Department of Neuroscience, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
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ABSTRACT |
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Connor, John A. and Robert J. Cormier. Cumulative Effects of Glutamate Microstimulation on Ca2+ Responses of CA1 Hippocampal Pyramidal Neurons in Slice. J. Neurophysiol. 83: 90-98, 2000. Glutamate stimulation of hippocampal CA1 neurons in slice was delivered via iontophoresis from a microelectrode. Five pulses (~5 µA, 10 s duration, repeated at 1 min intervals) were applied with the electrode tip positioned in the stratum radiatum near the dendrites of a neuron filled with the Ca2+ indicator fura-2. A single stimulus set produced Ca2+ elevations that ranged from several hundred nM to several µM and that, in all but a few neurons, recovered within 1 min of stimulus termination. Subsequent identical stimulation produced Ca2+ elevations that outlasted the local glutamate elevations by several minutes as judged by response recoveries in neighboring cells or in other parts of the same neuron. These long responses ultimately recovered but persisted for up to 10 min and were most prominent in the mid and distal dendrites. Recovery was not observed for responses that spread to the soma. The elevated Ca2+ levels were accompanied by membrane depolarization but did not appear to depend on the depolarization. High-resolution images demonstrated responsive areas that involved only a few µm of dendrite. Our results confirm the previous general findings from isolated and cell culture neurons that glutamate stimulation, if carried beyond a certain range, results in long-lasting Ca2+ elevation. The response characterized here in mature in situ neurons was significantly different in terms of time course and reversibility. We suggest that the extended Ca2+ elevations might serve not only as a trigger for delayed neuron death but, where more spatially restricted, as a signal for local remodeling in dendrites.
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INTRODUCTION |
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When brain tissue is subjected to ischemic insult
or mechanical trauma, there is a large release of glutamate from
damaged or otherwise directly affected cells that results in widespread exposure of all neurons in the affected region to high levels of
glutamate and other neurotransmitters (Benveniste et al.
1984; Globus et al. 1991
; Mitani et al.
1990
, 1994
). In these catastrophic models, the
glutamate exposure has long been thought to trigger excitotoxic
reactions involving intracellular Ca2+ in neurons,
resulting in cell death that extends far beyond the initially damaged
region (Choi 1988
; Manev et al. 1989
;
Rothman and Olney 1986
).
Glutamate-Ca2+-initiated processes have also been proposed
in models of more subtle neuron destruction such as amyotrophic lateral
sclerosis and Alzheimer's, among others (Loopuijt and
Schmidt 1998
; Mattson 1994
; Olney
1990
; Schousboe et al. 1997
).
The effects of excessive exposure to glutamate and the resulting
toxicity have been extensively studied in neurons isolated from
embryonic or neonatal brains maintained in tissue culture (Choi
et al. 1988; Dubinsky and Rothman 1991
;
Glaum et al. 1990
; Randall and Thayer
1992
; Vornov et al. 1991
) and to a lesser
extent, in completely isolated neurons from adult tissue (Chen
et al. 1997
; Connor et al. 1988
;
Wadman et al.1993). However, there are often
difficulties in drawing conclusions from these types of preparation
about the behavior of mature neurons in situ. Because of the
powerful ability of glia to remove glutamate from the
extracellular space (reviewed in Barbour and Hausser
1997
), it has proved more difficult to examine the effects of
glutamate in vivo or in brain slices in a controlled fashion. In the
present study, we exploited microiontophoresis of glutamate to deliver
locally high concentrations of this agonist to only a few cells in the
slice, thereby stimulating cells with no compromise of oxygenation and
no widespread excitation in the slice as with global superfusion. Using
this method, we investigated whether there are overt, cumulative
effects on Ca2+ homeostasis of local stimulation by
glutamate, and we explored the cellular regions in which these effects
are expressed in adult in situ neurons. We show that local applications
of glutamate not only cause the expected rapid increases in
intracellular Ca2+ that recover within seconds after the
application but, if applications are sufficiently large and repeated,
that they trigger disproportionately long-lasting increases in the
dendrites. These increases persisted up to 10 min before recovery was
attained. The long-persisting increases were little affected by
applying hyperpolarizing current during the extended response.
