J Neurophysiol 106: 1629 –1636, 2011.
First published July 13, 2011; doi:10.1152/jn.00403.2011.
Physiological evidence that D-aspartate activates a current distinct from
ionotropic glutamate receptor currents in Aplysia californica neurons
Stephen L. Carlson and Lynne A. Fieber
Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami,
Miami, Florida
Submitted 2 May 2011; accepted in final form 7 July 2011
invertebrate; cation channel; reversal potential; buccal ganglion; Nmethyl-D-aspartate
IN THE 1960S, D-aspartate (D-Asp)
was discovered to have physiological actions in neurons (Curtis and Watkins 1963; Davies
and Johnston 1976), but it was not until decades later with the
discovery of free D-amino acids in the tissues of several
organisms that the relevance of these compounds was understood (reviewed in D’Aniello 2007; Fuchs et al. 2005).
Whereas D-serine (D-Ser) has been the subject of much research
regarding its role in the nervous system, acting as an agonist at
the glycine binding site of NMDA receptors (NMDARs), there
have been comparatively few studies of the role of D-Asp.
D-Asp undergoes a transient increase in concentration in the
central nervous system (CNS) during embryonic development
in vertebrates, and then these levels rapidly decrease postnatally due to a rise in the concentration of D-aspartate oxidase
(Furuchi and Homma 2005). Although there has been considerable research on an endocrine role for D-Asp (D’Aniello
2007), there has been less exploration of a possible neurotransmitter or neuromodulatory role, despite its presence and the
machinery for its synthesis, release, uptake, and breakdown in
nervous tissue of a variety of organisms (D’Aniello 2007;
Homma 2007; Kim et al. 2010; Savage et al. 2001; Scanlan et
Address for reprint requests and other correspondence: L. A. Fieber, Division of Marine Biology and Fisheries, Rosenstiel School of Marine and
Atmospheric Science, Univ. of Miami, 4600 Rickenbacker Cswy., Miami, FL
33149 (e-mail: [email protected]).
www.jn.org
al. 2010; Waagepetersen et al. 2001; Wang et al. 2011; Wolosker et al. 2000; Yamada et al. 2006) and in neuronal processes
of Aplysia (Miao et al. 2005; Spinelli et al. 2006). Yet to be
demonstrated is the physiological and molecular description of
the ion channels activated by D-Asp.
Owing to their structural similarity, D-Asp and L-Glu both
purportedly activate ionotropic glutamate receptors (Huang et
al. 2005; Kiskin et al. 1990; Olverman et al. 1988). L-Gluactivated ion channels include subtypes activated by NMDA,
AMPA, and kainate. The majority of evidence gathered so far
would suggest that D-Asp is a ligand for NMDARs (Kiskin et
al. 1990; Olverman et al. 1988). Errico et al. (2008a, 2008b; in
press) found that D-Asp activated currents via NR2A–D receptor subunits and increased long-term potentiation through
NMDAR activity, yet it also activated currents independently
of NMDARs, suggesting a unique D-Asp receptor. Meanwhile,
Huang et al. (2005) found that D-Asp did not activate AMPA/
kainate or metabotropic glutamate receptors.
Miao et al. (2006) investigated the electrophysiological
responses to D-Asp in Aplysia neurons of the abdominal and
cerebral ganglia. They found that D-Asp induced depolarizations similarly to L-Glu; however, in some neurons D-Asp
responses had opposite polarity from L-Glu responses, similarly suggesting that D-Asp may have actions independent of
L-Glu and its target receptors.
The identified cells and cell clusters of Aplysia californica
provide a useful system for the study of D-Asp. Numerous
studies have confirmed the widespread presence of D-Asp in
each of the ganglia of the Aplysia CNS in multiple species such
as A. fasciata (D’Aniello et al. 1993), A. limacina (Spinelli et
al. 2006), and A. californica (Liu et al. 1998; Miao et al. 2005,
2006). L-Glu receptors are found throughout the Aplysia nervous system, including the well-studied NMDA- (Ha et al.
