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The Journal of Neuroscience, May 15, 1998, 18(10):3548-3553
Extrasynaptic Vesicular Transmitter Release from the Somata of
Substantia Nigra Neurons in Rat Midbrain Slices
Erica H.
Jaffe1, 2,
Alain
Marty2,
Albert
Schulte1, and
Robert H.
Chow3
1 Division of Molecular Biology of Neuronal Signaling,
Max-Planck-Institut für Experimentelle Medizin, Göttingen,
Germany D-37075, 2 Arbeitsgruppe Zelluläre
Neurobiologie, Max-Planck-Institut für Biophysikalische Chemie,
Göttingen, Germany D-37077, and 3 Membrane
Biology Group and Department of Physiology, University of Edinburgh
Medical School, Edinburgh, United Kingdom EH8 9AG
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ABSTRACT |
Substantia nigra neurons release dopamine from their
somatodendritic regions. A long-unresolved question is whether this
release occurs by exocytosis or by a nonvesicular mechanism. We used
carbon fiber microelectrodes in a brainstem slice to assay secretion from single cell bodies that had been cleared of connective tissue. Amperometry at the carbon fiber microelectrodes revealed unitary events
in ~90% of cells in resting conditions. These events had charge
integrals ranging from a few femtocoulombs to several hundred femtocoulombs (fC). Local glutamate application enhanced the event frequency by 3.5-fold on average and up to 10-fold in highly responsive cells, although the mean charge integral was not modified. Local application of a high K+-containing saline had
effects similar to those of glutamate. The frequency of resting and
stimulated amperometric events was much lower at 21-22°C than at
32-35°C. The addition of Cd2+ (50 µM), a blocker of voltage-dependent
Ca2+ channels, to the bath solution blocked the
stimulatory effects of glutamate. These results suggest that dopamine
is released from the somata of substantia nigra neurons by exocytosis
and that this mechanism is regulated by neuronal electrical activity. More generally, this study demonstrates the applicability of carbon fiber microelectrodes to the measurement of quantal monoamine secretion
in brain slices.
Key words:
substantia nigra; carbon fiber; exocytosis; dopamine; amperometry, pars compacta
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INTRODUCTION |
Over the past two decades
investigators have accumulated evidence that the classical view of
neurotransmission involving fast Ca2+-triggered
fusion of transmitter-containing vesicles at well defined synapsesmay
not be valid in the substantia nigra. The cell bodies and short
"basal" dendrites of these dopaminergic neurons reside in the pars
compacta and extend axons to the neostriatum (the nigrostriatal tract).
Longer dendrites are located in the pars reticulata. Surprisingly high
concentrations of dopamine are found not only in the regions of the
synaptic terminals in the neostriatum (where synaptic vesicles are
located) but also in the dendrites (Björklund and Lindvall,
1975 ). This finding led to the suggestion that dopamine may be secreted
not only at synapses in the neostriatum but also from dendrites
(Björklund and Lindvall, 1975). Subsequent studies have confirmed
the presence of Ca2+-dependent dopamine secretion in
the substantia nigra (for review, see Cheramy et al., 1981). In
addition, dopamine has been noted to have a local inhibitory action on
the firing of substantia nigra neurons (for review, see Lacey, 1993).
Despite the findings of local dopamine release and action, electron
micrograph studies have not revealed a significant number of vesicles
in the pars compacta and reticulata (Wassef et al., 1981), raising the
possibility of a nonvesicular mechanism of release.
Studies using the false neurotransmitter hydroxydopamine to localize
dopamine (Mercer et al., 1979) and using gold-tagged antibody to label
vesicular monoamine transporter-2 (VMAT-2, the vesicular monoamine
transporter that is found in neurons) (Nirenberg et al., 1996) have
shown that dopamine and VMAT-2 are found in membrane-delimited
compartments both in the somata and in the dendrites of substantia
nigra neurons. These membrane-delimited compartments include clear
vesicles of small size, large dense-core vesicles, tubulovesicular
structures tentatively assigned to the smooth endoplasmic reticulum,
and the trans-Golgi network. Thus, although early electron microscopy
studies did not find many synaptic vesicles (Wassef et al., 1981),
substantia nigra neurons do contain membranous organelles that could be
the vehicles of exocytosis.
