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Bjoern Ch. Ludwar, Colin G. Evans, Jian Jing and Elizabeth C. Cropper
J Neurophysiol 102:1976-1983, 2009. First published Jul 15, 2009; doi:10.1152/jn.00418.2009
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J Neurophysiol 102: 1976 –1983, 2009.
First published July 15, 2009; doi:10.1152/jn.00418.2009.
Two Distinct Mechanisms Mediate Potentiating Effects of Depolarization on
Synaptic Transmission
Bjoern Ch. Ludwar,1 Colin G. Evans,1,2 Jian Jing,1 and Elizabeth C. Cropper1
1
Department of Neuroscience, Mt. Sinai School of Medicine; and 2Phase Five Communications, New York, New York
Submitted 14 May 2009; accepted in final form 10 July 2009
INTRODUCTION
The selection of sensory inputs is an essential function
within the CNS. It is, therefore, not surprising that a variety of
regulatory and gating mechanisms have been described, operating at multiple loci and by multiple means (e.g., Büschges
and El Manira 1998; Rossignol et al. 2006; Rudomin and
Schmidt 1999). We study the regulation of sensorimotor transmission in an experimentally advantageous model system, the
mollusk Aplysia californica. More specifically, these experiments
focus on synaptic transmission between a feeding mechanoafferent, B21 (Rosen et al. 2000b), and motor neurons that receive
chemical excitatory input, the B8 neurons (Rosen et al. 2000b).
In previous work, we characterized a form of gating that is
membrane potential dependent. We demonstrated that active
spike propagation in B21 is dynamically regulated during
motor programs by centrally induced changes in membrane
Address for reprint requests and other correspondence: B. Ch. Ludwar,
Dept. Neuroscience Box 1065, One Gustave L. Levy Place, New York, NY
10029 (E-mail: [email protected]).
1976
potential (Evans et al. 2003). Specifically, B21 is a bipolar
neuron with a centrally located soma and two major processes
referred to as medial and lateral (Fig. 1A). The medial process
innervates the periphery, whereas the lateral process is the
primary point of contact with the B8 neurons (Rosen et al.
2000b). If peripherally generated spikes are to propagate from
the medial process to the lateral process, impulses must be
conducted through the relatively inexcitable somatic region
(Evans et al. 2007). This does not occur when B21 is at its resting
membrane potential. However, it does occur when B21 is centrally depolarized (Evans et al. 2003). Regulation of spike propagation is not unique to B21, i.e., it has been reported in several
other systems (e.g., Monsivais et al. 2005; Verdier et al. 2003).
In the current study, we characterize a second potentiating
effect of central depolarization on afferent transmission to B8.
We demonstrate that changes in membrane potential produce
graded increases in the background (i.e., resting) calcium
concentration in B21. These types of increases have been
described in other systems and generally potentiate spikemediated synaptic transmission (e.g., Awatramani et al. 2005;
Connor et al. 1986; Ivanov and Calabrese 2003; Kemenes et al.
2006). The B21–B8 synapse is, however, unusual in that
background increases in the intracellular calcium concentration
are virtually essential for synaptic transmission. When they are
pharmacologically blocked, and B21 is depolarized so that
spikes propagate, postsynaptic potentials (PSPs) in the B8
neurons are not recorded. In other systems, synaptic transmission generally occurs when spikes are evoked relatively close
to resting membrane potential. Taken together, previous data
and these results indicate that our preparation is unique in that
central depolarization exerts two effects that are both essential
for synaptic transmission. First it ensures that spikes actively
propagate to the lateral process. Second, it ensures that propagating spikes effectively induce transmitter release.
What is the unique functional significance of the two mechanisms that regulate B21 mechanoafferent transmission? To
approach this issue, we sought to determine whether they will
necessarily be simultaneously invoked, i.e., whether they are
induced in the same voltage range. We show that this is not the
case. Depolarizations of only a few millivolts alter spike
propagation, whereas central depolarizations that exceed ⬃10
mV are necessary to increase calcium concentrations and
induce chemical synaptic transmission. Although both effects
necessarily go hand in hand when afferent transmission to B8
is regulated, this is not likely to be the case for some of the
other neurons that receive afferent input from B21, i.e., cells
that are electrically coupled to B21 (Borovikov et al. 2000;
Rosen et al. 2000a,b). B21-induced coupling potentials are
apparent in these neurons at membrane potentials where chem-
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Ludwar BC, Evans CG, Jing J, Cropper EC. Two Distinct Mechanisms Mediate Potentiating Effects of Depolarization on Synaptic
Transmission. J Neurophysiol 102: 1976 –1983, 2009. First published
July 15, 2009; doi:10.1152/jn.00418.2009. Two distinct mechanisms
mediate potentiating effects of depolarization on synaptic transmission. Recently there has been renewed interest in a type of plasticity
in which a neuron’s somatic membrane potential influences synaptic
transmission. We study mechanisms that mediate this type of control
at a synapse between a mechanoafferent, B21, and B8, a motor neuron
that receives chemical synaptic input. Previously we demonstrated
that the somatic membrane potential determines spike propagation
within B21. Namely, B21 must be centrally depolarized if spikes are
to propagate to an output process. We now demonstrate that this will
occur with central depolarizations that are only a few millivolts.
Depolarizations of this magnitude are not, however, sufficient to
induce synaptic transmission to B8. B21-induced postsynaptic potentials (PSPs) are only observed if B21 is centrally depolarized by ⱖ10
mV. Larger depolarizations have a second impact on B21. They induce
graded changes in the baseline intracellular calcium concentration that are
virtually essential for the induction of chemical synaptic transmission.
