examined. based identification of all concentrations morphological vitro, exposure glutamate results of as 1 morphologically to vital jiM the indicated on the results TTX. with stains the were neuronal of incubation of In to BSS the with 3 of latter exposure order Ca 2 glutamate were examined mM Viability assessment by yielded neuronal of effects at an the soma, glutamate. method to (Fig room obtained buffers EC 50 medium higher 6.1). was of for 193 of of 194
During exposure to glutamate, neurons appeared swollen with vacuolated cytoplasm and their somata lost their phase brightness. These changes were observed even at concentrations that were not subsequently associated with wide-spread neuronal damage. In the majority of neurons, the early morphological changes associated with cell injury were completely reversed 4-6 hours after termination of exposure to glutamate (even at high concentrations), and the neurons regained their phase bright appearance. However, this morphological recovery was only temporary and many neurons were assessed as being dead 24 hours after the initial insult.
Face-up fetal cultures were exposed to excitotoxic glutamate concentrations using the extended exposure protocol, i.e. for 24 hours at 34°C in their N2 culture medium containing 2+.Mg Under these circumstances the 50EC of glutamate in the face-up cultures was 72 ± 11 .6 jiM but 45-50% of the fetal neurons were found to be resistant to glutamate toxicity up to a concentration of 1 mM. This is interesting in view of the observation that, in the vast majority of morphologically identifiable neurons, glutamate exposure evoked an increase in neuronal 2+].[Ca b. Postnatal Cultures:
Postnatal cultures displayed a significantly higher sensitivity to the neurotoxic effects of glutamate when compared to either type of fetal cultures. Using the extended exposure protocol, approximately 90% of the neurons were killed using a glutamate concentration of 1 mM. The 50EC for glutamate toxicity was 20.4 ± 9.7 pM, the lowest amongst the 50EC values in the three culture types tested. For example, only 68 ± 5.3% of the neurons survived an exposure to glutamate at a concentration of 12.5 pM (Fig 6.2).
and
slow,
over following
which
wash-out
population
glutamate
neurons d.
test).
8%
compared pM)
under
loperamide
higher than
using
varying
neurons
c.
[Ca 2 +] 1
Effects
magnitude
survival
the
increased
10%
and
unpaired
Using
our
was
Glutamate
survival
degrees
was
following
using
its
of
in
experimental
(3
to
to
under
maintained
of
changes
(25
the
fura-2
wash-out. rate
many
mM)
no-antagonist
determined
73
Antagonists:
the
neuronal
of
rate
t-test).
pM),
glutamate
the
with study
±
receptor
this
at
4-6
cases
extended
microfluorimetry,
8%
of
under
excitotoxic
were
room
early
p<0.05
hours
84
throughout
showed
A
conditions,
and
In
survival
complete,
at combination
±
antagonists
most
much
temperature, excitotoxic
(Fig
glutamate-treated
post-insult
regular
55
exposure
although
9%.
and
6.3A). a
±
neurons, effects
in
less
rapid
the
The
10%,
the
intervals
p although recovery the effective for conditions: exposure protocol. and of recovery VGCC presence The increase of and respectively [Ca 2 ] both this individual glutamate VGCC average during for they respectively, of cultures blockers, was than antagonists [Ca 2 j 1 varied a to to of MK8O1 of further both blockers then glutamate a a 3 neurons identified glutamate [Ca 2 j 1 high as 30-minute mM (p<0.001 (28 widely. improved nifedipine to followed tested (4pM) 18-24 glutamate steady-state using pre-insult ± achieved ameliorated of the and fetal 11 antagonists the in exposure and % hours unpaired in time survival by postnatal for (2 face-down both and neuronal a from pM) a values CNQX some course phase slightly after value to 35 cases, and when t to less ± time (40 195 of 196 Subsequent cell death also occurred after widely varying time intervals in different neurons. The final cell collapse was indicated by a very high fluorescence ratio followed shortly by loss of fluorescence intensity resulting from leakage of fura-2 out of the cell through the injured neuronal membrane. Several examples of the responses of individual neurons are shown in Fig 6.4. Also, neurons with higher peak responses to glutamate generally demonstrated an earlier collapse in the post-insult phase (Fig 6.5). When glutamate exposure and 2+][Ca measurements were conducted at 34°C similar findings, but with an accelerated time course, were observed and neuronal loss was complete within 18 hours after glutamate exposure compared to >24 hours at room temperature. A small number of cells (<5%) that responded to glutamate with an increase in 2+][Ca displayed a very early and usually complete recovery of 21+][Ca in the continued presence of glutamate. These cells remained viable for the duration of the experiment and it is likely that they were morphologically ‘neuron-like’ glia demonstrating a transient increase in 2+][Ca as a result of metabotropic glutamate receptor activation (Holzwarth et a!, 1994). II. EXCITOTOXICITY IN NEURONS LOADED WITH BAPTA AND ITS ANALOGUES The loading of neurons with the artificial 2Ca buffers BAPTA and di methyl-BAPTA (DMB) for excitotoxicity experiments was achieved by incubating the cultures with the cell permeant acetoxy-methyl ester, BAPTA AM or DMB-AM. The objective was to test the hypothesis that increasing neuronal 2+Ca buffering capacity would confer a degree of neuroprotection against excitotoxic insults believed to be mediated largely via 2Ca-dependent 197 processes. DMSO and the non-Ca-binding molecule, half-BAPTA (hBAPTA), were employed as controls for2 the vehicle and the BAPTA ‘nucleus’ respectively. The concentrations of the DMSO and hBAPTA-AM were selected to match the highest BAPTA or DM8 concentrations used. a. Fetal Cultures: Fetal cultures which have been maintained face-down were turned face up and loaded for 30 minutes with 60 pM of BAPTA-AM or DMB-AM at RT, before an exposure to 500 pM glutamate (30 minutes, RT). This treatment resulted in a significantly greater neuronal loss detected 8 hours later when compared to control cultures (39.1 ± 8.2% and 25.8 ± 13.6% survival for DMB- and BAPTA-loaded neurons respectively compared to 87.1 ± 2.7% and 86.6 ± 2.1% neuronal survival in the DMSO- and hBAPTA-treated groups). Reducing the concentration of buffer-AM used for loading from 60 pM to 20 jiM reduced, but did not abolish, the enhanced neuronal damage in buffer- loaded neurons (78.5 ± 5.5% and 79.7 ± 4.3% respectively, Fig 6.6) when compared to that seen in the control groups. A complete concentration response curve for glutamate toxicity was determined for both control and 20 pM BAPTA-loaded neurons. At all concentrations of glutamate tested, toxicity determined 24 hours after exposure was greater in the BAPTA-loaded cultures compared to the control cultures (Fig 6.7). The estimated 50EC for glutamate was reduced from 68.6 ± 23.1 pM in the control group to 16.7 ± 4.9 pM in the BAPTA-loaded group. b. Postnatal Cultures: Using the extended glutamate exposure scheme, postnatal cultures loaded using 20 pM BAPTA-AM or DM8-AM showed the same trend of 198 heightened glutamate toxicity in the buffer-loaded neurons when compared to the DMSO and hBAPTA control groups (Fig 6.8). c. Correlation with 2+Ca changes in BAPTA-loaded neurons: Using fura-2 microfluorimetry, the 21[Ca of neurons loaded with BAPTA was determined prior to, during and followingi a 30-minute exposure to glutamate (3 mM) at room temperature. The rates of increase and recovery of intracellular 2+Ca levels were much slower in the buffer-loaded neurons although the plateau of 21[Ca achieved was not significantly different when compared to the control jcultures. Fewer cells were capable of near-complete restoration of their pre-stimulation 2[Ca after wash out of the glutamate (28% and 73% of cells 6 hours afteri the glutamate exposure in the BAPTA loaded and control groups respectively), and neuronal collapse occurred earlier (Fig 6.3B); consistent with the observed increase in glutamate toxicity in buffer-loaded neurons reported above. Ill. THE ASSOCIATION BETWEEN CaBP IMMUNOREACTIVITY AND GLUTAMATE NEUROTOXICITY a. Fetal Cultures: The low incidence of CaBP neurons in fetal neuronal cultures grown in serum-free conditions made it impractical to study excitotoxicity in CaBP neuronal sub-population in fetal cultures. b. Postnatal Cultures: The relationship between CaBP immunoreactivity in postnatal cultures and the survival rate of these neurons under excitotoxic conditions was studied found the of CaBP VI. neuronal subject 100% number higher concentrations glutamate-treated population. 