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MICROSCOPY RESEARCH AND TECHNIQUE 46:370?379 (1999)
Correlated Measurements of Free and Total Intracellular
Calcium Concentration in Central Nervous System Neurons
LUCAS D. POZZO-MILLER,1,2* NATALIA B. PIVOVAROVA,2,3 JOHN A. CONNOR,4
THOMAS S. REESE,2,3 AND S. BRIAN ANDREWS2,3
1Department
of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Biological Laboratory, Woods Hole, Massachusetts 02543
3Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, Maryland 20892
4Department of Neuroscience, University of New Mexico, Albuquerque, New Mexico 87131
2Marine
KEY WORDS
endoplasmic reticulum; mitochondria; Ca2? imaging; electron probe X-ray microanalysis
ABSTRACT
Transient changes in the intracellular concentration of free calcium ([Ca2?]i) act as
a trigger or modulator for a large number of important neuronal processes. Such transients can
originate from voltage- or ligand-gated fluxes of Ca2? into the cytoplasm from the extracellular
space, or by ligand- or Ca2?-gated release from intracellular stores. Characterizing the sources and
spatio-temporal patterns of [Ca2?]i transients is critical for understanding the role of different
neuronal compartments in dendritic integration and synaptic plasticity. Optical imaging of
fluorescent indicators sensitive to free Ca2? is especially suited to studying such phenomena because
this approach offers simultaneous monitoring of large regions of the dendritic tree in individual
living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis
provides quantitative information on the amount and location of intracellular total, i.e., free plus
bound, calcium (Ca) within specific subcellular dendritic compartments as a function of the activity
state of the neuron. When optical measurements of [Ca2?]i transients and parallel EDX measurements of Ca content are used in tandem, and correlated simultaneously with electrophysiological
measurements of neuronal activity, the combined information provides a relatively general picture
of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available
with this approach, we review here results from our ongoing work aimed at evaluating the role of
various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and
termination of [Ca2?]i transients in dendrites of pyramidal neurons in hippocampal slices during
and after synaptic activity. Our observations support the long-standing speculation that the
dendritic endoplasmic reticulum acts not only as an intracellular Ca2? source that can be mobilized
by a signal cascade originating at activated synapses, but also as a major intracellular Ca sink
involved in active clearance mechanisms after voltage- and ligand-gated Ca2? influx. Microsc. Res.
Tech. 46:370?379, 1999. r 1999 Wiley-Liss, Inc.
INTRODUCTION
In neurons of the mammalian central nervous system
(CNS), transient changes in the intracellular concentration of free calcium ([Ca2?]i) following neuronal activity
regulate important functions such as neuronal excitability, dendritic integration, and synaptic plasticity (reviewed by Berridge, 1998). For free Ca2? to act as an
intracellular signal transducer, these transients must
be efficiently buffered to resting levels rather quickly,
but the processes that accomplish this are not well
understood. Depending on the magnitude of the [Ca2?]i
transients, neurons may employ any of several buffering, sequestering, and extrusion mechanisms, including binding to cytoplasmic proteins, ATP-mediated
uptake into smooth endoplasmic reticulum (ER), electrochemically driven uptake into mitochondria, and extrusion across the plasma membrane by a Ca2? exchanger
and a Ca2?-ATPase (reviewed by Carafoli, 1987; Miller,
1991).
r 1999 WILEY-LISS, INC.
Digital optical imaging of Ca2?-sensitive fluorescent
indicators has become the method of choice for measuring spatio-temporal changes of [Ca2?]i in individual
CNS neurons (reviewed by Connor, 1988; Connor et al.,
1994). Cultures of dissociated embryonic cells (Connor,
1986; Murphy et al., 1987), acutely isolated neurons
from young animals (Connor et al., 1988), and acute
brain slices (Llano et al., 1991; Regehr et al., 1989;
Tank et al., 1988), are the most commonly used in vitro
preparations for optical imaging of [Ca2?]i in the mammalian CNS. In acutely isolated neurons and dissociated cell cultures, individual neurons are easily identiContract grant sponsor: Grass Foundation; Contract grant sponsor: Lakian
Foundation; Contract grant sponsor: NIH Intramural Research Program; Contract grant sponsor: NIH; Contract grant number: NS35644.
*Correspondence to: Lucas D. Pozzo-Miller, Ph.D., Department of Neurobiology, University of Alabama at Birmingham, Civitan International Research Center 429-A2, 1719 6th Avenue South, Birmingham, AL 35294.
E-mail: [email protected]
Received 28 January 1999; accepted in revised form 7 May 1999
FREE AND TOTAL CALCIUM IN CENTRAL NEURONS
fied and the optical properties of the preparation are
optimal. However, in the case of dissociated cell cultures from embryonic tissue, the separation and subsequent random reaggregation of the cells, leads to a
neuronal network that lacks the characteristic features
of the intact brain region from which they were derived.