During motor programs, subthreshold depolarizations that increase calcium concentrations are observed during one of the two antagonistic
phases of rhythmic activity. Chemical synaptic transmission from B21 to
B8 is, therefore, likely to occur in a phase-dependent manner. Other
neurons that receive mechanoafferent input are electrically coupled to
B21. Differential control of spike propagation and chemical synaptic
transmission may, therefore, represent a mechanism that permits selective
control of afferent transmission to different types of neurons contacted by
B21. Afferent transmission to neurons receiving chemical synaptic input
will be phase specific, whereas transmission to electrically coupled
followers will be phase independent.
EFFECTS OF DEPOLARIZATION ON SYNAPTIC TRANSMISSION
tanks at 14 –16°C for several days. Animals were anesthetized by
injection of ⬃100 ml isotonic MgCl2. Depending on the experiment,
the isolated buccal ganglion, the buccal and cerebral ganglia, or the
buccal ganglion with attached radula nerve and subradula tissue
(SRT) were removed from the animal, and pinned out in a silicone
elastomer (Sylgard, Dow Corning, Midland, MI)-lined dish. Ganglia
were desheathed to facilitate intracellular recordings. During most
experiments, the preparation was continuously superfused with artificial seawater (in mM: 460 NaCl, 10 KCl, 11 CaCl2, 55 MgCl2, 10
HEPES, pH ⫽ 7.6) at 14 –16°C. In one set of experiments, the
preparation was superfused with high-Ca2⫹/low-Mg2⫹ artificial seawater (in mM: 495 NaCl, 10 KCl, 33 CaCl2, 10 MgCl2, 10 HEPES,
pH ⫽ 7.6) at 16°C. A capital “N” indicates the number of preparations, whereas a lowercase “n” indicates the number of observations.
In some experiments, we bath applied 10 ␮M nifedipine (No. 1075,
Tocris, Ellisville, MO). In other experiments, ethylene glycol tetraacetic acid, E-4378 (EGTA , Sigma Chemical, St. Louis, MO) was
intracellularly injected into the somatic region of B21 via pipettes
filled with a 250 mM solution.
A
medial
B
MN
B8
lateral
B21
B8
B21
C
1977
1.0
Electrophysiology
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0.4
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propagation
normalized
1.0
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0
25
50
75
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FIG. 1. A: schematic of the preparation. Mechanical stimulation of the
subradula tissue evokes spikes, which travel along the medial process, across
the central soma, and into the lateral branch where the main contacts between
B21 and follower motor neuron B8 are located. B: effects of depolarization on
B21–B8 transmission. Simultaneous recordings from B21’s lateral process and
B8 during peripheral activation of B21. The dashed line under the lateral
process trace indicates the resting membrane potential. The 1st 2 responses
were evoked at resting membrane potential. Thereafter B21 was progressively
depolarized by current injection into the soma (trace not shown). In each case,
2 responses are shown at each membrane potential. When B21 was peripherally activated at resting potential, spikes did not actively propagate to the
lateral process, and PSPs in B8 were virtually nonexistent. As B21 was
depolarized, the spike propagation failure was relieved, but initially PSPs in B8
were virtually undetectable (arrows). With further depolarization, PSP amplitude progressively increased. C: summary of data for the experiment shown in
B from 4 animals, each plotted with a different symbol. y axis data have been
normalized to maximum spike height or peak PSP amplitude. X axis data have
been normalized to the amount of depolarization that was needed to evoke
spikes centrally. The shaded region indicates the approximate point at which
spike propagation to the lateral process begins. Note that the B8 PSPs are still
virtually undetectable at this point. Also note the graded, progressive increase
in PSP amplitude as the holding potential was progressively increased.
ical synaptic transmission to the B8 neurons is not induced. We
suggest that differential control of spike propagation and chemical synaptic transmission may represent a mechanism that
permits selective control of afferent transmission to different
types of neurons contacted by B21.
METHODS
Preparation
Experiments were conducted with Aplysia californica (200 – 400 g)
obtained from Marinus Scientific (Garden Grove, CA) and held in
J Neurophysiol • VOL
Electrophysiological recordings were obtained with sharp glass
electrodes filled with 3 M KAc and 30 mM KCl, or 3 M KCl (typical
impedance 7 M⍀). Signals were amplified with a SEC-10 LX amplifier (⫻10 headstage, npi electronic GmbH, Tamm, Germany) in
discontinuous current clamp or bridge mode or an AxoClamp 2B
amplifier (Molecular Devices, Sunnyvale, CA) in bridge mode. Signals were then filtered (LP 1 kHz) with a Model 410 instrumentation
amplifier (Brownlee Precision, San Jose, CA) and sampled at 3 kHz
with a Power 1401 A/D converter (CED, Cambridge, UK) using Spike
II software (version 6.01– 6.09, CED). In the experiments to determine B21 depolarization during motor activity and experiments with
recordings from B21’s lateral process, signals were amplified with an
AxoClamp 2B or Getting 5A amplifier (Getting Instruments, Iowa
City, IA), filtered (LP 1 kHz) with a model AM502 differential
amplifier (Tektronix, Richardson, TX), and sampled with a Digidata
1322A (Molecular Devices) A/D converter using pClamp (version 9,
Molecular Devices) software. Data were analyzed with Spike II. Excel
(version 2002, Microsoft, Redmond, WA) was used for statistical tests
and Excel and CorelDraw (version 13, Corel, Mountain View, CA) to
plot the data and prepare the figures.
Motor activity was triggered in the isolated nervous system by
either intracellular stimulation of the cerebral buccal interneuron 2
(CBI-2) or extracellular stimulation of the esophageal nerve (EN) of
the buccal ganglion. For extracellular stimulation, the nerve was cut,
drawn into a suction electrode, and stimulated using the stimulus input
of a model 1700 amplifier (A-M Systems, Carlsborg, WA), a Grass
SIU5 stimulus isolation unit, and a Grass S48 stimulator (both
Astro-Med, West Warwick, RI). Stimulus artifacts were graphically
removed in the figure shown.