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METHODS |
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Coronal brain slices (350-400 µm thick) were prepared from
adult Sprague Dawley rats (Harlan; 5-10 wk old) by standard methods that optimize in situ visualization of neurons near the cut surface of
the slice (Aghajanian and Rasmussen 1989). Animals were
deeply anesthetized (0.85 mg/kg ketamine and 0.15 mg/kg xylazine) and perfused through the right cardiac ventricle. The perfusion solution was ice-cold and consisted of, in mM, 250 sucrose, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 6 MgCl2, 0.4 CaCl2, 10 dextrose, and 0.1 kynurenate. The perfusion solution was bubbled with 95%
O2, 5% CO2, as were all
solutions in the present study. After the perfusate was clear of blood
(
1 min), decapitation of the animal and dissection of the brain
began. Brain slices were made under the cold saline on a vibrotome (Ted
Pella). After preparation, the slices were warmed to 28°C over the
course of 30 min in artificial cerebrospinal fluid (ACSF) that
contained (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 MgSO4, 2 CaCl2, and 10 dextrose. The slices
remained under these conditions until use, at least 1.5 h. ACSF (31 ± 0.5°C, gassed with a mixture of 95%
O2/5% CO2) flowed at 1-2 ml/min through the
submersion chamber used to study individual slices.
Injection/recording microelectrodes were filled at the tip with 12 µM
fura-2 in 0.5 mM K-acetate. They were then back-filled with 3 M KCl/1 M
K-acetate. After cell impalement, filling to an adequate level of
fura-2 (100-200 µM; Molecular Probes, Eugene, OR) required 15-20
min, during which time a small negative current (200-400 nA) was
applied. After this filling time, enough KCl/K-acetate had diffused to
the tip to lower electrode resistance to ~100 M. Only cells with
input resistance >50 M
and steady resting membrane potentials more
negative than
60 mV after dye injection were used in the experimental
population. Holding current was adjusted to maintain the potential
between
65 and
75 mV under nonstimulated conditions. An Axoclamp 2A
amplifier in bridge mode was used to record voltage signals.
Orthodromic electrical stimulation was delivered via monopolar
electrodes and positioned in the stratum radiatum (50-100 µA, 100 µs applied current).
Ester loading of fura-2 followed the basic procedure of Regehr
and Tank (1991). To fill CA1 neurons, a large bore micropipette (tip ~15 µm) filled with fura-2/AM:DMSO (10 uM:0.3%) was
positioned in the alveus and pressure pulses (1 Hz, 0.5 duty cycle)
were applied for 15 or more minutes. Basal dendrites of CA1 neurons steadily accumulated fura-2 that diffused to cell bodies and apical dendrites. This positioning of the pipette allowed measurements to be
made in the cell body layer, distant from the loading site. Calcium
measurements were made by ratio imaging of fura-2 (Grynkiewicz et al. 1985
) using 350/380 nm excitation, an upright
microscope, and a cooled frame-transfer charge coupled device
camera system (Petrozzino et al. 1995
). For injected
neurons, fura-2 ratios were converted to [Ca2+] using in
vitro standards. Measurements with ester-loaded fura-2 were left as
ratios because of the following factors. First, nonspecific loading
leads to background from out-of-focus cells, which may include damaged
high-Ca2+ cells near the slice surface. Second, the
fluorescence signal includes a nonquantifiable component from the
unconverted ester form of fura-2/AM, which does not bind
Ca2+.