2006) and AMPA-type receptors that activate channels (Antzoulatos and Byrne 2004), as well as the invertebrate-specific
⫺
L- Glu-activated Cl channels (King and Carpenter 1989). In
addition, D-Asp current responses in Aplysia have been shown
to undergo changes associated with aging (Fieber et al. 2010)
and are subject to modulation by 5-HT similarly to AMPA
receptors (Carlson and Fieber, in press), suggesting this endogenous compound may play an important physiological role.
The goal of this study was to provide an electrophysiological
characterization of D-Asp-activated currents in S cluster neurons of the buccal ganglion (BSC), describing the electrophysiological properties of D-Asp current responses and comparisons to L-Glu-activated currents. BSC cells show a relatively
high preponderance of responses to D-Asp and are innervated
(Jahan-Parwar and Fredman 1976) by neurons believed to
0022-3077/11 Copyright © 2011 the American Physiological Society
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Carlson SL, Fieber LA. Physiological evidence that D-aspartate
activates a current distinct from ionotropic glutamate receptor currents
in Aplysia californica neurons. J Neurophysiol 106: 1629 –1636,
2011. First published July 13, 2011; doi:10.1152/jn.00403.2011.—DAspartate (D-Asp) activates an excitatory current in neurons of Aplysia
californica. Although D-Asp is presumed to activate a subset of
L-glutamate (L-Glu) channels, the identities of putative D-Asp receptors and channels are unclear. Whole cell voltage- and current-clamp
studies using primary cultures of Aplysia buccal S cluster (BSC)
neurons were executed to characterize D-Asp-activated ion channels.
Both D-Asp and L-Glu evoked currents with similar current-voltage
relationships, amplitudes, and relatively slow time courses of activation and inactivation when agonists were pressure applied. D-Aspinduced currents, however, were faster and desensitized longer, requiring 40 s to return to full amplitude. Of cells exposed to both
agonists, 25% had D-Asp- but not L-Glu-induced currents, suggesting
a receptor for D-Asp that was independent of L-Glu receptors. D-Asp
channels were permeable to Na⫹ and K⫹, but not Ca2⫹, and were
vulnerable to voltage-dependent Mg2⫹ block similarly to vertebrate
NMDA receptor (NMDAR) channels. D-Asp may activate both
NMDARs and non-L-Glu receptors in the nervous system of Aplysia.
1630
A DISTINCT EXCITATORY CURRENT FOR
contain D-Asp due to high D-Asp racemase immunoreactivity
(Scanlan et al. 2010; Wang et al. 2011).
MATERIALS AND METHODS
J Neurophysiol • VOL
IN APLYSIA
Dose-response data were expressed as a fraction of the current
amplitude in response to 10 mM D-Asp. Data were fit using the
equation I ⫽ Imax/[1 ⫹ (EC50/[agonist])nH], where nH is the Hill slope
coefficient (Verdoorn and Dingledine 1988). Curves and graphs for
desensitization and dose-response data were plotted using GraphPad
Prism (version 5.03; GraphPad Software, San Diego, CA).
Solutions. All reagents were obtained from Sigma-Aldrich (St.
Louis, MO). Extracellular solution (ECS) normally consisted of ASW
containing (in mM) 417 NaCl, 10 KCl, 10 CaCl2(2H2O), 55
MgCl2(6H2O), and 15 HEPES-NaOH, pH 7.6 (⬃physiological pH;
unpublished observations). Control intracellular solutions (ICS) contained (in mM) 458 KCl, 2.9 CaCl2(2H2O), 2.5 MgCl2(6H2O), 5
Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4. For ion substitution experiments, currents for determining the reversal potential were
first recorded for an individual cell for one condition (control or ion
substituted). The bathing solution was then switched for the alternate
solution, and a new current-voltage (I-V) relationship was determined
in that cell. For K⫹-replacement experiments, after currents in one
solution were recorded, the cell was patched again with a new pipette
containing the alternate intracellular solution, and 10 min were allowed for the new solution to dialyze the cell interior before current
measurements were made. For other ions, the intracellular solution
remained the same throughout the experiment.