Imaging
For imaging experiments, B21 was filled with Calcium Orange
C-3013 (Invitrogen, Carlsbad, CA) by electrophoretic injection. The
preparation was then transferred to a FNO1 fixed stage upright
microscope (Nikon Instruments, Melville, NY) equipped with Nikon
10X/0.30w Plan Fluor and 40X/0.80w NIR Apo objectives and ⫻0.5
camera adapter. The light from an X-Cite 120 PC metal halide lamp
(EXFO, Mississauga, Ontario, Canada) was filtered using an ET-CY3
filter set (Chroma Technology, Rockingham, VT). Fluorescence was
detected with a CoolSNAP HQ2 CCD camera (Photometrics, Tucson,
AZ). The image-acquisition rate depended on the resolution of the
image, with 30 image/s being typical for a 200 ⫻ 900 pixel area. The
fluorescence of the Ca2⫹-sensitive dye was measured in defined
regions of the image using NIS Elements AR (versions 2.10 and 2.30,
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0.8
1978
B. C. LUDWAR, C. G. EVANS, J. JING, AND E. C. CROPPER
Nikon Instruments), normalized, background subtracted, and exported
to Spike II using custom-written software that synchronized electrophysiological and optical data. The relative change in fluorescence
was calculated as dF/F0 ⫽ (F ⫺ F0)/F0 where F0 denotes the
background subtracted prestimulus fluorescence level (Yuste and
Konnerth 2005). Tests of the sensitivity and dynamic range of the
Ca2⫹ imaging setup were done with calcium calibration buffer kit 2
(C-3009, Invitrogen).
Mechanical stimulation
Some experiments required mechanical stimulation of the SRT to
evoke action potentials in the sensory neuron B21. To accomplish
this, a nonconductive tip was attached to a small loudspeaker which
was driven by a Grass SIU 5 stimulus isolation unit attached to a
Grass S48 stimulator (Astro-Med, West Warwick, RI). The tip was
positioned to lightly touch the SRT, which resulted in individual
action potentials being triggered in B21 by a 3-ms square pulse to the
loudspeaker (see also Cropper et al. 1996).
Effect of B21 depolarization on spike propagation and
synaptic transmission
When B21 is peripherally activated at its resting potential, PSPs
are not induced in the B8 neurons. In contrast, when B21 is
centrally depolarized and then peripherally activated, PSPs are
observed (Rosen et al. 2000a). The effect of membrane potential
is graded, i.e., as the holding potential is progressively increased,
PSP amplitude progressively increases (Evans et al. 2003) (also
see Fig. 1, B and C, bottom). Previously we characterized one
effect of depolarization on afferent transmission (Evans et al.
2003, 2007). We demonstrated that somatic depolarization modifies spike propagation in B21’s processes. In the current study,
we sought to determine whether other phenomena are additionally
invoked. In particular, we sought to determine whether we could
identify a mechanism that could account for the graded nature of
the effect of membrane potential.
To determine whether central depolarization impacts synaptic transmission beyond the point where spike propagation is
modified, we simultaneously monitored both spike propagation
in B21 and synaptic transmission to B8. These experiments
A1
A2
Do subthreshold changes in membrane potential alter
intracellular calcium concentrations in B21?
A number of well-characterized mechanisms for the graded
presynaptic regulation of transmitter release involve alterations
in calcium currents. For example, relatively steady-state, lowthreshold calcium currents can be induced and can modify the
strength of spike-induced synaptic transmission (e.g., Connor
et al. 1986; Ivanov and Calabrese 2003). To determine whether
subthreshold depolarizations alter intracellular calcium concentrations in B21, we electrophoretically injected it with
Calcium Orange, a Ca2⫹ indicator dye (Fig. 2A1, top). We then
centrally depolarized it via somatic injection of current pulses
(Fig. 2A1, bottom 2 traces). Depolarization resulted in a graded
increase in fluorescence (dF/F0) in the primary lateral process
(Fig. 2A, 1, top trace, and 2). For example, when the soma was
depolarized by 20 mV, we observed an average dF/F0 increase
of 1.3 ⫾ 0.3% (N ⫽ 5) in the lateral process. When the soma
was depolarized by 30 mV, we observed a 2.7 ⫾ 0.8% increase
6
dF/F0 [%]
5
100 µm
dF
4%
Vm
20 mV
Iinj
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3
2
1
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2 nA
0
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40
50
60
10 s
B21 soma depolarization [mV]
B1
B2
C
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dF
B
C
0.5%
A
A
20mV
Vm
100 µm
2 nA
Iinj
10 s
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FIG. 2. Subthreshold depolarizations increase the intracellular calcium concentration in B21. A1, photo: low-power (⫻10)
image of a neuron B21 after injection of the
Ca2⫹ indicator dye Calcium Orange. The
box on the lateral process marks where optical measurements were made in the recordings shown below. Traces: subthreshold current pulses of progressively increasing amplitudes were injected into the soma of B21
(Iinj). These pulses induced changes in membrane potential (Vm) and graded increases in
calcium fluorescence (dF). A2: group data
and fitted exponential functions from 5 experiments like the 1 shown in A1. Note the
graded increase in dF as pulse amplitude was
increased. B1: higher (⫻40)-power image of
a B21 neuron injected with Calcium Orange.
The boxes mark the regions imaged in B2.
Subthreshold current pulses were injected
into the soma of B21 (Iinj), which induced
changes in membrane potential (Vm) and
increases in calcium fluorescence (dF).