Iontophoresis pipettes (1 M Na-glutamate, ~10 M) were positioned
20-50 µm from the primary apical dendrite of the fura-2-filled neuron. Glutamate was ejected by five iontophoretic pulses (10 s
duration at intervals of 60 s), and the pipette was then
withdrawn from the slice. Control experiments demonstrated that
iontophoresis current itself had no effect on synaptic transmission,
membrane potential, or intracellular [Ca2+]. Recovery
times for Ca2+ transients are expressed as elapsed time
between termination of iontophoretic delivery of glutamate and 90%
recovery (0.9 ×
Ca2+peak) to
prestimulus levels.
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RESULTS |
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Conditions for extended Ca2+ response
Glutamate was administered via microelectrode iontophoresis using
a protocol that has been used for long-term potentiation (LTP)
induction (Cormier et al. 1993); 10-s pulses repeated
five times at a frequency of 1/min. The iontophoresis electrode was positioned in the mid region of the stratum radiatum. Although previously used for LTP induction, the purpose here was to administer a
stimulation protocol that, administered singly, had no known deleterious effects. We then explored the cumulative effects of multiple applications of this stimulus.
To assess the effective spread of glutamate away from the electrode, we
examined Ca2+ responses in a number of
neighboring cells filled noninvasively by fura-2/AM (see
METHODS). Figure 1 shows
responses in a field of cells to glutamate iontophoresis from an
electrode positioned in the lower right hand region of the field. The
amplitude of the iontophoretic current was increased with each pulse in
the train (0.83, 2.5, 3.3, 4.2, and 8.7 µA). The first (smallest) pulse elicited a small response only in cell 5,
but as the pulse amplitude was increased, responses were recruited in
neurons at greater and greater distances from the electrode tip. The
most distant cell (cell 1) showed no appreciable
response until the fifth pulse and then gave a large, rapidly
recovering response. Effective concentrations of other transmitters
such as dopamine are achieved at lower current intensities
(Nicholson 1995) as expected from a lower density of
uptake sites. It would appear, however, that the larger amplitude
ejection currents are necessary to stimulate neurons outside of a
radius of about 40-70 µm and that iontophoretic currents in >3 µA
are required to overcome the rapid glutamate uptake capacity.
Presumably the large ejection currents (glutamate quantities) are
necessary to overcome the glutamate uptake capacity of glia in the
vicinity of the ejection site. Because of the branching of dendritic
trees, it is not possible to place a more exact radius on the effective
diffusion of glutamate.
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After the fifth (largest) glutamate ejection cells 2 and 4 showed a much slower recovery rate than cells 1, 3, and 5. The positioning of cell 2 between the rapidly recovering cells 1 and 3 argues against the slow recovery reflecting merely the restoration of extracellular glutamate to its normal levels and for the possibility that factors intrinsic to the individual neurons are responsible. Heterogeneity of Ca2+ recovery within single neurons and on a much smaller spatial scale is shown in Figs. 5 and 6. At this time we cannot say whether the very different recovery rates are due to preexisting differences in the state of the cells or if they result from differences in mechanical or chemical manipulations during and after slicing. Response heterogeneity was also seen in fura-2-injected neurons, forcing our observations to be descriptive in nature.
A total of 27 neurons microinjected with fura-2 met the electrophysiological acceptance criteria outlined in METHODS). To assess the likelihood of delayed effects of the stimulation, 12 of these neurons were given one set of stimuli and then observed for at least 25 min during which time no further exogenous glutamate was applied. Ca2+ levels in nine of these neurons recovered to prestimulus levels within 1 min after termination of the fifth iontophoretic pulse and remained there for the duration of the observation period. The remaining three neurons failed to restore low Ca2+ levels, with two of these losing the fura-2 during the observation period, indicating membrane breakdown. We concluded that if a cell recovered from the initial stimulation it was unlikely that there would be a delayed change in Ca2+. Fifteen additional neurons recovered from the initial stimulus set and were given a subsequent stimulation set after a recovery period of at least 10 min.