In this paper we performed amperometric measurements with carbon fiber
microelectrodes to examine the mechanism of dopamine release from the
somata of substantia nigra neurons. Amperometry is a highly sensitive
electrochemical technique based on recording the redox current arising
from released oxidizable or reducible transmitters. When applied to
isolated, cultured neuroendocrine and neuronal cells, it can detect the
quantal (vesicular) release of catecholamine transmitters (Wightman et
al., 1991; Urena et al., 1994; Chen et al., 1995; Zhou and Misler,
1995; Chow et al., 1996). In slices of substantia nigra, related
voltammetric methods have been used to monitor increases in the
concentration of dopamine in the solution superfusing the slices (Rice
et al., 1994; Cragg et al., 1997). However, electrochemical detection
of neuroamine secretion in slice preparations at the single-cell level
has not proven feasible until now.
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MATERIALS AND METHODS |
Slice preparation. Rats 14-16 d old were decapitated
under Metofane anesthesia, and the brain was removed. A block of
midbrain containing the substantia nigra was cut out and fixed on a
vibratome. During cutting, slices were bathed in an ice-cold incubation
solution containing (in mM): NaCl 125, KCl 2.5, NaH2P04 1.25, NaHCO3 26, CaCl2 1, MgCl2 4, ascorbate 0.5, and glucose 10 and gassed with 95% O2/5% CO2. Coronal
slices 200 µm thick containing the caudal substantia nigra and the
accessory optic tract were cut and incubated at 32°C for 1 hr in the
above solution. Slices then were transferred to a recording chamber
where they were visualized with a 40× water immersion objective and
superfused (1.5× the bath volume/min) with a solution containing (in
mM): NaCl 125, KCl 2.5, NaH2PO4 1.25, NaHCO3 26, CaCl2 2, MgCl2 1, and glucose 10 and gassed with 95% O2/5%
CO2. Except where otherwise indicated, experiments were conducted at 32-34°C.
Carbon fiber microelectrode fabrication. The carbon fibers
(PAN T650, 5-6 µm diameter; Amoco Performance Products, Greenville, SC) first were heated at 390°C for 3 hr to remove sizing compound from the surface of the carbon. These fibers were used to manufacture two types of carbon fiber microelectrodes of comparable sensitivity and
noise. One type was prepared with polyethylene insulation (Zhou and
Misler, 1995; Chow et al., 1996). The other type used an insulation
method involving electrodeposition of an insulating organic "paint"
(EDP; Canguard, BASF Lacke und Farben AG, Muenster, Germany) (Schulte
and Chow, 1996). A 3 cm length of PAN T650 carbon fiber was attached to
a 5 cm length of 0.5-mm-diameter copper wire with a small drop of
conductive carbon paste. Then the carbon fiber, attached to the copper
wire, was threaded into a patch pipette that previously had been pulled
on a standard vertical patch-pipette puller to yield a tip having a
diameter of ~10 µm, until the carbon fiber passed out of the tip
and the copper wire lodged at the narrowed neck of the pipette tip. A
small drop of two-component glue was applied to the back end of the
pipette to fix the copper wire firmly in the pipette, and the glue was allowed to harden for several hours. SYLGARD (Rhône-Poulenc
Rorer, France) was applied to the tip region to hold the carbon fiber in place and to provide a watertight seal. An electrical connection to
a DC power supply was made to the copper wire extending from the back
of the patch pipette. Electrodeposition of the insulating paint was
performed by applying a constant voltage of 3-5 V for 2-4 min between
the carbon fiber dipped into EDP and a platinum counterelectrode.
