(Received for publication, November 3, 1994; and in revised form, December 1, 1994)
From the
Using single rat adrenal chromaffin cells, we examined the
coupling of action potential activity to quantal release of
catecholamines by combining perforated patch current-clamp recording
with electrochemical microcarbon fiber amperometry. Chromaffin cells
display steeper dependence of quantal release on action potential
frequency than many nerve terminals, as well as more desynchronized
release following an action potential. Also in contrast to neurons, in
chromaffin cells, a major chemical secretagogue (acetylcholine)
triggers potent quantal release even in the absence of electrical
activity. These findings are consistent with an hypothesis that a major
component of exocytosis from chromaffin cells involves diffusion of
Ca to secretion sites which are less well
co-localized with Ca
channels than those in nerve
terminals.
A defining feature of the nerve terminal of a synapse is that
the propagation of an action potential (AP) ()results in
rapid and highly synchronized, Ca
-dependent
exocytosis of up to hundreds of packets of neurotransmitter often
within 1 ms(1) . At several classical synapses, evidence
ranging from simultaneous pre-terminal and post-synaptic recording (2) to nerve terminal morphometry (3, 4) and
histochemistry(5) , suggest that this rapid and secure mode of
release results from intense Ca
entry through
voltage-dependent channels which are closely co-localized with a
population of ready-to-fuse Ca
-triggered vesicles.
These domains of close co-localization of Ca
channels
and vesicle secretion sites are known as ``active zones.''
The coupling of Ca
to vesicle fusion may occur
through a low affinity Ca
-binding protein which
requires local Ca
concentrations up to hundreds of
µM for its binding sites to be
filled(6, 7, 8) .
Chromaffin cells of the
adrenal medulla function analogously to post-ganglionic sympathetic
neurons. In response to splanchnic nerve activity and release of
acetylcholine (ACh) and peptide co-transmitters, chromaffin cells
secrete, into the circulation, catecholamines (CA) and small peptides (e.g. enkephalins). Like nerve terminals, chromaffin cells
have voltage-gated Ca channels and display APs when
depolarized (9, 10, 11) and exocytose their
humoral products in a Ca
-dependent
fashion(12, 13) . However, the kinetics of
depolarization-evoked release may be more delayed and prolonged in
chromaffin cells than in nerve terminals. In voltage-clamped chromaffin
cells, release begins milliseconds, rather than microseconds, after the
start of Ca
entry and continues for up to seconds
after Ca
entry ceases(14, 15) .
Investigation of the dynamics of quantal release of CA release from
single chromaffin cells conducting APs would be a very useful
counterpart of voltage-clamp studies, especially as APs permit more
physiological recruitment of Na
and Ca
currents than voltage-clamp pulses.
Carbon fiber amperometry permits real-time electrochemical detection of exocytosis of the CA content of single chromaffin granules with speed and sensitivity nearly approaching transmitter detection by post-synaptic membranes(15, 16, 17) . Using single rat chromaffin cells, we have combined perforated patch current-clamp recording, with amperometry, to examine in real time the dynamics of the direct coupling of quantal release of CAs to AP activity. Part of the data has been presented in abstract form(47, 48) .
The following modifications of the published technique for CFE fabrication proved useful in enhancing our success rate and increases the sensitivity of our CFE significantly. (i) After constructing the polyethylene-coated CFE according to Chow et al.(15) , the tip of the electrode was reheated to remelt the polyethylene tubing covering the carbon fiber. This extended the tip region of the electrode and avoided insulation defects which could provide leak pathways to ground and reduce the potential at the cut surface and the ability of the CFE to oxidize catecholamines. This extended tip of the CFE could be carefully recut several times. (ii) After each cutting, the exposed surface of the electrode was treated by insertion of the tip into 95% ethanol for 3-5 s. (iii) Prior to positioning near a cell, a given CFE, held at +780 mV, was inserted into the bath solution to measure its background current. CFEs displaying background currents initially >300 pA, but declining to <10 pA over 5-10 min, permitted the most sensitive and stable recordings of amperometric spikes (ASs). CFEs with background currents < 100 pA were withdrawn from the solution, recut, recleaned, and retested. With these modifications of the technique more than 90% of the CFE fabricated can be used for extended recording.