In Na⫹-replacement experiments, ASW was replaced with a solution containing (in mM) 417 N-methyl-D-glucamine (NMDG), 10
KCl, 10 CaCl2(2H2O), 55 MgCl2(6H2O), and 15 HEPES-HCl, pH
7.6, whereas ICS was replaced with solution containing (in mM) 458
KCl, 2.9 CaCl2(2H2O), 2.5 MgCl2(6H2O), 5 K2ATP, 10 EGTA, and
40 HEPES-KOH, pH 7.4.
In K⫹-replacement experiments, ASW was replaced with solution
containing (in mM) 417 NaCl, 10 NMDG, 10 CaCl2(2H2O), 55
MgCl2(6H2O), and 15 HEPES-HCl, pH 7.6, whereas ICS was replaced with a solution containing (in mM) 458 NMDG, 2.9
CaCl2(2H2O), 2.5 MgCl2(6H2O), 5 Na2ATP, 10 EGTA, and 40
HEPES-HCl, pH 7.4.
For Ca2⫹-removal experiments, ASW was replaced with a solution
containing (in mM) 417 NaCl, 10 KCl, 55 MgCl2(6H2O), 10 NMDG,
1 EGTA, and 15 HEPES-NaOH, pH 7.6. For Ca2⫹-addition experiments, ASW was replaced with a solution containing (in mM) 417
NaCl, 10 KCl, 65 CaCl2(2H2O), and 15 HEPES-NaOH, pH 7.6.
For Mg2⫹-replacement experiments, ASW was replaced with a
solution containing (in mM) 417 NaCl, 10 KCl, 10 CaCl2(2H2O), 83
NMDG, and 15 HEPES-HCl, pH 7.6, whereas ICS was the same as
control.
For Cl⫺-replacement experiments, ASW was replaced with a
solution containing (in mM) 417 Na-gluconate, 10 K-gluconate, 10
CaCl2(2H2O), 55 MgCl2(6H2O), and 15 HEPES-NaOH, pH 7.6,
whereas ICS was the same as control. Data were corrected for a
⫺17-mV junction potential arising in this solution.
Data analysis. Significance of within-group comparisons was assessed using Student’s paired t-test, and the unpaired (2 sample) t-test
was used for between-group comparisons. Analyses were performed
using Data Desk software (version 6.2; Data Description, Ithaca,
NY). Differences at P ⬍ 0.05 were accepted as significant. Data
are means ⫾ SD.
RESULTS
Electrophysiological responses of Aplysia neurons to D-Asp.
currents were present in 54.1% of cultured BSC
neurons examined (n ⫽ 37), 28.6% of non-BSC buccal neurons
(n ⫽ 7), 18.8% of abdominal ganglion bag cell neurons (n ⫽
16), 40.0% of unidentified cerebral ganglion neurons (n ⫽ 25),
45.8% of pleural ventral caudal neurons (n ⫽ 59), and 31.3%
of unidentified neurons of the pleural ganglion (n ⫽ 16). In
some neurons, pressure application of D-Asp elicited an action
D-Asp-induced
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Cell culture. California sea hares, A. californica (⬃300 –500 g;
⬃6 – 8 mo of age and sexually immature with no evidence of mating
and no egg masses in the cages), were obtained from the University of
Miami National Institutes of Health National Resource for Aplysia
(Miami, FL). Primary cultures of BSC cells were prepared according
to the methods described by Fieber (2000). Animals were anesthetized
for 1 h in a 1:1 mixture of seawater from the culture facility and 0.366
M MgCl2. Ganglia were then dissected out, and each was placed in a
5-ml solution containing 18.75 mg of dispase (Boehringer Mannheim
10165859001), 5 mg of hyaluronidase (Sigma H4272), and 1.5 mg of
collagenase type XI (Sigma C9407) in artificial seawater (ASW) and
placed on a shaker set to low speed for ⬃24 h at room temperature
(⬃22°C). Cells from specific ganglia or areas of ganglia were then
dissociated onto 35-mm diameter polystyrene culture plates (Becton
Dickinson, Falcon Lakes, NJ) that were coated with poly-D-lysine
(MP Biomedicals IC15017525) at 0.2 mg/ml sterile water for 25 min
and then rinsed twice in sterile water, dried, and UV-sterilized before
use. Cells were stored at 17°C until used in experiments 24 h later.