Coated carbon fibers were heat-cured at 195°C for 2 min. To enable
electrical connections, we soldered a gold-plated pin to the copper
wire at the back of the electrode. The tip of the carbon fiber was cut
with a scalpel blade under a dissection microscope before each
experiment.
Although both electrode types were used and both were able to detect
the quantal events from substantia nigra neurons, the EDP-insulated
electrodes were more convenient, because the tips were straighter and
passed more readily beneath the microscope water immersion objective.
Testing of microelectrode properties. Before the
experiments, all carbon fiber microelectrodes were tested for noise and
sensitivity. Their electrical noise was measured with the freshly cut
tip of the electrode dipped into the standard saline solution with a Ag/AgCl reference electrode. Only electrodes having noise lower than
200 fA root mean square at 1 kHz were used. The electrodes also were
tested for peak reduction currents in cyclic voltammograms in a
solution containing 1 mM K3Fe(CN)6
and 500 mM KCl, pH 3.0. A voltage ramp from +500 to  500
mV and then back to +500 mV was applied at 100 mV/sec between the
carbon fiber and a Ag/AgCl reference electrode. The cyclic
voltammograms routinely displayed the expected sigmoidal shape (see
Fig. 1), with peak currents in the range of 800 to 1000 pAnear the
value predicted for microelectrodes having an electroactive diameter of
~5 µm (Schulte and Chow, 1996). In the rare case of an electrode
not having a peak current within this range, the tip was recut.
Cleaning procedure. The mediocaudal region of the pars
compacta, near the accessory olfactory tract, was used in the present experiments. A very large proportion (>85%) of the neurons in this
structure has been shown to be dopaminergic (Yung et al., 1991). The
neurons were large, closely packed together, and had somata that were
either spindle-like or pyramidal (Yung et al., 1991). For recording, we
selected neurons located well within the pars compacta on the basis of
(1) healthy appearance (smooth membranes and nonvacuolated), (2)
superficial localization within the slice, and (3) presentation of a
smooth somatic surface without emerging dendrites. The experimental
chamber was rotated on the microscope stage until the edge of the
membrane area to be recorded from was perpendicular to the axis of the
cleaning pipette. Cleaning pipettes had tip diameters of 8-10 µm and
were filled with standard bath saline. They were mounted on the same
micromanipulator that served subsequently to hold the carbon fiber
microelectrode. Cleaning involved alternating positive and negative
pressure (Edwards et al., 1989). Only the selected side of the neuron
was freed of covering tissue, to minimize the risk of cell damage.
Recordings were initiated as quickly as possible after cleaning, after
the cleaning pipette had been replaced with the carbon fiber
microelectrode (see Fig. 1A).
Amperometric recording. A carbon fiber microelectrode was
mounted onto the headstage of the patch-clamp amplifier. The
microelectrode tip then was touched gently to a cell, as illustrated in
Figure 1A, and current signals were recorded under
voltage clamp at a holding potential of 720 or 800 mV. Sampling was at
5 kHz with low-pass filtering of 1 kHz. Traces shown in the figures
additionally have been filtered at 200 Hz.
The cell was stimulated by pressure-ejecting solution from a glass
pipette (tip opening ~3 µm) positioned in the vicinity of the
recorded neuron. Control experiments showed that the observed amperometric responses could not be ascribed to movements of the carbon
fibers because of solution flow, nor could they be attributed to
extracellular recordings of action potentials, which would have been
biphasic or principally downward deflections instead of the uniformly
upward inflections that were recorded.