Figure 6: AP-dependent and -independent pathways of stimulation of secretion by CCh in rat chromaffin cells. A, in this cell, sequential, extended but low intensity puffs of CCh resulted in bursts of APs accompanied by barrages of ASs which ended shortly after membrane repolarization. B, in another cell, sequential brief, but intense, puffs of CCh given after the cell desensitization of the cell to the depolarizing effects of CCh resulted in extended bursts of ASs. Not shown is that this cell responded with short, vigorous bursts of APs and ASs to several CCh puffs prior to these recordings.
Figure 1:
Simultaneous measurements of APs and
single vesicle release, measured as ASs in a rat chromaffin cell. A, results of a typical experiment of simultaneous recording
of trains consisting of 20 APs (membrane potential trace, V
) and barrages of ASs (amperometric current
trace, I
) in response to sustained depolarizing
current pulses (I
). B, histogram of
the number of APs preceding the occurrence of the first AS in a barrage
of ASs evoked by the AP train. Data were pooled from 13 trains evoked
in the cell depicted in A and 27 trains from five other cells
which were likewise stimulated by 5-s depolarizing current pulses and
produced similar AP trains. C, histogram of the time of
occurrence of AS events during and after 5-s-long AP trains. Data were
pooled from the 13 trains evoked in the cell depicted in A and
12 trains from two other cells showing similar pattern (frequency and
total number) of APs per train as well as similar numbers of ASs evoked
by train. Zero time marks the time of the peak of the first AP within a
train, typically 10-20 ms after the onset of depolarization. Note
the nearly 2-s tail of AS events after the cessation of the stimulating
current. D, cartoon of the cell stimulation/recording
arrangement featuring the patch pipette recording electrode attached to
the cell, the carbon fiber electrode within 1 µm of the cell
surface, and the drug application pipette positioned within 50 µm
of the cell surface. E, sample amperometric spike recorded at
high gain.
Since cells are of variable responsiveness, Fig. 2presents sample traces from two cells. In these cells, as in others, APs evoked at a frequency of 0.2-0.5 Hz produced very rare ASs. (Over this range of frequencies, the effect of an individual AP should be independent of that of others, in that even after a train of APs, ASs are usually no longer apparent by 2-5 s after cessation of a train.) However, increases in the frequency of stimulation dramatically increased quantal output. In Fig. 2A, which depicts a cell with high release efficiency, note that increasing the stimulus frequency from 0.2 to 1 and finally to 4 Hz increased the average number of quanta following an impulse from 0.37 ASs per AP to 1.63 ASs per AP and finally to 2.95 ASs per AP. In contrast, in Fig. 2B, which depicts a cell with lower release efficiency, note that raising the stimulus frequency from 0.33 to 4 Hz and then up to 12.5 Hz in a burst increased the number of quanta following an impulse from 1 AS per 20 APs to, on average, 3 ASs per 20 APs, and finally to 8 ASs per 10 APs.
Figure 2: Dependence of rate of quantal secretion on AP frequency. A and B depict sample traces of quantal secretion from two cells stimulated at varying rates. C depicts average quantal release per AP as a function of AP frequency in three rat chromaffin cells and compares it with post-synaptic potential amplitude as a function of AP frequency at the frog neuromuscular junction. Post-synaptic potential data were averaged from Magleby(23) , where the compound post-synaptic potential was measured extracellularly from a group of surface muscle fibers, and from Misler(24) , where the post-synaptic potential was measured from singly impaled muscle fibers. Data were normalized to the number of quanta released per impulse at 1 Hz. D presents, in an expanded scale, a burst of APs evoked by a sustained depolarizing current.
Fig. 2C depicts the normalized dependence of
quantal secretion on AP frequency seen with three chromaffin cells with
large numbers of events (total 300 events). Increasing AP frequency
from 0.2 to 4 Hz produces a 4-fold increase in the average number of
quanta released per impulse. The frequency dependence of chromaffin
cell secretion is strikingly steeper than the frequency dependence of
post-synaptic potential amplitudes at frog skeletal neuromuscular
junctions stimulated to conduct single APs in reduced
Ca/elevated Mg
solutions chosen to
minimize depletion of available quanta
(see data plotted
from Magleby (23) and Misler(24) ). However, other
nerve terminals, including individual post-ganglionic adrenergic
varicosities innervating smooth muscle of the vas deferens (25) and branched terminals innervating crayfish claw
muscles(26) , seem to show steeper frequency dependence of
release, more comparable with that seen from chromaffin cells.