Downloaded from jn.physiology.org on September 2, 2009
RESULTS
were performed without the dye usually injected in B21 to
facilitate the impalement of the lateral process because dye
injection potentially impacts synaptic transmission (van Hooft
2002). As expected, when B21 was peripherally activated at its
resting membrane potential, spikes did not actively propagate
to the lateral process and PSPs were absent in B8 (Fig. 1, B and
C). To induce spike propagation to the lateral process, we
centrally depolarized B21 by injecting current into the soma
(trace not shown) and then peripherally activated it. Interestingly, PSPs in B8 were still virtually absent (Fig. 1, B, arrows,
and C, bottom). To verify the integrity of the B21–B8 synaptic
connection in these preparations, we increased the depolarization in B21. PSPs did in fact become apparent (Fig. 1, B and
C). We measured B8 PSP amplitude for different amounts of
depolarization in B21 and, as previously reported, potentiating
effects of membrane potential were graded (Fig. 1C, bottom)
(Evans et al. 2003). These results suggest that signal transmission in B21 is not solely determined by the control of spike
propagation. An additional mechanism is invoked at membrane
potentials that are more depolarized than those that alter
propagation to the lateral process.
EFFECTS OF DEPOLARIZATION ON SYNAPTIC TRANSMISSION
A
B21 soma
B21 lateral
B4/5
CBI-2
B
B21 soma
B21:
B4/5
B8
I2 nerve
bn2 nerve
EN nerve
C
ΔF
3%
Vm
FIG. 3. Changes in membrane potential and calcium fluorescence in B21
during motor programs. A: experiment conducted in the isolated nervous
system in which a cycle of a motor program was induced by stimulating the
command-like neuron CBI-2. Note that centrally induced depolarizations were
observed in both the soma and lateral process of B21. B: experiment conducted
in the isolated nervous system in which a cycle of a motor program was
induced by stimulating a gut afferent, the esophageal nerve (EN). Note that
B21 was centrally depolarized. C: 2 cycles of an EN-evoked motor program in
an experiment in which we monitored both the somatic membrane potential of
B21 and the change in calcium fluorescence in the lateral process. Note the
phasic increases in the 2 signals.
J Neurophysiol • VOL
cium fluorescence in the lateral process (due to problems
impaling CBI-2 while using our imaging setup). We were,
however, able to record from the lateral process and measure
the magnitude of the centrally generated depolarization (so that
it could be related to the depolarization that was used to induce
increases in calcium fluorescence in imaging experiments such
as the one shown in Fig. 2A1). We found that the mean lateral
process depolarization was 13.6 ⫾ 0.9 mV (N ⫽ 6, n ⫽ 12),
which was not significantly different from the mean depolarization in the soma of 15.7 ⫾ 1.1 mV (N ⫽ 9, n ⫽ 20, t-test
P ⬎ 0.05 dF: 30; Fig. 3A, 1 and 2).
To relate the change in membrane potential in the lateral
process observed during the motor program to the experimental
depolarizations in imaging experiments, we determined the
lateral process length constant. These experiments were necessary because the changes in membrane potential monitored
during imaging were somatic. The lateral process length constant was, therefore, needed to determine how a somatic
change in membrane potential would impact the lateral process. To measure the length constant, we used animals that
were similar in size to those used in the previously described
experiments and inserted recording electrodes in the soma and
lateral process of B21, as well as a third electrode in the soma
for current injection. We measured the distance between soma
and lateral process recording sites and calculated ␭ ⫽ ⫺d/ln
(Va/Vb), with d being the distance between recording sites, and
Va and Vb being the voltage changes resulting from current
injection in the soma and lateral process, respectively. We
found ␭ to be ⬃390 ⫾ 59 ␮m (N ⫽ 6, n ⫽ 14). This calculation
indicates that it is necessary to depolarize the soma by ⬃30 mV
to induce a 14 mV depolarization 300 ␮m into the lateral
process. This is roughly at the midpoint of the lateral process
because the total average length is 622 ⫾ 20 ␮m (N ⫽ 4). As
noted in the preceding text, this change in somatic membrane
potential produces a significant increase in fluorescence (see
Fig. 2B).
In the second type of motor program experiment, activity
was induced via stimulation of the esophageal nerve (EN) (Fig.
3, B and C). These types of motor programs are primarily
egestive (Proekt et al. 2004) but are advantageous in that we
were able to directly image changes in fluorescence in the
lateral process. We compared somatic depolarizations during
the two types of motor programs, and found that they were
similar. During cycles of EN-evoked activity, they were on
average 12.5 ⫾ 2.6 mV (N ⫽ 6), which is not significantly
different from the 15.7 mV observed during cycles of CBI-2evoked programs (t-test P ⬎ 0.05, dF: 42). In calcium imaging
experiments, we did, in fact, observe changes in fluorescence in
the lateral process that corresponded to changes in the somatic
membrane potential of B21 (Fig. 3C). Taken together these data
suggest that motor program induced alterations in membrane
potential are sufficient to induce changes in the intracellular
calcium concentration of the lateral process of B21.
Is the effect of B21 depolarization on PSP size a result of an
increase in intracellular calcium?
The determine whether B21–B8 synaptic transmission is
impacted by changes in baseline calcium concentrations, we
took advantage of previous work that has demonstrated that
these types of increases can be blocked in Aplysia by nifedipine
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in dF/F0 (N ⫽ 5; Fig. 2A2). To determine whether changes in
calcium fluorescence are also observed in secondary and tertiary branches of the lateral process, we performed a second set
of experiments in which we imaged B21 using a ⫻40 objective
(Fig. 2B1). Again we observed increases in dF/F0 when subthreshold current pulses were injected into the soma of B21
(Fig. 2B2; N ⫽ 4).
During feeding motor programs B21 is depolarized within
the CNS via electrical coupling with pattern-generating interneurons (Rosen et al. 2000a,b). To determine whether these
depolarizations are likely to be sufficient for inducing changes
in calcium fluorescence, we performed two types of experiments. In one type of experiment, we induced rhythmic activity
by stimulating cerebral buccal interneuron 2 (CBI-2; Fig. 3A)
to obtain primarily ingestive motor programs (Jing and Weiss
2001). This type of feeding motor program has been most
extensively utilized for studies of the regulation of B21 mechanoafferent transmission. During CBI-2-evoked activity, we
were not technically able to directly monitor changes in cal-
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B. C. LUDWAR, C. G. EVANS, J. JING, AND E. C. CROPPER
(Edmonds et al. 1990). Importantly, effects of nifedipine have
been shown to be selective, i.e., calcium currents that contribute to synaptic plasticity are modified, whereas calcium currents that contribute to exocytosis are not (Edmonds et al.