The acute slice preparation retains much of the original
cellular and synaptic organization of the brain region in
situ, but the high degree of absorbance and scattering
produced by damaged tissue at the slice surface presents
limitations for the application of high-resolution optical
imaging of fluorescent signals. Organotypic slice cultures (reviewed by Ga?hwiler et al., 1997) are suitable
preparations for combined electrophysiological and optical imaging in individual CNS neurons. Hippocampal
slice cultures maintained in vitro over a period of
several days undergo a progressive thinning, thereby
reducing scattering and absorbance from extraneous
tissue while retaining much of the cellular and synaptic
connectivity of the hippocampal cortex in situ (Ga?hwiler
1984; Pozzo-Miller et al., 1993; Stoppini et al., 1991).
Pyramidal neurons in such slice cultures mature and
differentiate, acquiring morphological (Buchs et al.,
1993; Pozzo-Miller and Landis, 1993; Pozzo-Miller et
al., 1997; Zimmer and Ga?hwiler, 1984) and physiological (Charpak and Ga?hwiler, 1991; Ga?hwiler, 1984;
Llano et al., 1988; Malouf et al., 1990; Muller et al.,
1993; Petrozzino et al., 1995; Pozzo-Miller et al., 1993,
1995, 1996, 1999) characteristics similar to those in
acute slices from adult animals. Thus, slice cultures
offer a favorable preparation for optical imaging of
Ca2?-sensitive fluorescent dyes in an organotypically
preserved neuronal network (Connor et al., 1994;
Petrozzino et al., 1995; Pozzo-Miller et al., 1993, 1996,
1999).
It is well known that the preponderance of cellular
calcium (Ca) is in the bound form, with typical bound/
free ratios on the order of 103 (McBurney and Neering,
1987; Neher, 1995). Because cytosolic and intraorganelle pools of bound Ca are so large, they exert a
profound influence over the magnitude, shape, and
time course of [Ca2?]i transients. While free Ca2?
transients are the direct effectors of many important
neuronal functions, the bound Ca pool nevertheless
comes into play by, for example, siphoning large fractions of Ca2? currents away from the signaling pool or
by sequestering significant amounts of Ca within noncytosolic compartments. The magnitude and importance of such effects can vary widely, in accordance with
the concentrations, distributions, affinities, and kinetics of Ca2?-binding proteins, pumps, and leaks. Thus, it
is uniquely informative to measure how concentrations
of total (bound plus free) calcium ([Ca]) in specific
subcellular locations change in parallel with [Ca2?]i.
We illustrate this point here by using defined changes
in [Ca] to identify intradendritic Ca sequestration sites.
Energy-dispersive X-ray (EDX) microanalysis, carried out on cryosections of rapidly frozen tissues, was
the first and is still the most reliable method for
quantitatively measuring the concentrations of Ca, as
well as of other physiologically important elements
such as Na and K, within identified subcellular compartments. The physical principles and instrumentation for
rapid freezing (Heuser et al., 1976; Van Harreveld and
Crowell, 1964) and EDX analysis (Hall and Gupta,
371
1983) have been known and available for decades;
results obtained from the combination of these methodologies, as pioneered by the Somlyos and colleagues
(Shuman et al., 1976; Somlyo et al., 1977), have contributed much to our current understanding of the ionic
changes that underlie cell physiology. Nonetheless,
EDX microanalysis requires highly specialized instrumentation and faces several technical problems that
are peculiar to biological specimens. Thus, EDX results
have had to date only limited impact in the field of
neuronal Ca regulation. Over the last decade, however,
several major problems have been largely ameliorated
by significant advances in instrumentation and techniques. Among the most important of these are: (1) the
ability to prepare high-quality truly ultrathin cryosections of unfixed, rapidly frozen tissues that have structural detail comparable to conventional preparations
(Buchanan et al., 1993; Michel et al., 1992), while still
retaining native distributions of diffusible elements;
and (2) improved detectors and analytical microscopes
(e.g., Leapman et al., 1993) that can achieve, in a clean
environment, the high sensitivity and resolution necessary to characterize biologically relevant changes in
cellular [Ca].
In the present communication, we review our studies
aimed at examining the role and relative importance of
the diverse Ca2? buffering and extrusion mechanisms
in neurons of mammalian brain slices. To begin that
investigation, we first characterized the conditions
necessary to elicit widespread dendritic Ca2? entry by
using simultaneous digital imaging of Ca2?-sensitive
dyes and electrical recording from pyramidal neurons
in hippocampal slice cultures during high-frequency
afferent fiber stimulation. We then replicated those
conditions in parallel experiments, rapidly froze the
slices at defined times after the high-frequency stimulation of afferent axons, and carried out EDX microanalysis in ultrathin cryosections to determine the amount
and distribution of total Ca in pyramidal dendrites. The
results illustrate well the advantages of combining
microfluorometric digital optical imaging of intracellular free Ca2? concentrations using fluorescent dyes
with high-resolution EDX microanalysis of total Ca
concentrations at the electron microscopic level.