6.9B count significant populations toxicity CaBP population. glutamate by bFGF BSA-treated comparing EFFECTS show to In at of Another glutamate to of treatment (50 neurons view be cultures each the neurons high viable concentrations with a significantly pM is The At cultured greater OF of concentration to the way sampling of all p<0.001 control and the difference to neurons calculate concentration bFGF with was cultures 12.5, survival on of glutamate of 200 finding decline the neuronal neurons less 2.5-5 discerning TREATMENT cultures 50 more errors. pM glutamate was in normalized the was of than and between that both of ng/ml in glutamate) CaBP toxic toxicity small (see ratio responses the glutamate statistically cannot 200 that (Fig the the cases bFGF CaBP chapter in of concentrations pM. and treatment the 6.10), ON of difference to the and CaBP discussed be CaBPN (Fig the the GLUTAMATE ratio statistical resulted to Analysis used. bFGF-treated reliably CaBPN significant 3), with population differences control excitotoxic 6.9A). in of and neurons. neurons in untreated statistically The above, in 4-day performed subpopulations behavior of parameters tested, the cell CaBP results groups at TOXICITY relative increased cultures were count to postnatal insults. we glutamate In expression cultures the the between the studied significant presented since treated statistically of total are survival to mid-range compared Glutamate the sensitivity the hippocampal at therefore the and neuronal the with different respective the in CaBPN total in of in almost effects two Fig the of to was of 199 200 differences more obvious in the mid-range of the neurotoxic response (p DISCUSSION The experiments presented above were conducted to study the effect(s) of different concentrations of glutamate on the survival of cultured hippocampal neurons under a variety of conditions. Under all the conditions tested, glutamate induced a concentration-dependent increase in the frequency of neuronal death. However, the sensitivity of neurons to glutamate toxicity varied depending on the type and conditions of the cultures. In view of the relationship between glutamate-induced increases in 21+][Ca and neuronal death, we further examined the potential neuroprotective effect of the presence of CaBP or artificial 2Ca buffers. EXCITOTOXICITY IN DIFFERENT CULTURE TYPES All fetal neurons maintained face-down were vulnerable to a 30-minute exposure to glutamate at room temperature. A total loss of neurons could be induced by exposure to 1 mM glutamate. Inclusion of 0.8 mM 2Mg in the incubation medium reduced the sensitivity to glutamate and increased the estimated 50EC from 68 to 192 pM. A notable phenomenon was the complete reversal of the widespread glutamate-induced morphological changes (swelling, loss of phase brightness) shortly after the exposure to glutamate was terminated. Within 20-24 hours, these changes together with vacuolation and breakdown of neurites reappeared in neurons that were considered dead. Fetal and postnatal cultures maintained face-up were exposed (always in the presence of 2Mg to glutamate at 34°C for 24 hours before neuronal survival was assessed.i Two important differences were observed between the fetal and postnatal neurons. First, the neurotoxic 50EC of glutamate was significantly lower for postnatal neurons than for fetal neurons. Second, 202 almost half the fetal neurons were resistant to glutamate toxicity up to a concentration of 1 mM while nearly all postnatal neurons were vulnerable. Our cultured neurons therefore displayed a similar sensitivity to the toxic effects of glutamate previously reported for hippocampal (Rothman, 1985; Michaels and Rothman, 1990) and cortical neurons (Choi, 1985). The shift of the glutamate concentration response curve to the right in the presence of 2+Mg is also in agreement with a neuroprotective effect of 2+Mg (Rothman, 1983; Choi, 1987b; Garthwaite and Garthwaite, 1987). EXCITOTOXICITY IN BAPTA- AND DMB-LOADED NEURONS In both fetal (face-down) and postnatal (face-up) cultures, a marked increase in the sensitivity of neurons to glutamate toxicity was observed after neurons had been loaded with either BAPTA or DMB but not with the control compound hBAPTA. In fetal cultures increased 2+Ca buffering was achieved by the use of either 20 or 60 pM of the buffer-AM during the loading process. The use of a higher buffer-AM concentration was associated with a higher incidence of neuronal loss following a subsequent exposure to glutamate. In fetal and postnatal neurons, no differences were observed between the effects of loading neurons with BAPTA or DMB. A full concentration response curve was constructed for glutamate toxicity in neurons loaded using 20 pM BAPTA AM and a shift to the left was observed in the BAPTA-loaded compared to control neurons, with the 50EC reduced from 68.6 pM to 16.7 pM. Measurements of 21+][Ca in fetal ‘face-down’ neurons loaded with BAPTA were performed during a 30-minute exposure to 3 mM glutamate at room temperature and for up to 24 hours after the exposure. Similar to our finding in chapter 5, the rates of rise and recovery of 2+][Ca were much slower in the adding on activation result inactivation properties compounds. in concentrations in with DMSO effect neuronal concentrations an with BAPTA who detected 2 resting in BAPTA BAPTA- from buffer-loaded cellular control exposure Several BAPTA-buffers. any reported In in was to or individual general, or [Ca 2 +] loaded an loss the of earlier with of of energy neurons. even and not increase of several Ca 2 possibilities to BAPTA potential (> that VGCC hBAPTA-AM DMB-loaded seen of of neurons 500 in fura2) neurons as our +.dependent 80%) neurons, stores. formaldehyde the the loading a BAPTA-buffer pM in under results Fewer and rapid and Additionally, buffer-AM buffer-loaded the Ca 2 -induced was independent although glutamate were exist DMB NMDA Delayed total cultured the increase presumably neurons associated neurons. are eliminated K+ to a could conditions in Ca 2 + during consequence channels, (100 explain the channels. agreement derivatives for and our fetal in were neurons, of lead peak cell For influx pM) the 5 with the the use extended through any minutes. hippocampal the to injury, used capable example, mean possibility Ca 2 + were de-esterification and of effects enhanced and a enhanced Any with and control (e.g., of reduction for the injured [Ca 2 +1 1 may associated thus the responses recovery or neuronal of Moreover, those the of reduced the BAPTA, all accumulation recovering that play groups impose neurons glutamate. neuronal loading glutamate plasma of Ca 2 in and of the these a processes, the collapse Dubinsky of with were likelihood less DMB, by treated high a chelating effects the of Ca 2 -dependent (after membranes. further loss the to effects significant extensive our no neurotoxicity The AM of pre-exposure dibromo loss was following lower 15-17 with neurons (1993a) toxic observed while of latter stress would of the either than fura DIV) role 203 direct arachidonic relative effects Taylor, (Nistri an resulting Berlin, example, regulation chelation was Also, similar procedure contribute during mobile Ca 2 + Ca 2 + neurons, glutamate in the impaired not It Several the effects and influx chelation case is 1992), increase exposure of and 1993). energy significantly in also that Ca 2 + survival and BAPTA (Olson Cherubini, not to exposures a acid high-affinity refilling of and other reduction of the important 24 an could, associated very deficit chelation in loading effects release of extrusion/sequestration efflux eta!, hours enhanced on of [Ca 2 i studies neurons prolonged of neurons other directly worse 1990), used during 1P 3 -sensitive of 1990), of in may cells to (Van BAPTA has with the face-up