1990). To verify its effectiveness in our preparation, we bath
applied 10 ␮M nifedipine and imaged Calcium Orange signals
induced by somatic depolarization of B21 (Fig. 4A1). In the
presence of nifedipine, somatic depolarizations produced fluorescence increases that were on average only 12.3 ⫾ 3.9% of
control values (N ⫽ 5, t-test: P ⬍ 0.001; Fig. 4A2).
To study effects of nifedipine on synaptic transmission, we
peripherally activated B21 (to induce spiking) and centrally
depolarized it so that synaptic transmission was maximally
potentiated (Fig. 4B1, left). We then bath applied 10 ␮M
nifedipine (Fig. 4B1, right). The nifedipine significantly reduced B21–B8 PSP peak amplitude (N ⫽ 4, t-test: P ⬍ 0.001;
Fig. 4B3). PSPs became very small and almost indistinguishable from noise (as they are when spikes initially propagate and
synaptic transmission has not been potentiated by further
depolarization; e.g., Fig. 4B, 1, right, vs. 1B).
Because PSPs are so small in the presence of nifedipine, we
performed two additional manipulations to verify the selectivity of nifedipine in our preparation, i.e., to determine whether
the concentration of nifedipine used blocked the relatively
high-voltage-activated calcium channels that contribute to exocytosis (as well as the relatively low-voltage channels that we
hypothesize contribute to plasticity). In experiments such as
the one shown in Fig. 4B1 we additionally monitored synaptic
A1
A2
***
0
100
80
60
40
SE
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0
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control
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n=192
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B21
SE
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FIG. 4. Inhibitory effects of nifedipine on calcium fluorescence in B21, and on B21–B8 synaptic transmission. A1: changes in calcium fluorescence induced
by subthreshold depolarizing pulses were significantly reduced by nifedipine (10 ␮M, bath applied). A2: group data from 5 animals for the type of experiment
shown in A1. The data are normalized to the maximum response. B1: B21 was peripherally activated and centrally depolarized so that PSPs were induced in B8
(left). Nifedipine (10 ␮M, bath applied) significantly reduced PSP amplitude (right). B2: in the same preparation, B8 PSPs induced by stimulating a B4/5 neuron
were unaffected by nifedipine. B3: group data from 4 animals for the type of experiment shown in B1. C1: the experiment shown in B1 was repeated in
high-calcium/low-magnesium saline. Under these conditions, B8 PSPs were detectable without central depolarization of B21. This did not change when 10 ␮M
nifedipine was bath applied (top). In the same preparation, central depolarization increased PSP amplitude in the absence of nifedipine but not in its presence
(bottom). C2: averaged data from 4 experiments as shown in C1. Dot symbols plot B8 PSP size under control conditions; dash symbols plot PSP size after 30-min
bath application of 10 ␮M nifedipine. Note that nifedipine only affects B8 PSP size when B21 is depolarized.
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dF/F0
N=5
EFFECTS OF DEPOLARIZATION ON SYNAPTIC TRANSMISSION
DISCUSSION
Previously we demonstrated that spike propagation in neuron B21 is regulated so that spikes propagate to the lateral
process (an output region) when B21 is centrally depolarized
but do not propagate when B21 is close to its resting membrane
potential (Evans et al. 2003). In neurons where spike propagation is regulated, one might expect an all or none aspect to
A
control
B8
B21
B
2.5
***
N=4
n=212
2.0
1.5
1.0
SE
0.5
0.0
control
EGTA
FIG. 5. Effects of EGTA on B21-B8 synaptic transmission. A: B21 was
peripherally activated and centrally depolarized so that a PSP was evoked in
B8 (control). Ten minutes after injection of EGTA into B21 via a glass
capillary filled with 250 mM EGTA, the amplitude of the B8 PSP size was
dramatically reduced. B: group data for the type of experiment shown in A.
Note that EGTA produced a significant decrease in PSP amplitude.
J Neurophysiol • VOL
sensorimotor control (Segev and Schneidman 1999). This is,
however, not what we found in previous experiments that
studied synaptic transmission from B21 to its postsynaptic
follower, B8. Instead we found effects of membrane potential
to be graded (Evans et al. 2003). This study sought to determine how the graded regulation is mediated. We demonstrate
that subthreshold changes in membrane potential produce
changes in the intracellular-free calcium concentration in B21.
Our results suggest that these increases are virtually essential
for synaptic transmission. For example, nifedipine effectively
blocks both increases in calcium fluorescence induced by
subthreshold depolarizations, and B21 induced PSPs in B8.
Additionally, B21–B8 synaptic transmission is depressed when
the calcium chelator EGTA is injected into B21. Increases in
the intracellular calcium are a graded function of membrane
potential, and they are likely to account (at least in part) for the
graded effect of holding potential on B21 to B8 synaptic
transmission.
Graded effects of membrane potential on synaptic
transmission are observed in other systems
Graded effects of membrane potential on synaptic transmission have been described in a number of preparations (Marder
2006). In the retina and some invertebrate neurons, the threshold for neurotransmitter release is very close to resting potential, and release occurs in the absence of action potentials
(Heidelberger 2007). At the B21–B8 synapse, there is no
evidence for this form of graded transmission. Depolarizations
are not observed in B8 if spikes are not evoked in B21. Thus
changes in membrane potential do not induce significant
synchronous transmitter release in B21. Instead they modify
spike propagation and spike-mediated synaptic transmission.