Electrophysiology. Whole cell voltage-clamp and current-clamp
measurements were made with glass patch electrodes pulled from
thick-walled 1.5-mm- diameter borosilicate glass capillaries using a
Flaming/Brown micropipette puller (Sutter Instruments, Novato, CA).
Voltage and current data were collected, and whole cell capacitance
and series resistance compensations were made using an Axopatch
200B clamp amplifier with a capacitance compensation range of
1–1,000 pF, connected to a personal computer and Digidata 1200
analog-to-digital converter using pClamp software to record data and
issue voltage and current commands (Molecular Devices, Sunnyvale,
CA). Bath solutions were flowed onto cells during recording via a
6-bore gravity-fed perfusion system that dispensed solutions from
1-␮l micropipettes ⬃200 ␮m away from the cell. The environment
around the cell was adjusted to a new solution within 500 ms of
switching on the flow of the relevant gravity pipette. Solutions
containing agonist were briefly applied to the cell via a micropipette
attached to a picospritzer powered by N2 adjustable for pressure and
duration (Parker Hannifin, Cleveland, OH). The picospritzer pipette
tip was aimed at the cell and positioned at an angle of ⬃45° from the
perfusion flow but closer to the cell, ⬃ 30 ␮m from the cell body.
Unless otherwise noted, in all experiments D-Asp was applied via the
picospritzer for 100 ms at a concentration of 1 mM. The effective
concentration of agonist was estimated to reach the cell surface ⬍25
ms after the start of the 100-ms pulse of agonist. Dye-loading
experiments indicated that mixing of agonist with the gravity-fed bath
solution was minimal for the duration of the picospritzer pulse due to
the proximity of the picospritzer pipette to the cell and the relatively
high force of the picospritzer pulse compared with the bath solution.
To avoid desensitization, ⬃1–2 min were allowed between applications of D-Asp to individual cells.
To determine the time constants of activation and inactivation,
whole cell currents from five cells with comparable current amplitudes were fit using a double-exponential Simplex/SSE algorithm
(Axograph). Time constants for these fits were averaged (⫾SD) across
cells.
Desensitization experiments were performed using a two-pulse
protocol with the interpulse interval varied from 2 to 44 s. Current
amplitude of the second pulse was expressed as a fraction of the initial
current. The plot of the normalized average current amplitude as a
function of the interpulse duration was fitted using a single-exponential equation: It ⫽ [Imax ⫺ (Imax ⫺ I0)exp(⫺t/␶)], where It is the
current at time t, Imax is the current amplitude at infinite time, I0 is the
current at time 0 (directly after the first pulse), and ␶ is the time
constant.
D-Asp
A DISTINCT EXCITATORY CURRENT FOR
potential under current-clamp conditions (Fig. 1) that corresponded to an inward current near the resting potential under
voltage clamp (Fig. 1, inset), whereas D-Asp elicited only
subthreshold depolarizations in others (data not shown). Pres-
IN APLYSIA
1631
sure application of ASW was without effect on BSC neurons
(data not shown), indicating excitatory responses were due to
the presence of D-Asp.