glutathione was have cellular consider in glutamate in to be and protein or with loaded der our been cellular excitotoxic control an enhanced BAPTA-AM observed a indirectly, reported or following Zee experiments derangement inhibition BAPTA. fetal functions. DMB associated intracellular the kinase-C with capacity et cultures toxicity (Brodie neurons possibility a!, molecules. as during a glutamate-induced either events contribute excitotoxic or multitude The of 1 a 989) activation DMB-AM result and For protein with in (30 to not of former Ca 2 and buffer exposure buffer-loaded or handle example, the have Reed, of minutes exposed a during of The postnatal to of direct loss synthesis normal stores situations, the (using will most all effects cell (Dieter oxidative 1991) increase to of been presence the for accentuate non-chelating ATP to injury. neuronal hBAPTA-AM, cell (Richardson likely coupling 20 neurons). face-down glutamate. extended neurons of potentially et (Preston reported depletion volume pM while in a!, Ca 2 + stress, does For of [Ca 2 j, buffer-AM) 1 loss. between 993) the highly since the not and Other to and fetal and a be and 204 a functional fluorescent treatment ionophore, responses exposed been delayed prolonged resistance adult to inclusion free increased et hydrogen BAPTA increased Deyoetal, loading exacerbate Plasma latter confer a!, radical-mediated The observed Some hippocampal 1 two 990) resulted membrane in the to of presence enhanced of peroxide using collapse Ca 2 + electrical of and hepatocytes effects A23187 BAPTA ischemic studies the 1991). BAPTA loss and frog the loss in energy BAPTA in buffering of ventral in spinal hippocampal could slices, the (assessed of injury could analog have electrophysiological rat of in survival stimulation. (2pM) insults cell an membrane solutions thymocytes reduction deficit as motoneurons (Ueda enhance root) reported artificial has be Scharfman injury capacity. a and or 3 result under alleviated also by in days and 10mM and slices Ca 2 induced filling response the of A potential) the Ca 2 protective been the of a Shah, after similar before DNA loss variety For prepared and probe, increased activity + with glutamate the Ca 2 -mediated when associated integrity buffer in irradiation example, damage of Schwartzkroin 1992). intracellular to stabilizing (Tsubokawa the renal of glutamate-induced of Quin2, effects cytoplasmic a of dentate from in acetoxy conditions. prolonged lipid NMDA tubular (Kudo (e.g., neurons in buffering with (Story was gerbil hepatoma associated peroxidation effect recording cell hilar decreased eta!, eta!, methyl channels cells BAPTA (1989) also buffering exposure has et brains injury For neurons of of a!, 1990). found by 1992). been BAPTA cell Ca 2 + ester depolarizing example, with and 1 observed electrodes exposure that (Choi, which 992). (Carpenter input line capacity to suggested to during Quin2 of an using Finally, had Pre the has delay (Dypbukt the 1990). may Also, in that been Ca 2 + to also was the 205 in postnatal EXCITOTOXICITY other Ca 2 + neurons buffers effects rapid, the BAPTA, toxicity. neuronal neurons end BAPTA after neurons, [Ca 2 i neurons artificial experiments significant a recent pre-exposure of In the It agents buffers and glutamate experiments of may is is they these displayed Ca 2 + and (Kudo loss study termination neurons In a interesting artificial associated cell neuroprotection 30-minute contrast, well such and a may are during neurons death concentration-dependent buffers eta!, by depend those level unlikely exposure, AND were as well Tymianski Ca 2 early using 45-minute was to of glutamate. with 1990; in exposure yet in be had CaBP of more compare exposure the and buffers, upon cultured prevented. to the toxic Dubinsky prolongation cell a in Tymianski continued and marked be eta! post-exposure vulnerable EXPRESSION 24-hour cultured death the exposure ‘universally’ in to a the suggest to hippocampal themselves, substantial (1993b), neuronal glutamate (1993a) glutamate. recovery was [Ca 2 j 1 These presence excitotoxicity eta!, explants of to cell to an that the glutamate BAPTA populations glutamate. recovery neuroprotective. differences, death 1993b). early with responses results enhancement of [Ca 2 i the or neurons of of Prior [Ca 2 +]j potentiate those event biological mouse which glutamate, loading in protocol, of toxicity loading In recovery a and observed Even in examined [Ca 2 cultured with to and sustained occurs spinal cultured a was of the the effects the plateau substantial when of than cultured Indeed, the glutamate effect found toxic phase which neurons neurons. only opposite hippocampal in CaBP and CaBPU increase spinal loaded of the close effects hours in of these to that at spinal was present some with the confer with to in 206 of 207 neurons at all concentrations of glutamate tested. CaBP neurons were eliminated from the glutamate-treated cultures faster than CaBPN neurons such that the ratio of neurons expressing CaBP to the total neurons surviving the insult was reduced as the glutamate concentration was increased. Few studies have focused on the relationship between CaBP expression and neuronal vulnerability to excitotoxic stimuli in vitro. Mattson eta! (1991), using fetal hippocampal neurons, reported that CaBP neurons were more resistant to both glutamate-induced and 2Ca ionophore-induced injury than CaBPN neurons. Also CaBP neurons were more capable of regulating their 2+][Ca in the face of glutamate or 2+Ca ionophore exposure. An earlier preliminary report from our laboratory (Baimbridge and Kao, 1988) suggested that fetal CaBP hippocampal neurons were more resistant to glutamate toxicity. The cultures used in the latter study were, however, of the fetal face down variety maintained in serum-containing culture medium and had a higher incidence of CaBP expression than the cultures used in the experiments described here. In contrast with these results, Mockel and Fischer (1994) could not find any selective sparing of CaBP neurons in fetal hippocampal neurons using short exposure to glutamate, long exposure to NMDA or kainate, or to a combined glucose/oxygen deprivation. Our results, however, indicate that CaBP expression is associated with enhanced vulnerability to glutamate induced neurotoxicity. Conflicting results regarding the role of CaBP as a neuroprotectant in vitro are also observed in studies using in vivo models of ischemia and epilepsy (Sloviter, 1989; Freund eta!, 1990), as well as in studies on post-mortem human brains from patients with neurodegenerative disorders (lchimiya et a!, 1988; lacopino and Christakos, 1990b; Yamada eta!, 1990). Such a widely cultured TNF Similar during that hippocampal as neuroprotection (reviewed of survival cultured days glutamate the percentage the EFFECT expression multitude biological emphasizes varied BSA-treated bFGF, effect cell TNFa treatment A Postnatal after glucose observations association large type rate hippocampal OF neurons nerve in of systems. and of toxicity. the may of BFGF the Mattson number of neurons developmental and bFGF end neurons TNFB were deprivation neurons control neurons growth against difficulty therefore were on ON of treatment between The were associated Since other as of et treated neurons the GLUTAMATE cultures. CaBP. expressing factor studies well treated ischemic/hypoxic al, in excitotoxic bFGF of also have developmental bFGF-treated injury. CaBP 1993a). cell CaBP as determining with on (NGF) exposed reported NGF treatment with have with different changes the expression At expression Attenuated excitotoxic CaBP TOXICITY exposure all were sensitivity and bFGF enhanced For recently to glutamate by cultures example, (see effects tumor cause/effect rather changes protocol. and glutamate. capable displayed Mattson is chapter and reported was concentrations [Ca 2 excitotoxic likely survival of necrosis than (on was concentrations neuronal postnatal Cheng of taking performed etal to By vulnerability) a an stabilizing 3). significantly relationships A changes significant that be that of isolated factors recent (1989) eta! place We neuronal one these vulnerability growth neurons time, of after therefore part (1994) as study