Although graded effects of membrane potential on spikemediated synaptic transmission were originally described ⬎30
years ago (e.g., Graubard 1978; Nicholls and Wallace 1978;
Shimara and Tauc 1975; Tauc 1962; Waziri 1977), interest in
this phenomenon has recently been renewed. In part this is due
to the fact that although the phenomenon was originally identified in invertebrates, it has become apparent that it is also
likely to play a significant role in synaptic transmission in
vertebrates, e.g., in local cortical networks (Alle and Geiger
2006; Clark and Hausser 2006; Marder 2006). Most of the
synapses in the mammalian cortex are formed by nonmyelinated axons that are likely to be a millimeter or less from
somatic regions (Douglas et al. 1995). Recent measurements of
axonal length constants have indicated that they can be surprisingly long, and that somatic depolarizations can impact
spike-mediated synaptic transmission in hippocampal mossy
fibers and neocortical pyramidal axons (Alle and Geiger 2006;
Shu et al. 2006).
Additionally, recent experiments in invertebrates have identified a new function for plasticity that is based on persistent
changes of membrane potential. Studies of single-trial classical
conditioning have demonstrated that persistent subthreshold
depolarizations may be important for encoding a type of
long-term memory (Frost 2006; Fusi 2008; Kemenes et al.
2006).
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transmission at another synapse (Fig. 4B2). Nifedipine did not
significantly effect synaptic transmission. Second, we conducted experiments in a modified saline solution. We increased
the calcium concentration (to 33 mM, which is 3X) and
decreased the magnesium concentration (to 10 mM), to increase PSP amplitude and excitability. In this type of saline, B8
PSPs are detectable when B21 is peripherally activated at its
resting membrane potential (as well as when it is centrally
depolarized; Fig. 4C1). B8 PSPs continued to be detectable in
the presence of 10 ␮M nifedipine (Fig. 4C, 1, top, and 2). As
expected, when B21 was depolarized, nifedipine significantly
decreased PSP amplitude (Fig. 4C, 1, bottom, and 2). These
data indicate that synaptic transmission can in fact occur in the
presence of nifedipine in our preparation.
In a final set of experiments, we reduced the intracellular
Ca2⫹ concentration in B21 by injecting EGTA (ethylene glycol
tetra acetic acid) (Deitmer et al. 1986). EGTA is a chelating
agent with a high affinity for Ca2⫹. While reducing the
background calcium level, it is thought to be too slow to
have a direct effect on the calcium currents mediating transmitter release (Haugland 2005). Again, B21 was peripherally activated to evoke spikes and centrally depolarized so
that PSPs were maximally potentiated. EGTA was then
intracellularly injected into B21’s soma. Approximately 10
minutes later, PSP amplitude was significantly decreased
(N ⫽ 4, t-test: P ⬍ 0.001; Fig. 5, A and B). Taken together
the nifedipine and EGTA data indicate that B21 to B8
synaptic transmission is impacted by increases in baseline
calcium concentrations.
1981
1982
B. C. LUDWAR, C. G. EVANS, J. JING, AND E. C. CROPPER
Mechanisms underlying graded effects of membrane
potential on synaptic transmission
of synaptic transmission under physiologically relevant conditions.
J Neurophysiol • VOL
Possible functional significance of two mechanisms for the
regulation of B21–B8 synaptic transmission
B21 differs from other cells that have been studied in that
depolarizations that modify intracellular calcium levels also
modify spike propagation. Thus there are two distinct mechanisms that regulate B21 mechanoafferent transmission. A possible functional consequence of this arrangement is that it
enables selective control of afferent input to different types of
follower neurons. This report focuses on one type of follower
motor neuron, the B8 cells, which receive chemical synaptic
input. Other neurons that receive mechanoafferent input are
electrically coupled to B21 (Rosen et al. 2000a,b). Our data
suggest that B21 mechanoafferent transmission to electrically
coupled follower neurons may occur under conditions where
afferent transmission to chemical follower neurons does not.
We show that when B21 is peripherally activated and centrally
depolarized by only a few millivolts, spikes can propagate to
the lateral process. Under these conditions, coupling potentials
will be generated in electrically coupled “follower” neurons.
We also show that relatively small depolarizations that modify
spike propagation do not necessarily induce chemical synaptic
transmission to the B8 neurons. PSPs are only induced in the
B8 cells under conditions where there is a larger central
depolarization. Our data, therefore, suggest that B21 may
provide afferent input to electrically coupled motor neurons
under conditions where it does not induce PSPs in the B8
neurons.
Under physiological conditions there are two potential
sources of central depolarization in B21. During motor programs, B21 is depolarized via phasic input it receives from
feeding interneurons (Rosen et al. 2000a,b). In this report, we
demonstrate that these depolarizations are sufficient to both
modify spike propagation and induce chemical synaptic transmission. Consequently, postsynaptic follower neurons (the B8
neurons) will presumably receive afferent input during the
radula retraction phase of motor programs. Second, B21 is
centrally depolarized in the absence of rhythmic activity when
peripherally applied stimuli generate bursts of spikes in B21,
and coactivate other neurons of the radula mechanoafferent
cluster (e.g., Miller et al. 1994). Baseline changes in membrane
potential are relatively small under these conditions but are
within the range where we demonstrate spike propagation in
this study (i.e., ⬃3 mV from resting potential). Interestingly,
B21 is activated in this manner during the radula protraction
phase of motor programs (Borovikov et al. 2000). During
protraction, B21 is, therefore, likely to provide afferent input to
electrically coupled followers despite the fact that chemical
synaptic transmission to B8 does not occur.
ACKNOWLEDGMENTS
We thank F. S. Vilim for expert advice on technical aspects of the
experiments and K. R. Weiss for valuable comments on an earlier version of
this manuscript.