Figure 3:
Latency of quantal release after single
APs applied at 1 and 4 Hz. A presents a histogram of events
tabulated from the cell shown in Fig. 2A, stimulated at
1 Hz. The inset compares tabulated latencies of short delay
amperometric events recorded from this cell (open squares)
with the latencies of quanta contributing to the post-synaptic
potential recorded at the frog neuromuscular junction at a comparable
temperature (28) (closed squares). B presents
a segment of the record used to construct the histogram in A. C presents a latency histogram for AS events recorded from the
same cell after the AP frequency was increased to 4 Hz. The inset to this panel compares the distribution of short latency events
recorded under those conditions with the latency of quantal release
recorded from a frog neuromuscular junction bathed in an extracellular
solution containing 0.3 mM Ca and 1 mM Mg
and stimulated for 3 min at 10
Hz
. D presents a segment of the record used to
construct the histogram in C. Note the occurrence of AS events
many tens of ms after each AP. In the insets to A and C, the peak value of the histogram for the neuromuscular
junction was normalized to the peak value of the histogram for the
chromaffin cell. Axes labels and quantities in the insets for A and B were identical to those in the major
graph.
The insets to A and C compare the latency of quantal secretion seen
in chromaffin cells with that at a frog neuromuscular junctions bathed
in modified Ca/Mg
external
solutions. When the nerve terminal is stimulated by APs at 1 Hz,
release occurs almost exclusively within a window of 1-2 ms; no
tail of release is discernible (data from (28) ). Even when the
nerve terminal is stimulated by APs at 10 Hz for 3 min to increase
poorly synchronized quantal release, recorded as miniature
post-synaptic potentials, less than 5% of the total quantal release
occurred after the narrow window of release. (
)Similar data
are available for varicosities of post-ganglionic adrenergic fibers
innervating vas deferens smooth muscle(25) , as well as for
preganglionic cholinergic terminals innervating the superior cervical
ganglion(27) . Despite methodological differences, (see
``Discussion'') comparison of data suggests that the
distribution of latencies of release is broader and more dependent on
AP frequency in the chromaffin cell than in nerve terminals.
Fig. 4provides evidence that PDs ranging from
-35 to -20 mV contribute to sustained secretion. In this
cell, which displayed a 300 pA Na
current in
response to a voltage-clamp ramp (bottom left inset), note
that repeated 25-ms depolarizing current pulses, which induce single
APs at 0.2 Hz, at best trigger only one AS (left-hand panel).
In contrast, repeated 1-s current pulses of identical amplitude and
frequency, each of which provoke a single AP followed by a PD, trigger
a salvo of ASs (right-hand panel). These results suggest that
even small amplitude depolarizations applied for many ms may be
sufficient to evoke CA secretion. They are consistent with results of
studies on chromaffin cells from a variety of species demonstrating
that small, but sustained, increases in extracellular
[K
] induce
[Ca
]
-dependent CA secretion
which is reduced, but not abolished, by the Na
channel
blocker tetrodotoxin (30) .
Figure 4:
Effectiveness of PD in inducing secretion.
In this cell, depolarizing current pulses (from -20 pA to
+20 pA) imposed for 25 ms (left upper panel) induced
single APs, whereas those imposed for 1000 ms (right upper
panel) induced single APs followed by PDs. Note the enormous
enhancement in AS frequency during the sustained PD. Expanded scale
records of the starred regions (one asterisk and two asterisks) of the V trace are shown in the lower panels.
The inset to the left upper panel displays the whole
cell current evoked in this cell by a voltage-clamp
ramp.
Since a previous study of the
voltage dependence of secretion in bovine chromaffin cells, assayed by C(31) , was limited to potentials positive
to -10 mV, we performed several experiments in the voltage-clamp
mode to confirm the presence of Ca
currents and
depolarization-induced secretion in the range of the plateau potential. Fig. 5(middle vertical panel) demonstrates that a 10-s
depolarization to -20 mV is sufficient to produce 22 ASs as well
as a 140 femtofarads increase in C
. The bracketed data panels, collected 20 s before and after the
latter, demonstrate that increasing the depolarization to +10 mV
nearly triples the number of ASs (to 60) and nearly quadruples the
increase in C
(to 570 femtofarads). In three such
experiments, the measured C
increase was roughly
four times that predicted from the product of (i) the number of
preceding ASs and (ii) the C
increment of
2.5
femtofarads/vesicle recently measured in bovine chromaffin cells (12) . This discrepancy suggests that amperometry, in this
configuration, records only a fraction of the total number of
exocytotic events per cell, which in this experiment is
¼,
or even less, if significant endocytosis occurred during the 10-s
depolarizing pulse (see also (15) ). The inset to this
figure, compiled using data from another voltage-clamp experiment in
which Ca
currents were measured directly, presents
peak Ca
current as a function of membrane potential (V
). It demonstrates that Ca
current is sizable at -20 mV, but is significantly larger
at +10 mV.