FREE [Ca2?]i MEASUREMENTS USING
FLUORESCENT DYES AND DIGITAL OPTICAL
IMAGING WITH SIMULTANEOUS
ELECTROPHYSIOLOGICAL RECORDING
Pyramidal neurons in slice cultures had resting
membrane potentials (-57?2 mV, mean ? SEM), input
resistances (50 ? 8 M?; Fig. 1A), and resting levels of
[Ca2?]i (49 ? 5 nM) similar to those from acute hippocampal slices (Brown and Johnston, 1983; Regehr et al.,
1989; Schwartzkroin, 1975). In response to a depolarizing current pulse applied through the recording electrode, they exhibited the characteristic train of action
potentials, spike adaptation, and slow afterhyperpolarization (Fig. 1B). Figure 1C shows the [Ca2?]i transient
in a neuron dialysed with the Ca2? indicator bis-fura-2
via the recording pipette in the patch-clamp whole-cell
configuration, evoked by a train of action potentials in
response to a depolarizing current pulse. Figure 1C
(left) shows a fluorescence image (top) and a pseudocolor map of the [Ca2?]i (bottom) at 250 ms from the
Fig. 1.
Fig. 2.
FREE AND TOTAL CALCIUM IN CENTRAL NEURONS
beginning of the depolarizing pulse, computed using
the pixel-by-pixel ratio of bis-fura-2 fluorescence. Figure 1C (right) illustrates the time course of the [Ca2?]i
transient in two dendritic and one somatic location
(colored boxes in the fluorescence image) temporally
aligned with the train of action potentials, and the
depolarizing current injection (bar). The faster rise
time and larger amplitude of the dendritic [Ca2?]i
transients compared to those at the somatic location
provides additional evidence that the high (L-type)
voltage-dependent Ca2? channels activated by the membrane depolarization are indeed preferentially clustered in proximal dendritic processes (Westenbroek et
al., 1990).
Fig. 1. Membrane electrical properties and voltage-dependent
Ca2? transients in a pyramidal neuron in hippocampal slice culture. A:
Upper trace shows the membrane voltage response to a hyperpolarizing current pulse (lower trace) under current-clamp in the whole-cell
patch-clamp configuration, used to estimate the input resistance of the
neuron. B: Upper traces show the membrane voltage responses to
depolarizing current pulses (lower traces). The characteristic action
potential frequency adaptation is observed on the left trace. The
expanded view on the right shows the typical afterhyperpolarization
that follows action potential trains. C: Membrane voltage response
and associated Ca2? transients evoked by a depolarizing current pulse.
Left: Fluorescence image at 357 nm excitation (top), and a pseudocolor
map calculated from the pixel-by-pixel ratio of background-subtracted
fluorescence images obtained at 357 nm (isosbestic point, Ca2?insensitive) and 380 nm (Ca2?-sensitive) excitation (bottom). Intracellular Ca2? concentration traces shown on the right (at 50 ms frame
interval) were calculated from the regions of interests marked with
boxes in the fluorescence image after background correction from a
region over the slice but outside the dye-filled cell. Membrane voltage
trace shows the train of action potentials evoked by the depolarizing
current pulse (1 second, 0.1 nA; black bar). The membrane voltage
response was digitized at 10 kHz, acquired synchronously with Ca2?
concentration traces (at 50-ms interval), and is shown with the same
time base. The spatial location of the color traces of Ca2? concentration
correspond to the colored boxes in the fluorescence image (left, top). All
experiments were performed in normal extracellular buffer, and using
patch pipettes containing K?-based intracellular solution and 500 然
of the Ca2? indicator bis-fura-2 (Molecular Probes); the pipette can be
seen attached to the soma in the fluorescence image (top). All
simultaneous electrophysiological recording and microfluorometric
digital imaging of Ca2?-sensitive dyes were performed in pyramidal
neurons maintained in cultured hippocampal slices using sharp
microelectrodes or whole-cell patch pipettes in inverted or upright
microscopes (Axiovert, or Axioskop FS, Zeiss), using dry (20X, 0.5NA)
or water-immersion (63X, 0.9NA) objectives, and equipped with 12-bit,
digital, cooled charged-coupled device (CCD) camera systems (Series
200, or PXL-37; Photometrics; Petrozzino et al., 1995; Pozzo-Miller et
al., 1993, 1996, 1999). Alternating dye excitation light was provided by
filter wheels (Lambda-10, Sutter Instruments), or by a galvanometric
monochromator (Polychrome I, TILL Photonics). Electrophysiological
recordings were performed using current-clamp amplifiers (Axoclamp2A, Axon Instruments). Synchronized electrical and optical recording,
afferent stimulus trigger, and control of the cooled CCD camera and
galvanometric monochromator was performed using a single Power
Macintosh computer (9500/120 MHz), running custom-developed software (TI Workbench).
Fig. 2. Synaptically activated free Ca2? transient in a pyramidal
cell in hippocampal slice culture. A: Membrane voltage response to one
500 ms tetanus at 60 Hz given to presynaptic afferent fibers. Sharp
microelectrode recording under current-clamp at resting membrane
potential. B: Same evoked response as in A, but on a longer time scale
to show the prolonged afterhyperpolarization. C: Fluorescence ratios
of the fura-2 signals, before (a) and after (b? k) the tetanus, which
evoked the response shown in B. The tip of the sharp recording
microelectrode contained 10 mM of the Ca2? indicator fura-2 in 200
mM KCl, and was backfilled with 3 M KCl; it appears as blue wedge on
the lower left. The electrical and optical recording was performed as
described in Figure1. Modified with permission from Pozzo-Miller et
al. (1993).