simultaneously. [Ca 2 i (TNF) glutamate, neurons. event, using tested, increase depending over a less 6 of in factors injuries result to DIV, from complex a reported confer bFGF than 70% such tested in the i.e. fetal of in such the and that in of 208 the on 3 209 same group (Mattson et a!, 1993b) reported that bFGF treatment in cultured fetal hippocampal neurons reduced the expression of NMDA receptors and that this may account for its neuroprotective effect since 2+Ca influx through NMDA-receptor operated channels would be reduced. However, the experimental protocols in these experiments were very different from our own. For example, while the studies mentioned above exposed the neurons to excitotoxic insults in the presence of the respective growth factors, our results were obtained after 6 DIV from neurons that were treated with bFGF only for the first 3 DIV. In chapter 3 (Fig 3.3), we show that bFGF-treated neurons undergo an accelerated neuronal loss and are more vulnerable to glutamate toxicity after bFGF treatment is terminated and when most neurons have been induced to express the relatively long-lived CaBP. It is possible that the increased neuronal vulnerability after termination of bFGF treatment may reflect the presence of CaBP, which may out-last the other effects of bFGF treatment, or even a ‘rebound’ hyper-expression of NMDA receptors. On the basis of all the available evidence it would seem that the presence of certain growth factors can indeed protect neurons against excitotoxic injury. However, neurons which become dependent on the presence of bFGF may be more vulnerable to excitotoxicity following its removal. The outcome of an excitotoxic incident can thus be determined by the abundance of growth factors relative to the excitotoxic stress on neurons, a factor that may have strong impact on the design of therapeutic approaches to the control of acute and chronic neurological disorders of excitotoxic origin. standard using absence when the (solid Exposure extracellular Figure D > o presence unpaired bars) assessed 6.1: 100 120 20 40 of 60 80 deviation 0 of extracellular reduces Glutmate fetal Mg of t-test). 24 Control 1 . neurons pM of hours neuronj at Glutamate TTX) toxicity Mg’ least later. 3OpM to induced glutamate damage 12 in (open Inclusion fields fetal ‘I OOpM a bars). when Concentration concentrq,tion-dependent face-down from for of 300pM 30 compared Mg+ at Data minutes least neurons: points in 4 1 the mM coverslips to at represent that extracellular room protective 3mM induced temperature neuronal (** the p<0.01 effect medium in mean the loss of (in 210 ± 211 Figure 6.2: Glutamate toxicity in postnatal culture. Exposure of postnatal neurons to glutamate (24 hours, 34°C) in DMEM-N2 culture medium induced a concentration dependent neuronal loss (assessed at the end of the 24-hour period). Data points represent the mean ± standard deviation of at least 10 fields from at least 3 coverslips. 140 120 100 - 80 > > 60 C,) 40 20 0 Control 12.5pM 5OpM 200pM 800pM 3.2mM Glutamate Concentration earlier. A) recovery experimental delayed individual 15 minutes, pre-stimulation Figure B A Q In 4-., Q minutes, o (13 4-I (‘3 o fura-2 6.3: gradual 0.0 0.5 2.0 2.5 3.0 3.5 1.0 1.5 0.0 0.5 1.0 2.0 2.5 3.0 1.5 3.5 of RT, neurons Changes mean loaded RT) - - - - - - conditions, 1 0 0 pM levels increase similr ______ [Ca at TTX) fetal in different within +j [Ca 2 j 1 changes face-down ______ but resulted in was mean 2 using time BA hours not 5 5 during in PTA in [Ca.+] as BAPTA-loaded [Ca 2 j points neurons, an fter complete group Time glutamate increase represents glutamate (n=61 were (67 glutamate 10 (hours) 10 and in observed toxicity. neu neurons). neurons [Ca+J, final was the ro ns) exposure collapse progressive washed that (20 (67 B) pM neurons) 15 15 Under recovered occurred (3 off. BAPTA-AM, mM, collapse similar A but 30 to 212 of 20 20 the 7 334/380 3 terminal different [Ca 2 ] Figure ratio mM, dead 6.4: units). 30 in increase times cell. ratio minutes, individual Time to All of a course in cell panels value 34°C) [Ca 2 ] 1 , fetal collapse. of of have neurons displayed glutamate-induced 0 is is the widely the Note, treated same result widely in variable x-axis panel with of varied complete neuronal D, in (0 glutamate different that to patterns 20 the leakage death, hours) (solid rapid cells. of ‘recovery’ as and of horizontal drop assessed fura2 y-axis in the from and (0 bars, by 213 to + significant the neurons) bar). At 25 hours peak U Figure C-) C CD CD CD C D room seconds, first 1000 1200 Ca 400 200 Dotted 600 800 (not 625: 80 0 temperature, had + 0 recovery shown) minutes responses. Neurons solid line smaller represents triangles) after at of that Ca [Ca 2 +] 1 a the he corresponding collapse + 20 experiment responses and end the in glut.mate [Ca1 of neurons early Time glutamate to is time in changes shown. the that 40 response (3 (mm) (n= initial mM, exposure had 13 in near exposure 30 to neurons neurons). minutes, glutamate complete (solid 60 to that line, NMDA Note solid have recovery did n = that horizontal not higher 35 (20 show only 80 pM, 6 214 unpaired solid either loaded indicated. Neurons Figure (I) .? > bars) 10o 120 DMSO- 20 40 60 80 6.6: using 0 were t-test). resulted A BAPTA 60 subsequent or treated pM 0.3% DMSO hBAPTA-treated — 1 T and in BAPTA- neuronal with DMB 30 6OpM hBAPTA I DMSO minutes or promote loss Treatment 0MB-AM cultures or 2OpM BAPTA that Buffer-AM exposure glutamate was when (* 6OpM BAPTA Group p<0.05, significantly * to compared, *** for toxicity — 500 30 2OflM DMB I jiM minutes in higher glutamate 8 p<0.OO1 fetal hours U 6OpM DMB Glutamate at neurons. * in RT 500pM Control later, cultures - (at using as RT, to 215 p<0.001 coverslips treated deviation toxicity At (20 Figure all pM, — a ir > 0 C’, concentrations 6.7: with to 30 20 of 40 30 10 50 70 80 using 90 when the 0 BAPTA minutes neuronal DMSO left. unpaired assessed Control shifts (open at of survival RT, glutamate Glutamate t-test). the bars). 24 3OpM solid concentration-response hours of bars) a Data tested, minimum 100pM after was points Concentration the survival worse 300pM of glutamate represent 1 2 compared fields of BAPTA-loaded curve 1mM the exposure or from mean of to glutamate 3mM control at (*** ± least standard neurons cultures 4 216 coverslips represent hours, 34°C) Postnatal Figure (I) > > Co 100 120 20 40 60 80 were 6.8: 34°C) 0 cultures the (* BAPTA more * mean compared p <0.01, Control sensitive loaded and ± standard DMB * to * using * the Glutamate to p promote the <0.001). DMSO BAPTA-AM deviation neurotoxic or glutamate 1 2.5pM concentration hBAPTA-treated of or at effect DMB-AM least toxicity of 1 2 glutamate fields (20 in cultures. postnatal 4 uM, 5OpM from 30 exposure at Data minutes, neurons. least points (24 4 217 218 Figure 6.9: CaBP postnatal neurons are more sensitive to glutamate toxicity. A) When postnatal neurons used for glutamate toxicity experiments (24 hours, 34°C; Fig 6.2) were stained for CaBP reconstructed glutamate concentration response curves showed that p(+l neurons (open bars) were much more sensitive to glutamate than CaBPN nyrons (solid bars). B) As more CaBP neurons were lost compared to CaBP’’ neurons, the percentage of CaBP neurons decreased with increasing concentration of glutamate. The total number of neurons at higher glutamate concentrations (inset) is small and the ratio estimation may vary considerably as a result of sampling error (* * p<0.01, p<0.001 using unpaired t-test). 140 A 120 100 CD 80 > > I 60 C’) 40 20 0 Control 12.5pM 5OpM 200pM 800pM 3.2mM Glutamate Concentration B 40 +a Control OD Co 30 C) 12. 