Figure 5:
Voltage dependence of the secretion
monitored by both amperometry and C measurements
in a voltage-clamped rat chromaffin cell (see text for general
description of the figure). The vertical tick marks in the
trace depicting the voltage pulse represents the computer generated
stimuli used to track C
at the holding potential
of -70 mV. In each panel the sloping dashed line in the C
trace spans the time during which no measurement
was made. The inset (top, middle) presents a curve of
peak Ca
current versus voltage (50-ms
pulses). This cell was permeabilized with a pipette in which the KCl
and K
SO
content of the standard internal
solution were replaced by CsCl and CsSO
, respectively, to
insure the blockade of outward K
currents. No
correction was made for liquid junction potential as this may be
complicated by the existence of a Donnan potential (<8 mV for KCl
internal solution) in the perforated patch
configuration(19) .
Fig. 6A provides direct evidence that pulse applications of CCh, which elicit cell depolarization and trains of APs, also evoke barrages of ASs (n = 12). As with the case of direct depolarization, an AS barrage usually begins following the generation of several APs and continues for several seconds following cessation of the train and membrane repolarization. (Note that the AP train evoked by CCh is similar to that evoked by prolonged current pulses in Fig. 1). The electrical responses to CCh, as well as the closely following secretory responses, usually decline with repeated application of CCh. However, these cells still are able to generate APs and secrete in response to direct depolarization, suggesting that while ACh receptor response desensitized, depolarization-secretion coupling remained intact (n = 12).
In some cells, following desensitization to small pulses of CCh, large pulses of CCh, which failed to depolarize, nevertheless later triggered barrages of ASs which continued for tens of seconds (see Fig. 6B) (n = 5). Taken together, these results present evidence that at least two modes of cholinergically evoked secretion co-exist in single cells: one that is AP related and the other that is not AP related. This is consistent with results of classical studies on whole perfused adrenal glands(32) , as well as more recent studies on populations of isolated chromaffin cells, suggesting multiple pathways for cholinergic activation of secretion.
(ii) A second prominent feature is latency of quantal release after an AP. The latency of an amperometric spike, as we define it, is not identical to the latency of a post-synaptic potential. Longer time for diffusion of catecholamines from the granule core to the electrode, as well as transient vesicle fusion prior to the spike event(15) , might displace and broaden our latency histogram compiled at low AP frequency (i.e.Fig. 3A). Hence we are not certain whether the timing of early release detected within 10-20 ms after a chromaffin cell AP, substantially differs from the timing of quantal release, comprising the synchronized post-synaptic potential, detected within 1-2 ms after a nerve terminal AP. However, close inspection of the timing of quantal release after an impulse suggests two significant differences between a chromaffin cell and a classical synapse. (a) At the synapses stimulated at 1 Hz for short periods, quantal release following the post-synaptic potential, is barely detectable (i.e. the frequency of asynchronous miniature post-synaptic potentials is hardly above background). In chromaffin cells stimulated at 1 Hz, only 50% of events contribute to an early peak at 10-20 ms, whereas the remainder contribute to a tail of quantal release continuing for hundreds of milliseconds. (b) At a classical synapse, such as the neuromuscular junction, increasing AP frequency to 5-10 Hz for several seconds produces a just detectable increase in both post-synaptic potential amplitude and the frequency of miniature post-synaptic potentials. In contrast, in chromaffin cells, raising the AP frequency to 4 Hz shifts the latency distribution so that the peak and tail are difficult to distinguish, with only 20% of events occurring within 20 ms of the impulse. Thus, compared with the synapse, impulse-evoked release from chromaffin cells seems to combine a relatively depressed component of early synchronous release, which at synapses contribute to the post-synaptic potential, with a greatly enhanced component of asynchronous release which, in synapses, contribute to the increased frequency of miniature post-synaptic potentials seen during prolonged repetitive stimulation. Both components may contribute to the sustained release seen during a plateau depolarization.