373
Endogenous excitatory neurotransmitter released
from presynaptic afferent fibers within the slice can
elicit suprathreshold membrane depolarizations that
trigger regenerative Na?-dependent action potential
discharge in postsynaptic pyramidal neurons. Highfrequency stimulation of these afferent axons activates
several mechanisms of dendritic Ca2? entry in postsynaptic pyramidal neurons, including influx through locally-activated, low voltage-gated Ca2? channels (Magee et al., 1995) and through the N-methyl-D-aspartate
(NMDA) subtype of glutamate receptors (Malinow et
al., 1994; Perkel et al., 1993; Petrozzino et al., 1995;
Pozzo-Miller et al., 1996, 1999), as well as more widespread voltage-gated influx triggered by back-propagating, Na?-dependent dendritic action potentials (Jaffe et
al., 1992; Miyakawa et al., 1992; Spruston et al., 1995).
Figure 2A shows the electrical response evoked in a
postsynaptic pyramidal neuron filled with the Ca2?
indicator fura-2 via the recording microelectrode, by a
single high-frequency stimulation to the afferent presynaptic fibers indicated by the arrow; Figure 2B shows the
same response on a slower time scale. A series of action
potentials followed by a slow (?10 second) membrane
depolarization and a prolonged (?1 min) afterhyperpolarization, were evoked by the high-frequency afferent
stimulation. This robust, long-lasting action potential
firing was accompanied by a rapid change of [Ca2?]i in
the primary dendrites (to 290 ? 60 nM), and a delayed
[Ca2?]i transient in the somatic regions (to 220 ? 60
nM). Proximal dendrites were the first regions where
[Ca2?]i transients occurred (Fig. 2C). Panel b in Figure
2C shows an image acquired between the trigger of the
stimulus and 600 ms after the synaptic stimulation,
displaying the rapid dendritic [Ca2?]i response to synaptic stimulation. A somatic [Ca2?]i response was observed (panel c), including within the cell nucleus, only
several seconds after the initiation of the stimulation
(panel d). The [Ca2?]i response decayed in all dendritic
and somatic regions with a similar time course (panels
e to g), returning to resting levels 15 seconds after
triggering of the synaptic stimulus (panel h). This
decay corresponded to the time course of the slow
depolarization evidenced in the membrane potential
recordings (Fig. 2A and B). No changes in [Ca2?]i were
observed during the prolonged hyperpolarization, suggesting that a persistent intracellular free Ca2? signal
is not required to drive this process. The long-lasting
synaptic responses to tetanic stimulation in the slice
cultures might arise as a consequence of the particular
circuitry that has developed in the isolated slices after
weeks in vitro. Activation of the B subtype of ?-aminobutyric acid receptors (GABAB) has been implicated in the
agonist- (Ga?hwiler and Brown, 1985) and synapticallyinduced (Malouf et al., 1990) slow hyperpolarization
observed in pyramidal neurons in slice cultures. Interestingly, the time course of the long-lasting depolarizations induced by synaptic stimulation in our slice
cultures resemble those observed in response to bath
application of glutamate and selective metabotropic
glutamate receptor (mGluR) agonists (Charpak and
Ga?hwiler, 1991), indicating the involvement of mGluRs
in the prolonged depolarizations and [Ca2?]i transients
induced by afferent fiber stimulation in our cultures
(Pozzo-Miller et al., 1995, 1996).
374
L.D. POZZO-MILLER ET AL.
EXPERIMENTAL CONSIDERATIONS
REGARDING INTRACELLULAR
FLUORESCENT INDICATORS
Fluorescent indicator molecules for Ca2? ion concentration have revolutionized the study of intracellular
Ca2? transients in small neuronal processes. So far, two
methods have been used to introduce the indicator
molecules into living cells: bath-loading of membranepermeable ester derivatives (AM forms), and passive
diffusion of the membrane-impermeant forms (K? salts)
from a patch pipette (or iontophoresis from a sharp
microelectrode). The potential drawbacks of both techniques of indicator loading have been reviewed (Connor, 1988), and experimentally addressed (Connor,
1994). They include: (1) excessive Ca2? buffering, which
may affect normal neuronal processing; (2) association
with intracellular constituents, which may affect indicator binding and spectral properties; (3) alteration of
absorption and fluorescence spectra by physical factors
such as cytoplasmic viscosity; and (4) transport of
indicator molecules out of the cell or into intracellular
compartments. The self-loading of membrane-permeant forms adds another set of potential artifacts,
such as: (1) incomplete de-esterification, leading to a
mix of responsive and unresponsive indicator; (2) deesterification and trapping within organelles, resulting
in artifactual report of Ca2? concentrations; and (3)
photobleaching (more severe in bath-loading conditions, since after loading and de-esterification there is a
limited amount of indicator within the cell, as compared to the unlimited supply of indicator when diffusing from a patch pipette). If caution is exercised to
control some of these experimental drawbacks, i.e., by
careful calibration of the experimental setup, rational