5pM 0 0) 0) 20 Co 4-. C 0) IC-) 0) 10 a- 200pM 3200pM 0 0.245 least using concentrations bars), bars) exposed Postnatal neurons. Figure (I) - U 0 > D > G) 0 C’) 12 starting unpa,red bFGF-treated mm. 6.10: fields to 40 20 30 10 60 70 80 neurons 0 different Effect on from t-test). tested. the treated Control of at neurons concentrations sixth bFGF least HPF Data Glutamate with DIV. 3 is treatment were 1 points 2.5pM coverslips high bFGF When more power represent of (2.5 on compared glutamate 5OpM sensitive Concentration (* glutamate microscope nglml) p<0.05, the 200pM at to to for mean 0, BSA-treated glutamate toxicity 24 ** field, 1 ± p<0.Ol, hours and standard 800pM approximately in 3 toxicity at postnatal DIV cultures 34°C were error p<0.OO1 at (solid all of (open at 219 220 CHAPTER 7 GENERAL DISCUSSION AND CONCLUSIONS 221 The potential neuroprotective role of artificial 2Ca buffers and -2Ca binding proteins of the ‘buffer’ variety, such as CaBP, is still a matter of controversy (Mattson eta!, 1991; Dubinsky, 1993a; Tymiansky eta!, 1993b, Mockel and Fischer, 1994). The work presented in this thesis was conducted with the objective of testing the hypothesis that an enhanced Ca-buffering capacity in neurons, either by loading with BAPTA-like 2Ca buffers2 or by the natural expression of CaBP, improves the neuronal capacity to handle agonist- or depolarization-induced 2+Ca influx, and thereby confers a degree of resistance to glutamate-induced neurotoxicity. Primary cultures of dissociated hippocampal neurons were used for our experiments, but in view of the diverse tissue culture methods used by our laboratory and those of other authors, it was necessary to characterize in detail many aspects of our hippocampal neuronal cultures relevant to the subsequent interpretation of experimental results. All types of primary cultures tested displayed both EAA- and depolarization-induced increases in 2[Ca in morphologically-identified In ] neurons. both fetal and postnatal neurons NMDA, AMPA, kainate and glutamate induced increases in 2+][Ca that were sensitive to known EAA antagonists such as APV, 2Mg and MK8O1 for NMDA-evoked responses, and CNQX for AMPA/kainate responses. In the fetal cultures, a detailed characterization of VGCC was conducted and three types of 2+Ca channels; L, N and P, could be identified. By far the largest portion of the 2Ca response resulted from the activation of L-channels while N and P channels were equally responsible for the remainder. We can conclude therefore that our cultured neurons, regardless of culture method, were equipped with the basic voltage and agonist-operated channels required for experiments designed to examine mechanisms postnatal combination Ca 2 + and regardless evoked intracellular sensitive their prolonged signals Ca 2 + a! differences peripheral report small. almost by glutamate-, addition Ca 2 the (1992) intracellular NMDA role +..mediated An Ca 2 + efflux/sequestration stores by Our compared completely in interesting of we Ca 2 + neurons. Shmigol and fetal high (DRG) of content Ca 2 + receptor-associated findings between Ca 2 + NMDA- of can may where the Uneyama neurons an extracellular stores release neurotoxicity eliminate postnatal route be buffers to responses increase eta! lacking a difference central or contrast those part central few in quisqualate-induced of from (1994). compared eta! central of in Ca 2 + minutes mechanisms. any in observed neurons in neurons modulating the K-induced caffeine-sensitive (neocortical, with with our (1993) the believed was significant channels) maturational influx. neurons The cultured density higher those to after observed with displayed in but latter postnatal to DRG either These these of Ca 2 Such respect are peaks were hippocampal contribution of be with neurons. authors Murphy intracellular membrane neurons. entirely central in differences transient stores a responses changes Ca 2 labile influx an neurons. the and small to enhanced + have pattern their and a Calcium to and consistent have stores (Shmigol much caffeine-induced to Moreover, and excitotoxic Ca 2 + Ca 2 + in release Miller observed caffeine-releasable spontaneously between the The may the been of brain were faster efficiency responses Ca 2 responses latter channels Ca 2 + Ca 2 + be (1989), eta!,1994). since with filled stem) due also caffeine- fetal recovery substantial process. signal had responses regulatory the during this to very Harada of [Ca 2 +] 1 and and (VGCC evoked faster lost or a recent the was by the a In 222 et 223 Major differences were also observed between culture types with respect to neuronal survival, the percentage of neurons expressing CaBP, and in their vulnerability to glutamate toxicity. While a time-dependent neuronal loss was a common feature in all cultures, its extent was highly variable, being much greater in both fetal and postnatal cultures that were maintained face-up. Use of the “sandwich technique”, in which the neurons were grown on the face of a glass coverslip in contact with the bottom of the tissue culture dish, proved to be of some benefit. Fetal neurons grown in this way had significantly less initial cell loss and, on average, survived for a longer period. Brewer and Cotman (1989) attributed this effect to a reduction of oxygen toxicity in the limited diffusional space around the neurons although an equally valid argument is that the restricted ‘extra-neuronal space’ in the sandwich method provides a more suitable volume/density ratio for neurons to respond to secreted trophic (glial or neuronal) factors with enhanced survival. This argument is supported by our observations that face-up cultures are very sensitive to glial density and that neuronal survival is enhanced by the reduction of the medium volume/density ratio and the inclusion of bFGF in the culture medium. Regardless of the culture conditions, it is important to note that experiments performed on viable neurons after 7-10 DIV were done on a population that represented a selected fraction of those that were initially plated. While the total neuronal count declined as a function of time, the number of neurons expressing CaBP increased in both fetal face-down and postnatal face-up cultures. In the former, CaBP immunoreactivity was still limited to a minority of the total population but in the latter all neurons were CaBP after 12 DIV. In contrast, fetal face-up cultures displayed a continuous and disproportionate loss of CaBP neurons so that by 14 DIV CaBP 224 immunoreactivity could not be detected at all although many CaBPN neurons were still viable. The enhanced loss of CaBP neurons from fetal face-up cultures suggests that CaBP neurons may be more vulnerable to environmental stresses than CaBPN neurons. In the postnatal cultures, this may be offset by the presence of secreted trophic factor(s), possibly glial (see later), that were capable of inducing the expression of CaBP as evident by the increase in the number of CaBP neurons and by the enhancement of survival of these neurons. The enhanced, albeit limited, expression of CaBP in fetal neurons maintained face-down, and the improved survival of CaBP neurons contrast sharply with the accelerated loss of CaBP neurons in fetal face-up cultures, and lend more support to our suggestion that the major benefits of the sandwich technique can be attributed to an improved volume/density ratio which increases the effectiveness of glial control over the culture environment. In postnatal cultured neurons, bFGF treatment was associated with a significant enhancement of neuronal survival and a remarkable increase in CaBP expression, whereas in fetal neurons a significant effect was only observed when bFGF treatment was supplemented with culture medium harvested from postnatal cultures. Considering the potential mechanism(s) by which bFGF increases neuronal survival and CaBP content, it is unlikely that a direct effect on neurons is the only mechanism. Certainly, bFGF influenced the number and morphology of the glial cells and it is quite possible that factors released by glia, as a result of the action of bFGF, could be the major means of enhancing neuronal survival and CaBP content. Indeed, postnatal cultures plated face-up and at high density contained many more glial cells and, even in the absence of exogenous bFGF, most if not all neurons eventually expressed CaBP. It is likely therefore that glial-neuronal interactions are an important factor in survival and 225 differentiation of neurons in culture, and that the observed effects of bFGF are, at least in part, mediated via glial release of neurotrophic factors. We cannot however rule out the possibility that such factors could play a permissive role in association with a direct action of bFGF on neurons. A very interesting observation was made when