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RESULTS |
Spontaneous amperometric events
For this work we used neurons located in the mediocaudal region of
the substantia nigra pars compacta. This region contains a particularly
high density of tyrosine hydroxylase immunoreactive cells and yields a
correspondingly high level of dopamine release in bulk voltammetry
experiments (Yung et al., 1991; Cragg et al., 1997). In our first
attempts to record amperometric signals from substantia nigra cells, we
positioned carbon fiber microelectrode tips at the soma or dendrite of
the cells, but we were unable to measure convincing amperometric
events. Touching the carbon fiber microelectrode tip to the brain slice
tissue led to a dramatic decrease (by factors as large as 10 or more)
in voltammetric currents measured in solutions of test agents (Fig.
1B), suggesting that the lack of successful recordings was attributable to the reduction in
the sensitivity of electrodes because of contact with extracellular materials in the brain slices. We therefore decided that, before recording from a substantia nigra neuron, we would dissect away the
tissue overlying the somata, using a "cleaning pipette" (Edwards et
al., 1989).
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Figure 1.
Cleaning procedure for amperometric
recording in substantia nigra. A, Appearance of
substantia nigra neuron during amperometric recording. A substantia
nigra neuron from the pars compacta was partly freed from overlying
connective tissue with the help of a saline-filled glass pipette. A
carbon fiber electrode (EDP-insulated type) then was positioned to
attain contact with the exposed part of the neuronal soma. Scale bar,
10 µm. B, Cyclic voltammograms with a scan rate of 100 mV/sec were performed in potassium ferricyanide solution (see Materials
and Methods). The vertical excursion gives the limiting current, an
indication of the sensitivity of the electrode. The limiting current is
reduced by a factor of ~5 after contact with brain tissue.
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Just after a cell was cleaned, a freshly cut carbon fiber tip was
positioned in the recording chamber ~50 µm above the cell, and a
1-2 min control recording was taken. Then the fiber was pushed gently
into contact with the cell membrane, and a "cell contact" recording
of 2 min was taken. Figure 1A illustrates the appearance of a substantia nigra neuron after the cleaning procedure and the position of a carbon fiber during a recording. In many instances spontaneous amperometric events with variable shapes were
observed in the cell contact recording, as illustrated in Figure
2A. Rarely, there were
larger, more irregularly shaped events, such as the one shown in the
second row of Figure 2A. These may represent
multiples of the underlying quanta and/or another type of vesicle.
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Figure 2.
Spontaneous amperometric events recorded from a
substantia nigra neuron. A, Examples are shown of
spontaneous events recorded from a single cell. Notice the large
variability in size and time course of these events. B,
Shown is the distribution of the mean frequencies of spontaneous
amperometric events in the different cells in which events could be
recorded.
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Over a total of 27 cells that were assayed in control conditions from
14 preparations, 25 had spontaneous events, a percentage of 93%. The
frequency of the spontaneous events was generally low, from 0.1 to 30 events/min, and in most cases it fell within the range of 0.3-3
events/min (Fig. 2B).
Response to local glutamate and
K+ stimulations
Once the background activity was determined, the preparation
usually was stimulated in an attempt to modify the pattern of the
amperometric events. Figure 3A
illustrates typical examples of events recorded before and during a
30-sec-long local application of 0.3 mM glutamate. The
frequency of amperometric events, which was ~6/min before the
application, rose to 32/min during and after the application, a
fivefold increase (Fig. 3B). In several instances, repeated
responses to multiple glutamate applications (up to four) could be
elicited from the same cell. However, the responsiveness to glutamate
and the resting frequency slowly decreased during the recording (Fig.
3B,C).
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Figure 3.
Enhancement of the frequency of amperometric
events by glutamate applications. A, Sample recordings
at rest (top) and a few seconds after local application
of 0.3 mM glutamate (bottom).
B, Timing of amperometric events after three successive
applications of glutamate to the same cell. The three plots are
consecutive. The events illustrated in A are taken
before and during the first glutamate application to this cell. Each
event is represented by a vertical box. There are 17 events during the first application, which lasted 30 sec, as compared
with three events during the preceding 30 sec period. Note that the
effects of glutamate become weaker for the second and third
applications than for the first. C, Effect of glutamate
on event frequency. Results are cumulative from 10 applications
obtained in five cells. The results have been aligned with respect to
the start of the applications. Applications ranged between 27 sec
(solid line) and 47 sec (the end of the
dotted line) in duration.