use of indicator concentrations, and limited UV illumination, optical imaging of Ca2? concentration using
ratiometric fluorescent indicators will provide meaningful quantitative measurements of Ca2? dynamics in
MEASUREMENT OF TOTAL CALCIUM
CONCENTRATIONS WITHIN SUBCELLULAR
DENDRITIC ORGANELLES BY EDX
MICROANALYSIS IN THE ELECTRON
MICROSCOPE
EDX microanalysis ? performed as depicted schematically in Figure 3, and described in detail elsewhere
(Andrews et al., 1994; Buchanan et al., 1993; Leapman
and Andrews, 1991; Pozzo-Miller et al., 1997) ? was
used to obtain direct measurements of persistent
changes in the distribution of total Ca within dendritic
compartments of pyramidal neurons after synaptic
stimulation. We focused on the three subcellular compartments that comprise essentially the entirety of
these dendrites, namely, endoplasmic reticulum (ER),
mitochondria, and cytoplasm. These dendritic compartments can be readily identified in unstained, ultrathin
cryosections (e.g., Fig. 3E). EDX analysis of cryosections from unstimulated slices, rapidly frozen either in
control buffer or in the presence of the Na? channel
blocker tetrodotoxin (TTX, 5 然, to block action potential-dependent spontaneous synaptic transmission) revealed that the concentrations of Ca in all three dendritic compartments (Fig. 4) were comparable to basal
concentrations in a variety of other cells (Andrews et
al., 1988; Somlyo et al., 1985). The mean concentration
of Ca within dendritic ER ([Ca]ER) was 5.1 ? 1.1
mmol/kg dry weight. The units mmol/kg dry weight are
used throughout for total Ca concentrations because
these units follow naturally from EDX data processing
procedures. This concentration unit is approximately
related to a more familiar unit ? mmol/l of hydrated
cell volume ? according to the dry mass fraction of a
given cell compartment. For ER, the relationship [Ca]ER
(mmol/l hydrated cell volume) ? 0.25 x [Ca]ER (mmol/kg
Fig. 3. Schematic diagram illustrating the main steps in the EDX
microanalysis. Sequence starts at upper left and proceeds clockwise.
A: Following defined stimulation in the recording chamber, the filter
membrane supporting the slice culture on a Millicell-CM (Millipore)
culture plate insert is cut out, and using the membrane as a handle so
as to avoid touching the tissue, it is placed on an agar/gelatin pad
already mounted on a specially designed freezing disc that fits both a
modified LifeCell CF-100 cold-metal-block freezing machine, and the
arms of Leica cryoultramicrotomes. A few seconds before freezing,
extraneous is wicked from the tissue, again being careful not to touch
the slice. The assembly is magnetically mounted onto the freezing
machine and frozen at controlled times. Special culture plate inserts
with pre-attached nichrome stimulating wires, as well as custom
freezing stages to accommodate these inserts without the necessity of
cutting the membrane, have been designed for electrical stimulation of
the slices directly on the stage of the freezing machine. This permits
times as short as 1 second between delivery of synaptic stimulation
and the instant of impact on the freezing block. B: Frozen cultures are
transferred under liquid nitrogen to a Leica Ultracut S/FCS or
Ultracut 4E/FC4E cryoultramicrotome, mounted in en face orientation, trimmed to a 250 ? 250 痠 block face (maximum). Ribbons of
ultrathin cryosections (nominal thickness typically ?80nm) were
prepared from the superficial 5?20 痠 of the hippocampal slice
cultures. C: Ribbons were manipulated with an eyelash probe and
transferred onto carbon- and formvar-coated, 200-mesh, thin-bar,
copper EM grids. The grids were cryotransferred into an analytical
electron microscope, either a VG HB501 dedicated STEM or a LEO
912 Omega TEM equipped with Proscan 1024 ? 1024 slow-scan CCD
camera. Cryosections were freeze-dried in the microscope at a specimen stage temperature of ? ?100蚓 over the course of 1?2 hours, then
recooled to below ?170蚓 for imaging and analysis. D: Images of
freeze-dried cryosections are recorded under relatively low-dose conditions (?103 e-/nm2), using digital dark-field detection in the VG STEM
or the zero-loss signal and CCD detection in the LEO 912 (diagram
illustrates the VG STEM). E: Morphological detail can be quite good in
micrographs of satisfactory cryosections, as illustrated by the lowdose, digital dark-field STEM image of the proximal dendritic shaft of
a pyramidal neuron. Cytoplasm rich in microtubules (MT), elongated
mitochondria (M), and cisterns of ER in both long- and cross-section
(arrows) are easily identified. Micrographs are evaluated for tissue
integrity and freezing quality, and optimal areas of dendrites selected
for EDX analysis. The usual probe size for EDX microanalysis is
indicated by the dot within the box. Bar ? 1痠. F: Lastly, electron
beam intensity is increased to ?5 nA and focused to a small probe on
an area of interest, so that characteristic and continuum x-rays are
generated by inelastic collisions. Probe size is typically 100 nm or less,
depending on the size of the target organelle; the usual probe size for
analysis of ER content is indicated by the dot within the box on E.