bFGF treatment of postnatal cultures was discontinued. The result was an enhanced loss of viable neurons that may be due to the phenomenon of apoptosis reported previously in response to the withdrawal of NGF from cultures of peripheral neurons (Eichler and Rich, 1989; Edwards and Tolkovsky, 1994) or bFGF from cultures of midbrain neurons (Mayer eta!, 1993). It would appear that treatment of neurons with these growth factors results in an absolute requirement for their continued presence and a rapid induction of a programmed series of events leading to cell death if these growth factors are removed. Vulnerability of hippocampal neurons in vitro to glutamate-induced toxicity was another major aspect of variability between the types of cultures tested. In cultures maintained face-uD, postnatal neurons were much more sensitive to glutamate toxicity than fetal neurons. It is interesting to speculate that this heightened sensitivity in postnatal neurons is attributable to their larger depolarization- or NMDA-induced 2+Ca responses. Our earlier suggestion that these neurons may also have more efficient 2+Ca recovery mechanisms does not contradict this speculation since the contribution of 2+Ca recovery mechanisms would be more evident during a transient 2+Ca load than during an extended 20-24 hour exposure of glutamate. An interesting feature observed in fetal face-up neurons was that nearly half the neurons were resistant to glutamate toxicity, although all fetal face-up neurons responded to NMDA and glutamate exposures with an increase in their .j21[Ca It is possible depolarizing glutamate-induced glutamate-induced Ca 2 + gated from agreement receptors (Rothman, excitotoxic Also, reducing 34°C, temperature, glutamate excitotoxicity, In of maintained glutamate conducted, that initially process fetal different the activation Regardless in influx, Ca 2 MK8O1 face-down plated. postnatal which, resistance glutamate-induced is significantly was 1985; with process face-uD, glutamate had central monovalent influx and the Ca 2 + (a larger of the by In resulted of VGCC presence non-competitive Choi, neurons, NMDA cannot fetal Ca 2 toxicity neurons, the the of to widely being however influx when these the exposure age reduced glutamate 1987b). neurons blockers cationic fluxes in receptor-associated be largely induction in of accepted seems in during the compared toxicity. using neurons ruled postnatal Mg 2 + such vitro elimination excitotoxicity (and protocols maintained However, fluxes had mediated exposure NMDA to a a out when direct of 30-minute 20-24 in toxicity) notion be is some These to since glutamate-induced the neurons. the and a excitotoxicity both antagonist) comparison major employed of medium by hour a result neuroprotective protocol that face-down, subsequent two Mg 2 small most more Ca 2 + and an fetal glutamate mediator exposure Ca 2 results The increase of resulted contribution than likely during also and channels the used in was may involvement influx these experiments the postnatal reduced highly activation are 90% reflects exposure of for excitotoxicity to the in and not in estimated effect consistent the [Ca 2 glutamate through two a the and of most be after selective higher from neuronal the this the induction +1 models. valid neurons is against of of at effective exposure were in neurons voltage resulting type VGCC VGCC NMDA room EC 50 . EC 50 with because at survival injury. of of for the in 226 in to 227 associated with the activation of both NMDA and non-NMDA type of glutamate receptors. It is clear from our results that in cultured hippocampal neurons several important properties (e.g., 21+][Ca regulation mechanisms, CaBP expression and survival in vitro) are sensitive to a multitude of factors in the culture environment. These include the culture type (fetal vs postnatal), culture age (Dlv), orientation state (face-up vs face-down), the number of glial cells and growth factor availability. Other factors such as the plating density and the serum content of the culture medium are also major determinants of the neuronal culture properties but they have not been the object of a detailed assessment in our work. The sensitivity of vital neuronal characteristics to culture conditions complicates attempts to compare results obtained from different laboratories and precludes generalization and extrapolation of results to other experimental models using different species for cultured neuron preparation or using whole animals. Having established some of the basic properties of our cultured neuron preparation our next aim was to determine the effect of artificial 2+Ca buffers and the presence of CaBP on increases in 2[Ca +1 induced either transiently or in a manner which would, depending upon the degree of stimulation, lead to cell death by a process of excitotoxicity. During short exposures to NMDA or high K-induced depolarization, changes in 21[Ca in BAPTA- or DMB-loaded neurons displayed reduced peak responses andj slower rates of increase and recovery compared to control neurons confirming the role of BAPTA (and DMB) as effective and fast intracellular 2+Ca buffers. Characteristically, the recovery phase was slow and regulation postnatal compared postnatal associated and interesting stimuli of however, that an relative used the effects buffer, expressing seem responses comparison even change responses neurons, neurons prolonged CaBP increase neuronal postnatal may in larger therefore are the of activity our in was after recoveries CaBP have that both to is CaBP of [Ca 2 +] to in with when causing expression CaBP. experiments, in than markedly these postnatal phenotype. quite neurons a note CaBP the the during resulted fetal very of that prolonged may CaBPN compared density stimulus neurons Ca 2 + different that were evident While [Ca 2 1 and supports short there have electrophysiological loaded different CaBPN of neurons larger from similar postnatal recovery control BAPTA or CaBP It of nature are and, therefore (Chard displayed has to responses to is by membrane other with our peak major possible, linger their neurons. a in is to although been in values most higher (tens earlier fetal possibly mechanisms. many etal, CaBPN CaBP a coincidental Ca 2 + CaBPN BAPTA-like differences at been discontinued. fast are likely and of a in however, Ca 2 + net 1993; cases, conclusion This in higher responses and milliseconds), experiments masked control postnatal reconstructed just postnatal neurons fetal counterparts influx acts difference complete channels Lledo one incomplete changes concentration between buffer neurons It as values that of by must aspect that Unlike had neurons. were a eta!, Ca 2 + neurons. the fast the where compared or recoveries a between be higher in neuronal in while in these the relatively observed Ca 2 + potential of a high 1993). BAPTA-loaded these recoveries, Fig in fetal mentioned, reduction a kinetics depolarizing CaBP For in the 7.1. larger peak responses affinity buffering neurons, buffer-loaded neurons to fetal Ca 2 + ease rates when It long in buffering neurons It Ca 2 + is change of both would in while neurons and also Ca 2 of stimuli of Ca 2 the such effect and were fetal + 228 in 229 During prolonged exposure to glutamate, BAPTA- and DMB-loaded neurons demonstrated slower rates of change in 2+][Ca compared to control neurons indicating that sufficient buffer was available in the cytoplasm to slow down changes in 2+][Ca even during these ‘unphysiological’ conditions of stimulation. Unlike the situation during shorter exposures, peak 2+Ca responses were not reduced in buffer-loaded neurons, leading us to conclude that active 2+Ca regulatory mechanisms determine the peak 2+Ca responses during longer stimuli without a. contribution from the passive intracellular buffering systems. This result is particularly interesting when considering the potential use of BAPTA-like buffers to protect against Ca-mediated neuronal injury believed to play a major role in the in vivo excitotoxic2 circumstances which are almost exclusively of a prolonged nature (e.g., minutes of ischemia). In a small set of experiments, fetal CaBP neurons exposed for several minutes to glutamate or NMDA displayed the same characteristic