Figure 2 illustrates cellular responses to two stimulus sets tracked at five locations, one somatic and four dendritic. Figure 2, left, shows the rapidly recovering response typically evoked by the initial stimulus set. Although in many cases the recovery progressively slowed with the course of the stimuli, there was no clear discrepancy between these recovery times and the times that might be needed to clear the extracellular space of glutamate and to buffer/sequester and pump out the intracellular Ca2+increase. Both glutamate uptake and Ca2+ regulation might be expected to slow somewhat with the cumulative demand. Note that recovery in the main portion of the dendrite is somewhat more rapid than in the soma, but that recovery in the distal dendrites requires the greatest time.
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Behavior different from this occurred when the subsequent stimulus set was applied. A recovery period of 15 min was given between the stimulus episodes shown. Figure 2, right, shows responses of this same neuron to a second set of glutamate pulses delivered at approximately the same location as the first set. Although there are significant differences in the time course of Ca2+responses in the soma and proximal apical dendrite between the two episodes (increased maximum amplitude and slowing of recovery, especially after the fifth pulse), the greatest difference is seen in the more distal apical dendrites. Instead of mirroring the soma/proximal dendrite response, the more distal dendrite Ca2+ levels plateau or "hang" for several minutes after termination of the last stimulus pulse before recovering to their prestimulus levels in a relatively rapid fashion. This plateau-rapid recovery waveform was typical of the cells studied. A vestige of the long dendritic response appears as a shoulder on the recovery phase of the soma/proximal dendrite response.
The range of recovery times observed in these 15 neurons is summarized in Fig. 3. One of the neurons failed to recover after the second stimulus set whereas three of the neurons showed no increase in response duration with the second pulse (recovery, 1 min). Of these latter cells, one produced a prolonged response after four stimulus sets and a second cell failed to recover after a third stimulus set.
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Figure 4 shows electrophysiological data that demonstrate that the extended Ca elevations and the induction of LTP were not related; that is, potentiation could be induced without an extended response and the ultimate formation of an extended response did not depend on the prior induction of potentiation. Excitatory post synaptic potentials (EPSPs) in response to stimulation of the stratum radiatum, before and after a single set of glutamate pulses, are shown superimposed for each cell. For both examples, Ca2+ levels had returned to prestimulation levels at the time the "Post" records were made. A subsequent glutamate stimulation set gave rise to an extended response in both cases. In Fig. 4A, paired pulse stimulation was delivered to a single pathway. The response shows clear potentiation of the initial and paired (facilitated) EPSPs. In Fig. 4B, two-pathway stimulation was delivered to a second cell via two electrodes positioned in stratum radiatum. No potentiation or significant depression was seen for either pathway. Analysis of EPSPs during the maintained responses was not attempted because of complications raised by maintained depolarization.
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Extended response results from intracellular not extracellular signalling
A primary concern in experiments such as these is whether the
extended Ca2+ elevations result from interesting
changes in the properties of the neurons or from unexpectedly long
persisting glutamate elevations in the extracellular space, even though
the long duration of the responses would make this seem unlikely. We
judged that the clearest way of separating the two possibilities was to
demonstrate that large differences in intracellular
Ca2+could exist in different dendritic branches
that were very close together. Diffusion times should be on the order
of a few seconds for distances of several µm in the extracellular
space (Nicholson 1995) and, more importantly, the local
region would have been subject to about the same initial concentrations
of glutamate during the stimuli. Figure 5
shows data from branching tertiary dendrites arising from a common
stalk. Morphology of the dendrites and recording locations are shown
using fura-2 fluorescence (top). There were no detectable
changes such as beading as a result of stimulation. Two stimulus sets
were given to the cell; the first was followed by rapid recovery of
Ca2+ levels whereas recovery from the second was
much more extended and is shown in the plots of Fig. 5. All regions
started at approximately the same levels and gave robust
Ca2+ increases during the five iontophoretic
stimuli. Regions 1 and 2 showed partial recovery
after the first stimuli; however region 3 showed no
appreciable recovery during the interpulse intervals and maintained the
longest Ca2+ elevation in the several minutes
after removal of the iontophoresis electrode. Even the most rapidly
recovering region (region 1) required 5 min before baseline
Ca2+ levels were reset.