Emitted X-rays are captured for ?100 sec (total dose ?108 e-/nm2) by
an ultrathin-window energy-dispersive detector (Tracor Micro-ZHV or
Oxford Pentafet), and sorted by a dedicated multichannel analyzer
with associated electronics to produce an EDX spectrum that, like the
one illustrated here, is essentially a plot of X-ray counts vs. energy. All
biologically interesting elements between C (Z ? 6) and Ca (Z ? 20)
produce X-rays at characteristic energies within the energy range
0?10 keV (see labels on F), which can be detected, identified, and
quantified. Subsequent processing of spectra, as described (Shuman et
al., 1976; Buchanan et al., 1993; Pozzo-Miller et al., 1997), provides
compartmental concentrations of elements in units of mmol/kg dry
weight.
living neurons and their finest processes, such as
dendrites and spines.
FREE AND TOTAL CALCIUM IN CENTRAL NEURONS
dry weight), so that 5.1 ? 1.1 mmol/kg dry weight for
control [Ca]ER becomes ?1.3 mmol/l. These relationships are discussed in more detail in Buchanan et al.
(1993).
Following the stimulation procedures outlined above
for measurements of free [Ca2?]i, afferent fibers were
given either a single high-frequency train of stimuli, or
four such tetani at 30-second intervals. In slices rapidly
frozen 3 minutes after a single tetanus, i.e., at a time
well after the decay of free Ca2? transients (see Fig. 2
375
and Petrozzino et al., 1995; Pozzo-Miller et al., 1993),
[Ca]ER had increased to ?15 mmol/kg dry weight in 40%
of dendritic ER cisterns; this population of cisterns had
a mean of 41 ? 7 mmol/kg (Fig. 4). The Ca content of the
remaining 60% was unchanged. We have proposed that
the 40% of cisterns showing large Ca increases must
represent a buffering component of the ER. It is noteworthy that responsive and non-responsive ER cisterns can
exist quite close to each other within the same dendritic
segment (Pozzo-Miller et al., 1997). Given the intercon-
Fig. 3.
376
L.D. POZZO-MILLER ET AL.
Fig. 4. Histograms comparing Ca concentrations within hippocampal dendritic organelles 3 minutes after different stimulation
protocols. Only Ca-accumulating ER takes
up large amounts of Ca following synaptic
stimulation by 1 or 4 action potential-inducing trains (50 Hz, 1 second) to afferent axons
(1 Train and 4 Trains). Ca2? uptake was
graded with increased stimulation (1 Train
vs. 4 Trains), and was inhibited by blocking
synaptic transmission with tetrodotoxin (4
Trains ? TTX) or by blocking SERCA pumps
with thapsigargin (4 Trains ? TG). In the
presence of thapsigargin Ca2? uptake was
diverted to dendritic mitochondria, within
which Ca was localized as small ??hot spots,??
termed inclusions, contained within an otherwise low-Ca matrix. Total Ca content in ER
cisterns returned to resting levels after a
15?30 minute recovery period (4 Trains ?
Rec). There were no large changes in the Ca
content of any other organelle at this time
point. Asterisks indicate values that were
significantly different (P ? 0.05) from appropriate controls. Modified from Pozzo-Miller et
al. (1997).
nected nature of the dendritic ER network, it is likely
that, at least in some instances, these functionally
distinct cisterns are actually in physical continuity.
This possibility provides further support for the emerging realization (Golovina and Blaustein, 1997; Meldolesi and Pozzan, 1998) that functional regio-specialization of the ER, as exemplified by the terminal cisternae
of skeletal muscle sarcoplasmic reticulum, may be more
common than is implied by the continuity and structural homogeneity of the ER network. In contrast to the
large increase in Ca within some ER cisterns, the
concentration of cytoplasmic total Ca ([Ca]cyto), increased only slightly (not significant). At this time
point, there was no significant change in the Ca concentration of dendritic mitochondria ([Ca]mito), nor in any
other analyzed element in any cellular compartment
(Pozzo-Miller et al., 1997).
Three minutes after 4 tetani at 30-second intervals,
[Ca]ER increased to a mean of 72 ? 8 mmol/kg, in the
45% of ER cisterns that exceeded 15 mmol/kg (Fig. 4).
The Ca concentrations achieved in the most avidly
accumulating cisterns occasionally exceeded 100 mmol/
kg, which is an extraordinarily high level. As with the
single afferent train, there was a residual pool of
non-responsive ER cisterns, which was reduced only
slightly by the recruitment of cisterns into the responsive pool; this suggests that inactive cisterns do not
have the capacity to accumulate Ca. There was still no
increase in [Ca]mito, and only a modest rise in [Ca]cyto at
this time point. Following four tetani at 30-second
intervals in the presence of 5 然 TTX, no Ca uptake
occurred in any cellular compartment (Fig. 4), demonstrating that Ca sequestration in ER was a strict
consequence of synaptic activity. In addition, [Ca]ER
completely returned to basal levels after a recovery
period of ?15 minutes from the last high-frequency
stimulus (Fig. 4). These results identify a specific
component of ER as the high-capacity Ca buffer in
hippocampal dendrites. This finding is largely consistent with previous work demonstrating that the ER is a
major physiological sink for Ca (Henkart et al., 1978;
Horikawa et al., 1998; Somlyo et al., 1985; reviewed in
Carafoli et al., 1995; Meldolesi and Pozzan, 1998;
Pozzan et al., 1994). However, three new findings
emerge. First, the levels of [Ca]ER attained by some
cisterns in hippocampal neurons is extraordinary, reaching levels otherwise found only in the terminal cisternae of skeletal muscle sarcoplasmic reticulum (Somlyo
et al., 1977). Even so, this large accumulation is apparently within the range of normal physiological activity
for these dendrites, since the associated electrical responses and [Ca2?]i transients (?500 nM) are well
within physiological levels (Fig. 2), the parallel elevation of cytoplasmic total Ca is in comparison quite small
(Fig. 4), and ER Ca sequestration was reversible (Fig.