features observed in shorter stimuli, i.e., higher peak and delayed recovery. In view of the effects of buffer loading or CaBP expression on the peak of 2+Ca responses during prolonged stimuli, and the prolonged and slow recovery process under the same circumstances, it is probably not surprising that glutamate-toxicity was enhanced in both fetal and postnatal neuronal cultures loaded with either BAPTA or DMB, as well as in postnatal CaBP neurons compared to the corresponding control cultures. In buffer-loaded neurons, an increased 2+Ca load, inferred from the unchanged peak 21+][Ca in the presence of a higher intraneuronal buffering capacity, would impose a greater demand on cellular energy stores in order to restore pre-stimulation 2[Ca and is likely a major factor underlying the enhanced cell death in buffer-loaded neurons. The increase in ]2[Ca in neurons during the exposure to BAPTA-AM or DMB-AM 230 is unlikely to be contributing to the enhanced glutamate toxicity observed in buffer loaded neurons since control cultures exposed to hBAPTA-AM displayed a similar increase in 2+]j[Ca without a subsequent enhancement of glutamate- induced excitotoxicity. The contribution of other ‘non-chelating’ effects of BAPTA or DMB to enhanced glutamate toxicity cannot, however, be excluded. With respect to the increased vulnerability of neurons which express CaBP we can only conclude that there is a clear association between these factors but a causal relationship cannot be established on the basis of either our experiments or, indeed, those of Mattson eta! (1991). In the light of our findings it would be appropriate to return to the question, should 2Ca buffering be neuroprotective? The hypothesis that an enhanced 2+Ca buffering capacity of neurons will protect them against 2+Ca mediated neuronal injury is based on the assumption that the peak concentration of 2[Ca during excitotoxicity is the sole determinant of the cell injury, and that 2Caj buffering will reduce this peak and thereby reduce or eliminate the cytotoxic consequences. While our findings refute the assumption of a reduced peak response, other flaws are still inherent in the hypothesis. The most obvious of these are, first, the possibility of an impairment of 2Ca +mediated activation of 2Ca +recovery (i.e., direct or indirect negative feed back loops) as a result of reduced peak 2+Ca levels and, second, the fact that the presence of an artificially-high cytoplasmic 2Ca buffering capacity will slow down and prolong the 2[Ca ‘11recovery phase. More subtle flaws become obvious when the high temporo-spatial organization of the 2Ca signal as well as the speed and mobility of the exogenous and endogenous 2+Ca buffers are considered. Disruption of the spatial properties of the 2+Ca signal (such as microdomains and 2+Ca waves) by a fast and 231 mobile buffer can be harmful (by increasing stray signalling and by disrupting 2Ca tmediated physiological processes). Also, the loss of the temporal precision of a 2+Ca signal can lead to a distortion of the appropriate time relationship between the 2+Ca signal and other 2Ca +..activated second messenger systems such as 31P and DAG. As a result of this complexity, the effects of additional 2Ca buffering on a particular biological system are essentially unDredictable and probably very cell-specific (depending on such factors as endogenous buffers mobility and affinity, artificial buffer immobilization as well as the nature of the time/space organization of the 2+Ca signal). In attempting to arrive at valid conclusions concerning our data we must be mindful of the pitfalls and limitations of the methods used. Examples have already been cited with respect to the tissue culture methods, and some comments regarding our use of imaging technology are also warranted. Attempts to understand the effects of enhancing the 2Ca buffering capacity in neurons have frequently made use of a number of fluorescent 2+Ca imaging techniques. Unfortunately, measurements and conclusions based on these techniques may not reflect the complexity of the 2Ca signal because of the limited spatial and temporal resolution of imaging systems available at present (msec, pm) compared to the resolution of the 2+Ca signalling process (psec and nm). Furthermore, the introduction of fluorescent 2+Ca probes, particularly if used at high intracellular concentrations, into the cell can distort the 2+Ca signal to be measured since these probes would act as fast, mobile and high-affinity 2Ca buffers with a particularly disruptive effect on the fast, short-range 2+Ca processes such as neurotransmission possibly in a manner similar to that of the parent molecule BAPTA (Adler eta!, 1991). During 2Ca judicious selected selected the ‘higher’ being in loops vulnerability vitro- vivo number neuroprotection modulation All link that [Ca 2 +] Additionally, average where Ca 2 + functionally responses, cell these functional measured models Despite may Another in as of properties channels many animal of have vivo the neuronal physiological much [Ca 2 i use technical be culture of high organisms. is will useless, these highly preclude of of Ca 2 been serious consequences this changes not as models, these the and is depend affinity and (e.g., on population conditions. the function reliable. criticisms limitations at:the localized imaging vital in the we in problem methods parameter successful vulnerability the enhanced in and the Ca 2 + on Ca 2 Neuroprotection have inherent level [Ca 2 +]j neighbourhood by ‘hot’ the The not is and +..mediated in of of should in limited not probes enhancing Extrapolation of can vitro. the cell imaging based in studying spots (such very Ca 2 predictions the cell to attempted vivo of test be connectivity a be neuronal by properties. can neuronal fast. on particular very as in buffering processes treatment technology taken the processes should the the the the [Ca 2 +] of also useful Also, from fact intracellular of to immediate cellular improvement possible into somata saturate, development the measure -which cell thus that capacity) The the all or such and if account may effect and the function, of membrane Ca 2 in be it extensive role in rather our as is is in vitro vicinity be and right evaluated changes Ca 2 + appropriate a of not neuroprotection vitro on of or initiated. measurements buffering in measure in become specific than results questions Ca 2 deterioration the any and reproducible vitro release potential) models of feed in overall attempt the the membranous in buffers to [Ca 2 +] to of capacity. whole changes back view neurites. sites, a the the the are large well in of to of and in in of in and 232 a a neurons molecular changes knockout, requires a neuronal restricted to neuronal better might can buffer channels, effects mediated distribution FUTURE ‘and attempted new asked. causal a associate capacity So The simple’ pharmacological also understanding mobility on as far, the relationship in Answers characteristic death to biological use cellular gene versus DIRECTIONS fast controls be neuronal in could ability the the hypotheses to order very of with short-range (e.g., in knockout, study target reporting aDoroDriately ‘deep’ functions. excitotoxic to be to helpful specific for to techniques properties. such between of dextran-conjugation) a manipulate tools. of give selectively (e.g., very neurons the slow the about of questions gene in insight processes, subcellular spatial powerful associative resistance modulating functional Fast Also, events. the Ca 2 + designed such the induction that Achieving the expression Ca 2 -mediated buffers into in aspects in mediated can express as expression tool vitro vivo initiated domains the role or neuronal-targeted buffers or the be or and in vulnerability and models this possible mode incidental of vital of transfection. Ca 2 the CaBP of processes. slow Ca 2 CaBP possibly objective in to CaBP (site-specific of selective before of the processes manipulate can signal either CaBP buffers mechanisms action signalling in phenomena. vicinity and be to neurons in by will in spontaneously and a Altering mRNA modulation used vivo The any of using can isolation disease that require immobilization) a may of subcellular and, particular use experiments drug. to have has influx of buffers antisense may test the lead of Establishing eventually, action process) been from the differential of CaBPN trigger artificial or specific to or Ca 2 - use that release Ca 2 + other a of are of 233 recovery observed fetal and fetal is face-up Dlv, in than initial cells, culture CaBP of GENERAL changes under requirement under particularly the culture agonist-induced 4. face-up face-down all 3. that 2. 