Ca2+ levels at region 3 remained
elevated for at least 3 min after levels in the parent (region
1) and parallel (region 2) branches had reset to
prestimulus values. The physical separation of these regions
across the extracellular space is <30 µm. It is extremely unlikely
that appreciable gradients of glutamate could exist for such a long
time over so small a distance unless some very unusual and
undemonstrated properties of glial uptake and release of glutamate are
invoked. Ca2+ profiles along the branched dendrite at three
different time points of the response are shown in Fig. 5,
right. It can be seen from these that all regions of the
dendrite are responsive to glutamate during the applications, and that
only a part of the lower branch shows the long-lasting Ca2+
elevation.
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Although both branches involved in the extended response shown in Fig. 5 were tertiary dendrites, such differences in the time course of recovery were not limited to dendrites of the same size. Figure 6 shows data taken from a primary dendrite and a small side branch during the recovery phase following a second stimulus epoch. Fig. 6, right, shows a picture of the neuron (fura-2 fluorescence, 380 nm excitation) and the regions from which Ca2+ measurements were taken. The small dendrite branch reached much higher levels than the main dendrite during stimuli, but after the plateau period shown in the initial portion of the plot abruptly recovered. Levels in the main dendrite and another branch increased and remained at higher than normal Ca2+ levels for the duration of the measurements. Again there were only ~10 µm separating the different measurement regions, and all regions showed robust responses to the glutamate application. Thus the difference in time course of the response was present in dendrites that were widely different in size as well as in small spine-bearing dendrites. The responses observed were indicative of secondary processes set off within the dendrites and not of persistent significant differences in extracellular glutamate concentration.
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Voltage dependence
Microelectrode penetration was sometimes lost during the course of
these experiments. In general this did not affect the condition of the
cell as judged from Ca2+ levels and the ability
to respond to glutamate pulses. Electrical recordings were maintained
in 16 of the neurons analyzed. During the first set of iontophoretic
pulses, cells depolarized to approximately 10 mV and recovered,
between pulses, more rapidly than the Ca2+ levels
illustrated in Fig. 1. During the prolonged Ca2+
response, membrane voltage remained depolarized to levels between
50
to
30 mV. Recovery of Vm to normal resting levels always preceded the
restoration of Ca2+. Depolarization to
10 to 0 mV by current injections lasting many minutes have been analyzed
previously. Such depolarizations generate large
Ca2+ increases in CA1 and CA3 pyramidal neurons,
exceeding 5 µM in many cases (Perkel et al. 1993
,
Pozzo-Miller et al. 1996
). These increases show partial
recovery during the depolarization, and in the absence of synaptic
stimulation or glutamate application, concurrent with the period of
high Ca2+, resting levels of
Ca2+are restored within 1-2 min after return to
resting potential. The depolarization-repolarization cycle can be
repeated a number of times. Therefore, simple depolarization and the
associated Ca2+ increase are insufficient to
generate the type of extended responses reported here.
In four experiments, a negative holding current was applied to bring
the neuron soma voltage to 80 mV. In the example shown in Fig.
7, hyperpolarization brought a partial
recovery to the soma Ca2+ level but caused
relatively little change in dendrites remote from the soma. While this
behavior might seem unexpected, it would follow from a model in which a
persisting current (carried partly or totally by
Ca2+) was being generated in the dendrites
because of the glutamate exposure. This current would depolarize the
soma as well as the dendritic tree and activate voltage-gated Ca
channels (VGCCs) in the soma. Hyperpolarizing the soma would shut VGCCs
there but might have little or no effect on the dendritic influx, given observations from other laboratories (see DISCUSSION).