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In total, 19 cells were stimulated with 0.3 mM glutamate
under the conditions of Figure 3. For each experiment the event numbers were determined for control periods, before and between stimulations, as well as for test periods during and shortly after glutamate applications. The mean frequency obtained when all experiments were
pooled together rose from 2.1 events/min during resting periods to 7.2 events/min during stimulations. The difference between these two values
was significant, showing that amperometric events were sensitive to
glutamate applications (Table 1). In
individual experiments it was not always clear whether glutamate was
excitatory because of the small total number of events that sometimes
was obtained (occasionally 0). Of 14 cells in which this total number was five or larger, 11 showed a clear increase in frequency
(test/control frequency ratios  2). Thus it can be concluded
that at least 11 of 19 equaling 58% of the cells responded to
glutamate. In addition to the increase in event frequency illustrated
in Figure 3, glutamate applications often led to a gradual and
reversible increase in the background current (data not shown). This
effect was attributed to the cumulative increase in the concentration of the released substance after liberation from distant sites located
either in the recorded cell or in its neighbors.
To reveal the kinetics of the effects of glutamate application (Fig.
3C), we aligned responses to 10 glutamate stimulations (0.3 mM) from five particularly responsive cells at the start of
the applications, which ranged from 27 to 47 sec in duration. In these
cells the glutamate applications increased the event frequency on
average by 10-fold, from 2.4 events/min to 24 events/min. The peak of
the response was attained 9 sec after the start of the stimulus. Over
the next 20 sec of maintained stimulation, the response subsided by
approximately twofold. On cessation of the stimulation the frequency
returned to control values with a time constant of 6.2 sec (determined
by aligning the responses with the offset of the applications). The
rather slow kinetics of onset and of recovery of the glutamate-induced
stimulation may reflect the slow diffusion of glutamate through the
slice tissue.
In another series of experiments the cells were stimulated locally by
using puffer pipettes loaded with a solution containing 100 mM K+. The high K+
stimulations led to a strong increase in the frequency of amperometric events, similar to that obtained with glutamate (see Table 1).
Event characteristics at rest and after
glutamate stimulation
Histograms of half-widths and charge integrals of the individual
events are shown in Figure 4,
A and B. The modal values of the half-width and
the charge integral were 3.5 msec and 4.5 fC, respectively. As
illustrated in Figure 4C, there was no significant difference in the charge integral distribution of spontaneously occurring events and glutamate-evoked events (Kolmogorov-Smirnov test).
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Figure 4.
Summary histograms of amperometric event
characteristics (498 events). A, Charge integral.
B, Half-widths (with 200 Hz of low-pass filtering).
C, Comparison of cumulative charge histograms for
spontaneous and for stimulated events. Both histograms have been
normalized. There was no significant difference between the two
curves.
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The response to glutamate depends on voltage-dependent
Ca2+ entry
Bulk release of dopamine in the substantia nigra can be blocked by
manipulations that block voltage-dependent Ca2+
entry into neurons (Geffen et al., 1976; Cheramy et al., 1981; Rice et
al., 1994). Likewise, it seemed plausible that glutamate and
K+ stimulations increased the frequency of
amperometric events by promoting voltage-dependent
Ca2+ entry into substantia nigra neurons. We
therefore examined whether glutamate-induced stimulation would be
sensitive to the inorganic Ca2+ channel blocker,
cadmium (Cd2+). In the presence of
Cd2+ (50 µM) the frequency of
spontaneous events was 0.57 event/min, smaller than in normal solution
(2.1 events/min). Although the difference was not significant at the
p < 0.05 level, this result suggests that blockage of
Ca2+ channels leads to a decrease of the background
frequency of amperometric events. The mean event frequency during and
shortly after stimulation in Cd2+ was 0.40 event/min, significantly different from the value obtained in control
stimulation (see Table 1). This result shows that Cd2+ effectively blocks the responsiveness of
amperometric events to glutamate. Indeed, there was no indication of an
enhancement of event frequency by glutamate in
Cd2+-containing saline. The lack of responsiveness
to glutamate could not be attributed to the preparations, because the
results of three cells interleaved in the Cd2+
experiments gave resting and stimulated frequencies of 1.4 and 5.3 events/min, close to the overall average, and in each cell a clear
response to glutamate was apparent.