GRANTS
This work was supported by National Institute of Mental Health Grant
MH-51393. Some of the Aplysia used in this study were provided by the
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In some systems where changes in membrane potential
potentiate synaptic transmission, mechanisms are largely calcium independent, i.e., subthreshold presynaptic depolarizations have no effect on resting or evoked calcium signaling
(e.g., Scott et al. 2008). Other systems are similar to the
B21–B8 synapse in that elevations in background (or resting)
calcium levels are clearly important (Awatramani et al. 2005;
Connor et al. 1986; Ivanov and Calabrese 2003; Kemenes et al.
2006). In systems where calcium-dependent effects have been
demonstrated, subthreshold depolarizations open relatively low-voltage-activated calcium channels (Awatramani et al. 2005;
Ivanov and Calabrese 2003). These channels are presumably
distinct from the channels that directly trigger exocytosis. It
has been hypothesized that background calcium interacts with
a high-affinity sensor that is distinct from the exocytosis
calcium sensor (Ivanov and Calabrese 2003, 2006a,b). The net
result is a potentiation of synaptic transmission (Awatramani et
al. 2005; Ivanov and Calabrese 2003; Shapiro et al. 1980).
Our data support the idea that a similar mechanism operates
at the B21–B8 synapse in that nifedipine effectively reduced
potentiating effects of depolarization on synaptic transmission.
In Aplysia, nifedipine blocks a calcium current that does not
contribute to synchronous synaptic transmission that is evoked
at “resting” potential, i.e., without a baseline depolarization
(Edmonds et al. 1990). At resting potential, spike-induced
transmitter release is mediated by a second type of current that
is rapidly inactivating and dihydropyridine insensitive (Edmonds et al. 1990; Eliot et al. 1993). B21 differs from other
Aplysia cells in the extent to which the two types of currents
impact synaptic transmission. In other cells, when spikes are
triggered at resting potential, PSPs are observed in follower
neurons (Shapiro et al. 1980). In contrast, when B21 is depolarized to the point where spikes propagate but where there is
very little induction of the dihydropyridine-sensitive current,
PSPs are virtually absent in B8. While PSPs can be observed
under high extracellular calcium conditions, the probability of
release in B21 under normal conditions is likely to be low
relative to other cells that have been studied. Our imaging
technique did not allow us to directly detect the dihydropyridine insensitive current, which is likely to be spatially highly
restricted (Ivanov and Calabrese 2003).
Previous experiments that characterized the dihydropyridine-sensitive current in Aplysia were limited in that they were
conducted under conditions that did not permit an evaluation of
its functional significance. For example, the current was induced via stimuli that were not designed to mimic a physiological input. With a somatic voltage clamp, the current did not
become apparent until approximately ⫺30 mV (Edmonds et al.
1990) (a membrane potential that is relatively high for the
induction of a “background” current). Our data indicate that
when depolarization is induced in a more physiologically
relevant manner (i.e., via distributed synaptic input within the
CNS), increases in intracellular calcium concentrations are
observed a little over 10 mV from B21’s resting membrane
potential (which is generally approximately ⫺65 mV). Thus
our data indicate that the dihydropyridine-sensitive current in
Aplysia can play a significant role in determining the strength
EFFECTS OF DEPOLARIZATION ON SYNAPTIC TRANSMISSION
National Resource for Aplysia of the University of Miami under Grant
RR-10294 from the National Center for Research Resources.
REFERENCES
J Neurophysiol • VOL
Ivanov AI, Calabrese RL. Spike-mediated and graded inhibitory synaptic
transmission between leech interneurons: evidence for shared release sites.
J Neurophysiol 96: 235–251, 2006b.
Jing J, Weiss KR. Neural mechanisms of motor program switching in Aplysia.
J Neurosci 21: 7349 –7362, 2001.
Kemenes I, Straub VA, Nikitin ES, Staras K, O’Shea M, Kemenes G,
Benjamin PR. Role of delayed nonsynaptic neuronal plasticity in long-term
associative memory. Curr Biol 16: 1269 –1279, 2006.
Marder E. Neurobiology: extending influence. Nature 441: 702–703, 2006.
Miller MW, Rosen SC, Schissel SL, Cropper EC, Kupfermann I, Weiss
KR. A population of SCP-containing neurons in the buccal ganglion of
Aplysia are radula mechanoafferents and receive excitation of central origin.
J Neurosci 14: 7008 –7023, 1994.
Monsivais P, Clark BA, Roth A, Häusser M. Determinants of action
potential propagation in cerebellar Purkinje cell axons. J Neurosci 25:
464 – 472, 2005.
Nicholls J, Wallace BG. Modulation of transmission at an inhibitory synapse
in the central nervous system of the leech. J Physiol 241: 157–170, 1978.
Proekt A, Brezina V, Weiss KR. Dynamical basis of intentions and expectations in a simple neuronal network. Proc Natl Acad Sci USA 101:
9447–9452, 2004.
Rosen SC, Miller MW, Cropper EC, Kupfermann I. Outputs of radula
mechanoafferent neurons in Aplysia are modulated by motor neurons,
interneurons, and sensory neurons. J Neurophysiol 83: 1621–1636, 2000a.
Rosen SC, Miller MW, Evans CG, Cropper EC, Kupfermann I. Diverse
synaptic connections between peptidergic radula mechanoafferent neurons
and neurons in the feeding system of Aplysia. J Neurophysiol 83: 1605–
1620, 2000b.
Rossignol S, Dubuc R, Gossard JP. Dynamic sensorimotor interactions in
locomotion. Physiol Rev 86: 89 –154, 2006.
Rudomin P, Schmidt RF. Presynaptic inhibition in the vertebrate spinal chord
revisited. Exp Brain Res 129: 1–37, 1999.
Scott R, Ruiz A, Henneberger C, Kullmann DM, Rusakov DA. Analog
modulation of mossy fiber transmission is uncoupled from changes in
presynaptic Ca2⫹. J Neurosci 28: 7765–7773, 2008.