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DISCUSSION |
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The data demonstrate glutamate-stimulated Ca2+ elevations in mature CA1 neurons that outlast focally applied glutamate stimulus by considerable periods. This effect was rarely seen after a single LTP-inducing stimulation that we have here termed "moderate stimulation," but generally was expressed only for repeated stimuli of this type. It would therefore appear that LTP and the maintained Ca elevations are separate or at least only partially intersecting phenomena. There were very significant differences in the response time course observed among neighboring cells responding to the same focal glutamate application. In some cases, as in Fig. 1, the time course differences are difficult to ascribe to amount or distribution of extracellular glutamate, because a neuron showing a prolonged Ca2+ elevation was sandwiched between two rapidly recovering neurons. Large time course differences were pursued on a much finer spatial scale by showing that different dendrites on the same neuron, separated by only a few µm, showed responses of significantly different time course. These findings indicate that the extended responses are not caused by unexpectedly long persistence of glutamate in the extracellular space. The extended Ca2+ elevations occurred in the presence of normal in vitro oxygenation and glucose. Therefore, we ascribe the cause of the response to glutamate activation and not to compromised metabolic capacity of the neurons involved.
Ca2+ increases that were transient and not
immediately lethal, but unexpectedly long, were our main concern
because this response may recapitulate, in a limited way, what occurs
in situ during an ischemic insult or other events that release
significant quantities of glutamate in the important CA1 region. During
the ischemic insult itself, CA1 neurons experience intracellular Ca
increases in the micromolar range (Silver and Erecinska 1990,
1992
), but the great bulk of them do not die until 2 to 4 days
later. In fact, using the gerbil 5 min occlusion model (Kirino
1982
), our laboratory has shown that resting
Ca2+ levels on the day after an ischemic insult were the
same as in control neurons (Connor et al. 1999
). This
behavior would indicate that high intracellular Ca2+ is a
trigger for the following degenerative events, but high ambient Ca2+ is not an enduring factor in the times nearer
to cell death (see also Dubinsky 1993
). This in situ
behavior points up a shortcoming of in vitro models. When put under
conditions of anoxia with glucose deprivation for periods of 5-10 min,
neurons in hippocampal slice do undergo large intracellular
Ca2+ increases (Lobner and Lipton 1993
;
Mitani et al. 1993
; Tanaka et al. 1999
),
but these changes seldom reverse and the cells show signs of rapid
deterioration. We also observed irreversible changes in the experiments
in the present study, but this population was not emphasized because of
the difficulty in establishing the actual cause of death in the acute
slice preparation.
The initial study showing prolonged Ca2+ responses to
exogenous glutamate in CA1 neurons (Connor et al. 1988)
was carried out in acutely isolated neurons. This and a subsequent
study demonstrated that in neurons pretreated with the protein kinase
inhibitor sphingosine, the prolonged response, but not transient Ca
increases, was blocked (Wadman and Connor 1992
). This
finding suggested that the response was not the result of simple
rundown of Ca2+ pumping or sequestration caused by the
repeated Ca loads, but of kinase-mediated processes triggered by the
glutamate receptor excitation and the resulting Ca2+
influx. In the acutely isolated neurons, the response, once
established, did not subside, but high Ca2+ levels
persisted until the neuron died, usually within 15-20 min. Persisting
elevations of Ca2+ were also shown to occur in small
dendrites and spines of CA3 neurons after repeated orthodromic tetani
to the associational-commissural pathways (Müller
and Connor 1991
). Although less well studied than the CA1
neurons, delayed death also occurs in CA3 pyramidal neurons after more
prolonged ischemic insults (Hatakeyama et al. 1988
;
Yanagihara et al. 1985
). Subsequently, studies done on
neurons in tissue culture also demonstrated that prolonged
Ca2+ elevations followed repeated exposures to glutamate
(Randall and Thayer 1992
; Weiss et al.
1993
). Sphingosine preexposure also blocked the extended
response in culture (Weiss et al. 1993
). The occurrence
of a secondary response in tissue culture neurons was shown to be an
early marker of cells that would die after glutamate exposure
(Tymianski et al. 1993
).
Unlike the acutely isolated or cultured neurons, the fully developed
neurons in slice studied here did not show appreciable Ca2+
recovery before the long plateau response started. The plateau was
either already developed after the final stimulus or it did not occur.