Influence of temperature on spontaneous and evoked
amperometric events
Ordinarily, recordings were performed at 32-35°C. However, a
series of experiments was performed at 21-22°C to investigate the
influence of temperature in resting and stimulated conditions. The mean
frequency of amperometric events was only 0.16 event/min in
unstimulated cells at 21-22°C, 13 times lower than at 32-35°C (see Table 1). Likewise, in the presence of glutamate the event frequency was significantly lower than in control experiments. After
stimulation by glutamate the frequency rose to 1.0 event/min; however,
because of the low frequency of events, the difference to the resting
frequency did not reach statistical significance. Control experiments
at the usual temperature were interleaved between the low-temperature
experiments and gave results close to the overall mean. These results
indicate that performing experiments at room temperature drastically
reduces the resting and stimulated frequency of amperometric
events.
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DISCUSSION |
The present work demonstrates that discrete amperometric signals
can be recorded at the somata of substantia nigra neurons in brain
slices. Two experimental manipulations were found to be essential for
the successful recording of such events: the cell had to be cleared of
surrounding tissue, and the preparation had to be warmed above 30°C.
These procedures should be applicable to other brain regions.
In previous studies of cultured cells, such as adrenal chromaffin
cells, unitary amperometric events have been attributed to vesicular
transmitter release (Wightman et al., 1991; Urena et al., 1994; Chen et
al., 1995; Zhou and Misler, 1995; Chow et al., 1996). In our
experiments, several properties of the amperometric events argue
strongly for an exocytotic mechanism and against a carrier mechanism.
First, some events reached an amplitude of 20 pA, corresponding to a
release rate of at least 6 × 107
molecules/sec. Single transmembrane transporters are unlikely to
function at such a high rate. Second, the pulsatile nature of the
signals would require the abrupt submillisecond or millisecond turning
on and off of the transporteruncharacteristic behavior for carriers.
Third, there is no obvious mechanism by which glutamate could activate
such a carrier. Finally, the earlier report that GBR 12909, a selective
dopamine uptake inhibitor, increases levels of dopamine in the
extracellular fluid strongly argues against a primary role of
(reversed) uptake in release of dopamine (Cragg et al., 1997). Rather,
it appears that the primary role of the transporter is to terminate
dopamine action by the removal of the transmitter from the
extracellular fluid. We therefore conclude that the amperometric events
correspond to exocytosis.
We have no direct evidence about the nature of the oxidizable substance
that was detected. However, because dopamine is the major catecholamine
neurotransmitter that is known to be contained in, and released from,
substantia nigra neurons (Rice et al., 1994; Cragg et al., 1997), it is
most likely that the amperometric events represent released dopamine.
In the pars compacta of the substantia nigra, the proportion of
dopamine-containing cells was estimated at 85% by Yung and colleagues
(1991), in reasonably good agreement with the proportion of cells in
the present study that displayed amperometric events (93%). It would
be valuable in future experiments to examine how inhibitors of the
reuptake mechanism for dopamine (and other catecholamines) affect the
size and time course of the individual amperometric events.