Segev I. Computer study of presynaptic inhibition controlling the spread of
action potentials into axonal terminals. J Neurophysiol 63: 987–998, 1990.
Segev I, Schneidman E. Axons as computing devices: basic insights gained
from models. J Physiol 93: 263–270, 1999.
Shapiro E, Castellucci VF, Kandel ER. Presynaptic membrane potential
affects transmitter release in an identified neuron in Aplysia by modulating
the Ca2⫹ and K⫹ currents. Proc Natl Acad Sci USA 77: 629 – 633, 1980.
Shimara T, Tauc L. Multiple interneuronal afferents to the giant cells in
Aplysia. J Physiol 247: 299 –319, 1975.
Shu Y, Hasenstaub A, Duque A, Yu Y, McCormick DA. Modulation of
intracortical synaptic potentials by presynaptic somatic membrane potential.
Nature 441: 761–765, 2006.
Tauc L. Site of origin and propagation of spike in the giant neuron of Aplysia.
J Gen Physiol 45: 1077–97, 1963.
Hooft JA van. Fast Green FCF (Food Green 3) inhibits synaptic activity in rat
hippocampal interneurons. Neurosci Lett 318: 163–165, 2002.
Verdier D, Lund JP, Kolta A. GABAergic control of action potential
propagation along axonal branches of mammalian sensory neurons. J Neurosci 23: 2002–2007, 2003.
Waziri R. Presynaptic electrical coupling in Aplysia: effects on postsynaptic
chemical transmission. Science 195: 790 –792, 1977.
Yuste R, Konnerth A. Imaging in Neuroscience and Development. Cold
Spring Harbor, NY: Cold Spring Harbor Press, 2005.
102 • SEPTEMBER 2009 •
www.jn.org
Downloaded from jn.physiology.org on September 2, 2009
Alle H, Geiger JRP. Combined analog and action potential coding in hippocampal mossy fibers. Science 311: 1290 –1293, 2006.
Awatramani GB, Price GD, Trussell LO. Modulation of transmitter release
by presynaptic resting potential and background calcium levels. Neuron 48:
109 –121, 2005.
Borovikov D, Evans CG, Jing J, Rosen SC, Cropper EC. A proprioceptive
role for an exteroceptive mechanoafferent neuron in Aplysia. J Neurosci 20:
1990 –2002, 2000.
Büschges A, El Manira A. Sensory pathways and their modulation in the
control of locomotion. Curr Opin Neurobiol 8: 733–739, 1998.
Clark B, Häusser M. Neural coding: hybrid analog and digital signalling in
axons. Curr Biol 16: R585–588, 2006.
Connor JA, Kretz R, Shapiro E. Calcium levels measured in a presynaptic
neurone of Aplysia under conditions that modulate transmitter release.
J Physiol 375: 625– 642, 1986.
Cropper EC, Evans CG, Rosen SC. Multiple mechanisms for peripheral
activation of the peptide-containing radula mechanoafferent neurons B21
and B22 of Aplysia. J Neurophysiol 76: 1344 –1351, 1996.
Deitmer JW, Eckert R, Schlue WR. Changes in the intracellular free calcium
concentration of Aplysia and leech neurons measured with calcium-sensitive
microelectrodes. Can J Physiol Pharmacol 65: 934 –939, 1986.
Douglas RJ, Koch C, Mahowald M, Martin KAC, Suarez HH. Recurrent
excitation in neocortical circuits. Science 269: 981–985, 1995.
Edmonds B, Klein M, Dale N, Kandel ER. Contribution of two types of
calcium channels to synaptic transmission and plasticity. Science 250:
1142–1147, 1990.
Eliot LS, Kandel ER, Siegelbaum SA, Blumenfeld H. Imaging terminals of
Aplysia sensory neurons demonstrates role of enhanced Ca2⫹ influx in
presynaptic facilitation. Nature 361: 634 – 637, 1993.
Evans CG, Jing J, Rosen SC, Cropper EC. Regulation of spike initiation and
propagation in an Aplysia sensory neuron: gating-in via central depolarization. J Neurosci 23: 2920 –2931, 2003.
Evans CG, Kang T, Cropper EC. Selective proagation in the central
processes of an invertebrate neuron. J Neurophysiol 100: 2940 –2947, 2008.
Evans CG, Ludwar BCh, Cropper EC. Mechanoafferent neuron with an
inexcitable somatic region: consequences for the regulation of spike propagation and afferent transmission. J Neurophysiol 97: 3126 –3130, 2007.
Frost W. Memory traces: snails reveal a novel storage mechanism. Curr Biol
16: R640 – 641, 2006.
Fusi S. A quiescent working memory. Science 319: 1495–1496, 2008.
Graubard K. Synaptic transmission without action potentials: input-output
properties of a nonspiking presynaptic neuron. J Neurophysiol 41: 1014 –
1025, 1978.
Haugland RP. The Handbook (10th ed.). Carlsbad, CA: Invitrogen CA, 2005.
Heidelberger R. Mechanims of tonic, graded release: lessons from the
vertebrate photoreceptor. J Physiol 585: 663– 667, 2007.
Ivanov AI, Calabrese RL. Intracellular Ca2⫹ dynamics during spontaneous
and evoked activity of leech heart interneurons: low-threshold Ca currents
and graded synaptic transmission. J Neurosci 23: 1206 –1218, 2000.
Ivanov AI, Calabrese RL. Modulation of spike-mediated synaptic transmission by presynaptic background Ca2⫹ in leech heart interneurons. J Neurosci
23(4): 1206 –1218, 2003.
Ivanov AI, Calabrese RL. Graded inhibitory synaptic transmission between
leech interneurons: assesing the roles of two kinetically distinct lowthreshold Ca currents. J Neurophysiol 96: 218 –234, 2006a.
1983