1. age-dependent and and number these the age viable (>90% Compared After Fetal In of associated in observed the vulnerability conditions. SUMMARY effects primary [Ca 2 fetal (Dlv), conditions for neurons remarkable availability cultures an neurons of and an after +j neurons. neurons initial of orientation to appropriate in cultures in with postnatal peak growth neuronal this 3 fetal both AND postnatal These maintained are to loss is Dlv). of with the expressing glutamate an Ca 2 + initial CaBPN. neurons, fetal Following CONCLUSIONS of growth of factors isolated differentiation include state face-up all loss CaBP hippocampal control loss responses and neurons viable using that (face-up factors. in toxicity postnatal of postnatal CaBP. the event short cultures vivo since CaBP neurons is neurons the is culture significantly that transient much or but to neurons, sandwich vs are In it in neurons cultured a is display postnatal face-down), more are from neurons highly distinct type being vitro, unlikely faster much Ca 2 + likely survival, all (fetal technique CaBP evidence lower does dependent display neurons than culture phenotype. continues neurons, that higher responses, one vs the not in (<50% CaBP postnatal), expression depolarization of fetal by density maintained types satisfy of than display on many this 12 an and expression neurons. a by those tested, DIV. increase the increase variety the by of an 3 of glial DIV) 14 In 234 the recovery reach exposures buffers reduced extracellular BAPTA accelerated medium develop evident neurons additional effect postnatal Ca 2 + induced that, when Postnatal recovery 6. 5. with the compared regulatory on also or by a from rate With when excitotoxicity. In times same certain neurons. dependence neurons factors DMB to the the vitro, neuronal effectively K, time of glutamate. transient postnatal maturation bFGF significant are levels rise are to the mechanisms cellular bFGF is necessary still are entirely fetal and This greater treatment effects loss as also for greatly enhances reduce exposures cultures. peak neurons. these effect elements However, is increase of the more consistent with observed for of fetal [Ca 2 +]j, result sustained prolonged. the of is prior is the BAPTA vulnerable both combined at non-buffer Postnatal neurons of rate in in Both the full least in loading cultured postnatal an when the with and an of peak expression of otherwise-small presence compared partially rise increased survival into In these a a neurons with to bFGF with prolonged loaded [Ca 2 j 1 both buffering neurons of glutamate-induced cultures postnatal the [Ca 2 1 results the treatment of instances mediated and of to sensitivity neurons, treated presence this responses acetoxymethyl the DMB. to expression action, recovery. which lead effect neurons, induced either growth bFGF with by the is us although may of discontinued. namely to of an NMDA effects. eventually to prolongation factor conditioned bFGF by glutamate- neurotoxicity bFGF These changes indirect of conclude then prolonged esters CaBP a or their also since on secrete This CaZ+ high fetal in in of 235 of an is 236 7. In both fetal and postnatal cultures, enhancing the 2Ca buffering capacity by loading neurons with BAPTA or DMB is associated with a significant increase in their vulnerability to glutamate-induced excitotoxicity. 8. In the presence of extracellular 2+,Ca acetoxymethyl derivatives of BAPTA, DMB or the non-calcium binding molecule hBAPTA evoke a rapid increase in 2[Ca that recovers rapidly and completely on removal of the ester. This jincrease is most likely mediated via the activation of VGCC. 9. In both fetal and postnatal cultures, both NMDA- and high extracellular K+induced increases in 2[Ca +11are significantly larger in CaBP neurons than in CaBP neurons. In fetal neurons, these larger responses are significantly prolonged with a slow and incomplete recovery of 21[Ca In contrast, larger 2+Ca responses in postnatal CaBP neurons are.i associated with much faster and complete recoveries compared to those observed in the CaBP neurons. 10. CaBP postnatal neurons are significantly more vulnerable to glutamate-mediated excitotoxicity when compare to their CaBPN counterparts. An increased vulnerability of CaBP neurons is also observed in fetal face down cultures that fail to recover after a transient exposure to NMDA or high extracellular K+. 11. An increased intraneuronal 2Ca buffering capacity either by artificial 2+Ca buffers, such as BAPTA or DMB, or in neurons that express CaBP does not confer neuroprotection. Rather, both are associated with an increase in the vulnerability of these neurons to glutamate-induced excitotoxicity in vitro. ci: 0 and Figure 0.5 3.0 3.5 2.0- 2.5 4.0 1.0- 1.5- CaBP(+ 0 7.1: imulated , in I I 1 50 response Ca 2 to 100 changes I a short-duration Time in 150 I fetal (s) neurons: (25 200 seconds) I control, Control CaBP(+) BAPTA-loaded exposure 250 BAPTA-loaded to 300 NMDA. 237 238 ABBREVIATIONS FWS: EPSP: FOR: EthD: ER: FBS: EGTA: EMEM: EAA: EC 50 : DRG: DMSO: DNA: DMB: DIV: DMEM: DAG: CMF CV: DHP: CNQX: CgTx: CBP: cAMP: CaBPN: CaBP: CaBP: Ca 2 BSS: [Ca 2 +]: BSA: ATP: bFGF: Ara-C: BAPTA: APV: APH: AMPA: ADP: AM: ACPD: AR: 5-FDU: +: BSS: Funnel Excitatory Free Fetal Ethedium Endoplasmic Ethyleneglycol-tetraacetic Eagle’s Excitatory Ligand Dimethyl Dorsal Dulbecco’s Deoxyribonucleic Day(s) Cresyl 5,5’-dimethylBAPTA Diacyl Ca 2 /Mg 2 Dihydro-pyridines 6-cyano-7-nitro-quinoxaline-2,3-dione Omega-conotoxin Ca 2 -binding Cyclic Not Calbindin-D28K Immunoreactive Buffered Intracellular Bovine Ionized Basic Arabinoside-C Adenosine a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic 2-Amino-7-phosphonoheptanoate Adenosine 2-Amino-5-phosphonovalerate Acetoxymethyl 1 Arachidonic 5-fluoro-deoxyuridine 1-amino-i ,2-bis-(O-aminophenoxy)ethane-N,N,N’,N’-tetraacetic immunoreactive oxygen bovine fibroblast glycerol Adenosine violet root in web concentration minimal serum calcium salt sulfoxide vitro homodimer post-synaptic ,3-cyclopentane-dicarboxylic amino triphosphate diphosphate ganglion modified spider ionized serum radical free reticulum acid Nissi solution protein albumin essential ester growth for acids BSS acid monophosphate GVIA stain calcium CaBP for Eagle’s evoking factor CaBP potential medium acid concentration medium half the maximal acid response acid acid possible 239 240 GFAP: GliaI fibrillary acidic protein GIuR: Glutamate receptors GLUT: Glutamate hBAPTA: N-(o-methoxyphenyl)-imino-diacetic acid HBSS: Hank’s BSS HK: High-potassium (50 mM) containing medium HS: Horse serum HVA: High-voltage activated iGluR: lonotropic glutamate receptors IHC: lmmunohistochemistry :31P Inositol triphosphate KA: Kainic acid LDH: Lactate dehydrogenase LGCC: Ligand-gate 2Ca channels LVA: Low-voltage activated mGIuR: Metabotropic glutamate receptors mRNA: Messenger ribonucleic acid nAchR: Nicotinic acetyl choline receptors NBQX: 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline NGF: Nerve growth factor NMDA: N-methyl-D-aspartate NO: Nitric oxide NSE: Neuron-specific enolase NT-3: Neurotrophin-3 PBS: Phosphate Buffered saline PCP: Phencyclidine PDL: Poly-D-lysine P1: Phasphatidyl inositol PIP: Phasphatidyl inositol phosphate 2PIP Phasphatidyl inositol diphosphate PK-C:: Protein kinase-C PL-A2: Phospholipase-A2 PV: Parvalbumin QA: Quisqualic acid ROl: Region of interest SE: Standard error SEM: Standard error of the mean SN: Substantia nigra SR: Sarcoplasmic reticulum TC: Tissue culture TNF: Tumor necrosis factor TTX: Tetrodotoxin VGCC: Voltage-gate 2Ca channels 241 BIBLIOGRAPHY Banker Baker Baker Baimbridge Baimbridge Baimbridge Baimbridge Baimbridge Baimbridge Augustine Auer Andressen Albers Allen Adler Loligo. 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