Zhou and Misler (1995) recorded amperometric events from the somata of
cultured neurons from superior cervical ganglia and attributed the
signals to presynaptic release from overlying nerve terminals rather
than to somatic release. We do not believe that the signals we recorded
in the substantia nigra originated from presynaptic nerve terminals;
whereas projections of serotonin- and noradrenalin-containing neurons
have been described in the substantia nigra, the density of these
projections is very low, and, furthermore, most projections lead to
synapses with dendrites in the pars reticulata rather than with pars
compacta somata (Nedergaard et al., 1988) (for review, see Lacey,
1993). In addition, it is unlikely that the few catecholamine- or
indolamine-releasing nerve terminals would have survived the extensive
"cleaning" procedure that preceded recording. Thus, our recording
of amperometric events from >90% of the cells we studied is difficult
to reconcile with the idea that the signals arise from serotonergic or
noradrenergic synaptic terminals. We therefore argue that the signals
were coming from the somata of the recorded cells. Consistent with this
proposal, somatic exocytosis of dopamine and of other neurotransmitters has been reported in molluscan neurons and in peripheral neurons in
culture (Dan et al., 1994; Chen et al., 1995; Huang and Neher, 1996).
The charge distribution histogram for amperometric events has a peak at
4.5 fC and a long tail component that may indicate a separate vesicle
population with a much larger content; however, the frequency of larger
events was too low to allow for the confirmation of this idea. The peak
value of the charge histogram corresponds to 2.8 × 104 elementary charges. Because dopamine has two
redox sites susceptible to oxidation, the corresponding number of
dopamine molecules is 14,000. Charge distributions previously obtained
for dopamine release from somata of Planorbis neurons are
biphasic, with a small and a large vesicle population containing
~40,000 and 800,000 molecules per vesicle, respectively (Chen et al.,
1995). Likewise, serotonin release from leech neurons revealed two
vesicle populations with contents near 5000 and 80,000 molecules per
vesicle, respectively (Bruns and Jahn, 1995). In the latter study it
was suggested that the small and large quantal sizes originate from
small clear vesicles and from large electron-dense vesicles,
respectively. On the basis of this earlier work, it appears plausible
that the main population of quanta in the present work also corresponds
to small, clear vesicles, whereas the tail in the distribution may be
attributable to larger, electron-dense vesicles.
In the present study, applications of glutamate or
K+ enhanced the frequency of amperometric events.
Earlier bulk release studies showed that K+-evoked
dopamine secretion from the substantia nigra depends on the presence of
extracellular Ca2+ ions (Geffen et al., 1976;
Cheramy et al., 1981; Rice et al., 1994). Likewise, the present finding
that Cd2+ blocks the response to glutamate indicates
that the intracellular Ca2+ concentration tightly
controls the event frequency. The notion of extrasynaptic
Ca2+-dependent exocytosis is in accord with recent
studies showing that Ca2+ concentration elevation
enhances vesicular release from the somata of isolated snail neurons
and of mammalian neurons in culture (Dan et al., 1994; Chen et al.,
1995; Huang and Neher, 1996).
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FOOTNOTES |
Received Sept. 15, 1997; revised Feb. 23, 1998; accepted March 2, 1998.
This work was supported by a Royal Society Research Grant (to R.H.C.)
and the Deutsche Forschungsgemeinschaft (SFB 406), and by grants from
the Deutsche Forschungsgemeinschaft (to E.J.), from the European
Community Training and Mobility Program (to A.S.), and from the Howard
Hughes Medical Institute (to R.H.C.). We gratefully acknowledge the
kind support of Professor Walter Stühmer (Max Planck Institute
for Experimental Medicine, Division of Molecular Biology of Neuronal
Signaling).
Correspondence should be addressed to Dr. R. H. Chow, Membrane
Biology Group, Department of Physiology, University of Edinburgh Medical School, Edinburgh, UK.
Dr. Jaffe's present address: Instituto Venezolano de Investigaciones
Cientificas, Caracas, Venezuela.
Dr. Schulte's present address: Department of Physiology, University of
Edinburgh Medical School, Edinburgh, UK.
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Top
Abstract
Introduction