It is not clear whether this represents a physiological difference in
neurons of the different preparations (slice vs. culture or acutely
isolated) or differences in the stimulus protocols. In the rat ischemia
model, Silver and Erecinska (1990) reported the
development of a delayed or secondary Ca2+ increase after
reperfusion. This ischemia-induced increase was irreversible although
the observation required long impalement with ion sensing electrodes,
which may contribute to cell deterioration. Future work will
investigate different glutamate application protocols in the slice preparation.
Recent electrophysiology studies employing whole-cell current
measurements in acutely isolated CA1 neurons have demonstrated the
developments of a persisting inward current after prolonged stimulation
by glutamate, N-methyl-D-aspartate, or high
intracellular Ca2+ (Chen et al. 1997, 1998
).
Ca2+ influx is a significant component of this current, and
continuing Ca2+ influx feeds further increase of the
current. A point of particular interest is that the persisting current
showed only a weak dependence on membrane voltage. Thus there are
similarities in the responses but further experiments will be required
to make better comparison. The use of zinc as a blocker (Chen et
al. 1998
) presents problems for Ca2+ indicator
experiments in that there is a finite membrane permeability to zinc,
and this ion binds with high affinity to
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid family indicators (fura-2, etc.) as with other blocker ions, e.g.,
cadmium. Interpretation of fluorescence signals is not
straightforward (Connor et al. 1987
). Whereas the
membrane current in the acutely isolated neurons, once established,
persisted until obvious cell death (Chen et al. 1988
),
the response analyzed here, if not pushed too far, recovered. Assuming
that the underlying membrane current is the same, this difference may
be one of degree of stimulation or may reflect differences in the
physiological state of acutely isolated neurons and those in slice.
Isolated neurons on which Ca2+ measurements were made
(Connor et al. 1988
; Wadman and Connor 1992
) also did not recover from multiple stimuli once a
secondary response was established. We note that the distal dendritic
tree, which in the experiments here was most sensitive to the response development, is generally lost in the acute isolation of neurons.
There is no shortage of potential factors leading to cell death that
might be activated by prolonged Ca2+ elevations. Among them
are certain members of the caspace family, (Fink et al.
1998; Namura et al. 1998
), phosphatases such as
calcineurin (Rao et al. 1997
, Wang et al.
1999
), or more complex cascades involving Ca-activated factors
such as Ca-calmodulin-kinases (Aronowski et al. 1992
;
Churn et al. 1992
; Picone et al. 1989
)
and mitogen-activated protein kinase (Murray et al.
1998
).
The appreciable differences in the Ca2+ responses of
different dendrites of the same neuron also illustrate the fact that
secondary processes activated by glutamate need not involve the whole
cell but can occur rather independently in different parts of the same neuron. This might imply that the responses illustrated in the present
study could be used in, among other things, dendritic "pruning."
That is, the long Ca2+ responses might selectively activate
degradative enzymes in these selected dendrites for a sufficient period
to promote their destruction whereas in uninvolved dendrites there
would be no such activation. The extended duration of the
Ca2+ response provides a second dimension for the
Ca2+ signaling. That is, in addition to the absolute
magnitude of the signal, which can be quite large in nondestructive
activity, reaching into the 30-50 uM range during LTP induction
(Petrozzino et al. 1995), there is the second dimension
of time; the signal may be several minutes rather than several seconds.
If the cascades triggered by an initial Ca2+ increase have
downstream steps that are also biased by ambient Ca2+
levels, then the ultimate outcomes could be very different depending on
whether Ca2+ is high or low when subsequent steps in a
cascade are reached.
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ACKNOWLEDGMENTS |
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We thank Dr. William Shuttleworth for helpful comments on the manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grant R01 NS-35644.
Present address of R. J. Cormier: Dept. of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110.
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FOOTNOTES |
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Address reprint requests to J. A. Connor.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 May 1999; accepted in final form 28 September 1999.
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REFERENCES |
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