(iii) A third feature is that ACh appears to
induce secretion via multiple pathways. Our data now show that, in
single rat chromaffin cells, transient release of packets of CAs is
closely related, in time, to CCh-induced electrical activity. Release
begins during the course of an AP train, which rides on a plateau level
depolarization and ends within 1-3 s after cessation of the
train. However, more intense cholinergic stimulation sometimes triggers
release of a similar number of quanta, although over a more extended
duration, even after the depolarizing response has desensitized. Hence,
at least in some cases, agonist-induced release, from chromaffin cells,
in the absence of significant depolarization or APs, can be as potent
as AP-induced release. This is in direct contrast with the situation at
nerve terminals, where depolarization-induced secretion far outweighs
any direct agonist-induced release. It is widely appreciated that CCh
is a mixed nicotinic and muscarinic agonist and that both classes of
agonists induce CA release from perfused adrenals. It is likely that
the transient secretory response which is temporally related to
depolarization is due, in large part, to the activation of nicotinic
ACh receptor (nAChR) channels(10) , cell depolarization, and
Ca entry through voltage-activated Ca
channels(11) . It is also likely that more sustained CA
secretion, which occurs with minimal or no depolarization, after
desensitization of nicotinic receptors, is due to activation of
muscarinic receptors, second messenger cascades, and release of
Ca
from intracellular
stores(33, 34, 35, 36) .
Although separately
explicable in other ways, taken together, the several features of
AP-induced release which we have examined are consistent with the loose
co-localization scheme referred to above. Qualitatively, such a scheme
might contribute significantly to (i) the widely distributed latency of
occurrence of release at 1 and 4 Hz (Fig. 3), by resulting in
variable delay times due to Ca diffusion; (ii) the
frequency dependence of release in response to single APs (Fig. 2); and (iii) the slow buildup of secretion during a train
of action potentials and the delay in cessation of secretion thereafter (Fig. 1, B and C), as Ca
slowly builds up at and then diffuses from these sites. (
)If the distances between Ca
channels and
secretion sites were randomly distributed, secretion sites with similar
Ca
affinities located closer to channels would be
activated at shorter latencies and following less intense
Ca
entry than the more distantly located ones. Hence
short latency events, occurring within several milliseconds, would
constitute the major fraction of total quantal release during low
frequency AP activity (
1 Hz), whereas long latency events,
occurring over many tens of milliseconds, would make a more prominent
contribution at higher AP frequencies. Qualitatively, this is what we
have observed (see Fig. 3). Physiologically, even a limited
number of domains where secretion sites are close to Ca
channels might be useful in insuring some CA secretion at low
frequency stimulation. Last, extending this loose co-localization
argument, some secretion sites might also be near enough to
Ca
-storage organelles for Ca
released by the action of second messenger systems from these
structures to reach threshold levels for triggering exocytosis. Such a
scheme might account for agonist-evoked secretion in the absence of
electrical activity as we have seen with carbamylcholine application (Fig. 6). However, other factors which might contribute to our
observations include (i) frequency-dependent enhancement of
Ca
entry evoked during the action potential, as seen
in pituitary nerve terminals (42) and (ii)
Ca
-dependent recruitment of vesicles into a
``pre-docked,'' ready-to-fuse pool(43, 44) .
Recently two groups have provided evidence in bovine chromaffin
cells for both ``hot spots'' of Ca channels(45) , using pulse laser fluorescent imaging, and
hot spots of quantal release of CA(46) , using ultrafine
amperometry. It is not known how coincident these two types of hot
spots actually are. However, it is interesting to consider that data
supporting hot spots and those supporting loose co-localization might
be encompassed by an arrangement where individual clusters of secretion
sites are concentric with clusters of Ca
channels but
extend outward over a larger surface area, hence making the average
distance between Ca
channels and secretion sites in
chromaffin cells larger than in the ``active zones'' of
exocytosis of some well studied nerve terminals.
Note Added in Proof-Since the submission of this paper, membrane capacitance measurements have been published suggesting the presence of a small pool of vesicles releasable within ms after triggering a brief action potential-like depolarization (Horrigan, F. T., and Bookman, R. J.(1994) Neuron13, 1119-1129).