4). Second, there is a clear functional distinction between Ca-sequestering and non-sequestering elements
of ER, even though cisterns expressing this difference
are structurally and spatially indistinguishable. The
invariant Ca content of the non-responsive ER pool
under conditions that induce large changes in both free
and total intracellular calcium concentration suggests
that this pool is not capable of Ca accumulation. These
findings support the concept that regional specialization of the ER is an important and common feature of
many cell types, including neurons. Finally, the recovery phase of [Ca]ER has a surprisingly long lifetime, on
the order of 15?30 minutes. Since ER stores will
presumably be loaded and primed for Ca2?- or ligandgated release during this phase (see Pozzo-Miller et al.,
1996), it is possible that ER cisterns carry a memory
trace of previous synaptic activity over the time frame
of minutes.
Our results detected negligible mitochondrial accumulation at the described time (3 minutes after synaptic
stimulation) and conditions of synaptically-mediated
Ca2? entry, which is interesting and perhaps surprising
in view of increasing evidence that mitochondria can
play a major role in neuronal Ca2? regulation (Babcock
et al., 1997; Friel and Tsien, 1994; Herrington et al.,
FREE AND TOTAL CALCIUM IN CENTRAL NEURONS
377
Fig. 5. Estimated time course curves illustrating hypothetical temporal relationships
between intracellular free Ca2? and total Ca
concentrations in various dendritic compartments of pyramidal neurons following synaptic activity-evoked Ca2? influx. The diagram
emphasizes the following points: (1) both mitochondria and a subset of ER can respond
with a large increase in their Ca content; (2)
in both organelles, the increased Ca load
evoked by Ca2? entry persists long after the
elevation and decay of the [Ca2?]i transient,
with the ER Ca load being particularly longlived; and (3) ER and mitochondria appear to
accumulate and release Ca in distinct, and
perhaps co-operative, time domains. Traces
for total Ca concentrations were generated by
extrapolating smooth curves between real
data points at 0, 0.5, 1, 3, and 15 minutes. The
trace for free Ca2? concentration was drawn
after representative responses from real bisfura-2 imaging experiments using an acquisition rate of 20 frames-per-second. Note different scales and units on Y-axes.
1996). Mitochondrial Ca2? transport can in fact be
brought into play, as revealed the action of thapsigargin, an inhibitor of ER Ca2? (SERCA) ATPase pumps
(Fig. 4). Ca sequestration activity of the ER was abolished by incubation with 10 然 thapsigargin, and
under these conditions Ca sequestration is diverted to
mitochondria, many of which exhibited large focal
increases in Ca (Fig. 4). Furthermore, when hippocampal slices were rapidly frozen 30 seconds after a single
high frequency stimulus, i.e., just after termination of
the free dendritic Ca2? transient (see Fig. 2 and
Petrozzino et al., 1995; Pozzo-Miller et al., 1993), both
the ER and mitochondria show significant increases in
Ca content as compared to unstimulated controls, with
[Ca]mito ?5 mmol/kg dry weight in approximately 50%
of mitochondria, even while [Ca]ER continues to rise
(Pivovarova et al., 1997). It appears that the ER and
mitochondria of these pyramidal neuron dendrites may
engage in a coordinated interaction to ensure control
over [Ca2?]i. The emerging view of neuronal calcium
homeostasis, illustrated schematically in Figure 5, is
similar to previous models (Lawrie et al., 1996; Rizzuto
et al., 1993, 1994) and supports the idea that the ER
and mitochondria participate in a highly choreographed interplay, each dominating neuronal Ca2?
regulation over specific [Ca2?]i and time regimes, as
determined by the characteristics of their respective
pumps, channels, and transporters.
Ca2? buffering is another aspect of neuronal Ca2?
regulation that has received recent attention (Neher,
1995). While a detailed discussion of this topic is
beyond the scope of this article, we would like to point
out that EDX measurements of total Ca concentrations,
specifically [Ca]cyto, are potentially useful in this context. Because, as mentioned, EDX directly detects the
?99% of Ca, which is not in the free Ca2? pool, tandem
measurements of [Ca]cyto and [Ca2?]i can be used to
infer Ca2? binding ratios, as well as the dependence of
this parameter on cellular Ca loads. As an example, it is
straightforward to calculate the bound/free Ca ratio of
dendritic cytosol at rest as follows: an average [Ca]cyto of
0.7 mmol/kg dry weight (Fig. 4) is equivalent to 120 然,
calculated as described (Buchanan et al., 1993; and
assuming a cytoplasmic water fraction of 85%); given
that [Ca2?]i ??0.1 然, the bound/free Ca ratio is
?1200. During activity-dependent Ca2? influx, the
most dramatic changes occur in the free Ca2? pool. For
example, stimulations that increase [Ca2?]i by about
one order of magnitude above resting levels (from ?0.1
to ?1然) will increase [Ca]cyto only a factor of about 4
above the normal range (from ?1 to ?4 mmol/kg dry
weight). The reason(s) for this decrease in bound/free
Ca ratio (from ?1,000 to ?500) with increased Ca2?
influx is an open question, although we may speculate
that it is due, at least in part, to a transition into a
non-linear buffering regime and/or to a significant
reservoir of tightly bound Ca. Future experiments will
hopefully clarify the cellular basis for the apparently
dynamic relationship between free and total Ca.
TECHNICAL ASPECTS OF CALCIUM
ANALYSIS IN THE ELECTRON MICROSCOPE
The studies reviewed here represent the first direct
correlation of the total Ca concentration in Ca2?regulating subcellular compartments with the physiological activity of central vertebrate neurons. Although EDX microanalysis is a well-established
technique (Somlyo et al., 1985) for quantitatively measuring elemental concentrations with subcellular resolution, the refined level of analysis demanded in studies
such as this one have been greatly facilitated by recent
technical advances in biological EDX (Buchanan et al.,
1993; Leapman and Andrews, 1991). These advances
have largely eliminated the problems that plagued
many early attempts at biological microanalysis using
immature techniques and instruments. Modern EDX
analysis now offers accurate and reliable performance,
with a demonstrated sensitivity of fewer than 50 atoms
of Ca in very small (?104 nm2) analytical volumes (Shi
et al., 1996).
For measuring total Ca in biological specimens, there
are only a limited number of alternative approaches.
Secondary ion mass spectrometry (SIMS; Chandra et
al., 1989) can detect low elemental concentrations but
378
L.D. POZZO-MILLER ET AL.
has spatial resolution that limits subcellular analysis.
Another emerging technique is electron energy loss
spectroscopy (EELS; reviewed by Leapman et al., 1994),
which has been experimentally demonstrated (Leapman et al., 1993; Shuman and Somlyo, 1987) to be 3?4
times more sensitive than EDX for Ca analysis, with
better spatial resolution. The improved sensitivity of
EELS translates to a time savings of ?15X for equivalent statistical precision, making it feasible to quantitatively map, rather than just point probe, Ca distributions at subcellular resolution. Mapping provides an
intuitively useful structural context for analytical data,
and eliminates the need to subjectively choose a few
locations for analysis in the hope that these are representative of the cell as a whole. There are two complementary approaches to element mapping in the electron microscope, STEM/EELS spectrum imaging (Hunt
and Williams, 1991; Jeanguillaume and Colliex, 1989),
and energy-filtered transmission EM (EFTEM; Grohovaz et al., 1996; Ottensmeyer, 1986). While both have
been used with some preliminary success, STEM spectrum-imaging would appear to have an advantage for
applications involving physiologically relevant concentrations of Ca, since Ca produces complex signals with
low peak/background ratios that require the full range
of spectral information to quantify properly (Leapman
et al., 1993).
CONCLUSIONS
The correlated application of optical digital imaging
of intracellular free Ca2? concentrations using fluorescent dyes combined with high-resolution EDX measurements of the subcellular distribution of total Ca has
directly demonstrated that in dendrites of hippocampal
pyramidal neurons, a subset of specific ER cisterns is
the major subcellular compartment responsible for Ca
sequestration in the minutes following neuronal activity. They also demonstrate that the Ca load associated
with dendritic Ca2? transients persists for many minutes following the decay of these transients, and that
within this time frame, the ER is the dominant sequestration compartment. The results have further characterized the following properties of the ER network in
relation to Ca2? homeostasis: (1) ER Ca2? buffering is
dependent on and graded with neuronal activity; (2) it
is extremely powerful yet still reversible, even in extreme instances of repeated high-frequency afferent
activity; and (3) it is dependent on a SERCA-type Ca2?
pump. The last observation suggests that ER Ca sequestration and inositol-1,4,5-inositol trisphosphate- and/or
calcium-induced Ca2? release may be alternative functions of the same organelles. Taken together, these
observations support the long-standing speculation
(Henkart et al., 1978) that the dendritic ER not only
acts as an intracellular source of Ca2? that can be
mobilized by a signal cascade originated at activated
synapses, but also as the major intracellular sink
involved in active clearance mechanisms following voltage- and ligand-gated Ca2? influx.
ACKNOWLEDGMENTS
We thank M.F. O?Connell for expert cryosectioning, J.
Chludzinski, R. Sun, and L. Verselis for technical
assistance, Dr. T. Inoue for image acquisition and
analysis software, and Dr. R.D. Leapman for invaluable
advice and discussions. This work was supported by
Fellowships at the Marine Biological Laboratory from
the Grass Foundation and the Lakian Foundation
awarded to L.D.P.-M., the NIH Intramural Research
Program, and NIH Research Grant NS35644 awarded
to J.A.C.
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