Under voltage-clamp conditions, some BSC neurons responded to both D-Asp and L-Glu in alternate applications of
these agonists. Whole cell currents induced by D-Asp and
L-Glu were inward at negative voltages, e.g., ⫺60 and ⫺30
mV, and outward at depolarized voltages, e.g., 60 mV (Fig. 2,
A and B). D-Asp- and L-Glu-activated currents possessed a
similar I-V relationship. L-Glu-activated currents reversed at
⫺0.4 ⫾ 9.3 mV (n ⫽ 7), whereas D-Asp-activated currents
reversed at 7.7 ⫾ 9.0 mV (n ⫽ 37) in ASW medium and KCl
intracellular solution. There was no significant difference in
reversal potential between D-Asp- and L-Glu-activated currents; however, currents induced by D-Asp displayed a slight
outward rectification compared with those induced by L-Glu
(Fig. 2, A and B). In 71 cells in which both agonists were
tested, 18 (25.4%) responded to both L-Glu and D-Asp, whereas
35 (49.3%) responded only to L-Glu and 18 (25.4%) responded
only to D-Asp, which was significantly different from our
expectation (␹2 goodness of fit, P ⬍ 0.05). Currents activated
by L-Glu also displayed a slower time course of activation and
inactivation compared with those activated by D-Asp (Fig. 3, A
and B; Table 1). Once pressure-applied agonist reached the cell
membrane, evidenced by an inflection of the current baseline,
D-Asp currents reached peak activation significantly faster than
L-Glu currents (P ⬍ 0.05, 2-sample t-test). To determine
whether duration of agonist exposure affected maximum current or current decay, we compared spontaneous and steadystate current decay. Both spontaneous inactivation in response
to a brief, 5-ms pulse of agonist and steady-state inactivation in
response to 3-s pulses of agonist were also significantly faster
Fig. 2. BSC whole cell currents in response to
pressure application of D-Asp and L-Glu.
A: D-Asp-evoked currents in a single BSC cell
at different holding potentials (1 mM; 100 ms
as indicated by horizontal bar). Graph indicates average (⫾SD) current-voltage (I-V) relationship (n ⫽ 55). B: L-Glu-evoked currents
in a BSC cell at different holding potentials (1
mM; 100 ms as indicated by horizontal bar).
Graph indicates average (⫾SD) I-V relationship (n ⫽ 8).
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Fig. 1. Action potential in a buccal S cluster (BSC) neuron evoked by pressure
application of D-Asp (1 mM; 100 ms). Membrane potential (Vm) ⫽ ⫺35 mV.
Inset: D-Asp-activated whole cell current in the same BSC cell at Vm ⫽ ⫺60 mV.
D-Asp
1632
A DISTINCT EXCITATORY CURRENT FOR
D-Asp
IN APLYSIA
for D-Asp than for L-Glu (P ⬍ 0.05, 2-sample t-test), but
spontaneous and steady-state inactivation times for D-Asp were
not different, suggesting D-Asp currents rapidly desensitize.
Time constants for activation and inactivation reflected the
differences in the kinetics of D-Asp- and L-Glu-evoked currents. The fast and slow activation time constants, ␶F and ␶S, of
D-Asp currents were significantly shorter than those for L-Glu
(P ⬍ 0.05 for ␶F and P ⬍ 0.01 for ␶S, 2-sample t-test).
An important distinction between currents elicited by D-Asp
and those elicited by L-Glu, in addition to the greater vulnerability of D-Asp currents to desensitization, was their recovery
from desensitization. Whereas D-Asp-activated currents rapidly
desensitized during repeated applications of agonist, L-Gluactivated currents recovered from apparent desensitization
promptly and potentiated over the same time course that
produced desensitization in D-Asp currents (Fig. 3C). D-Asp
currents recovered from desensitization and returned to maximal amplitude at interpulse intervals of ⬃40 s (Fig. 4A).
Half-time potential (T1/2) for recovery was 13 s, whereas ␶ was
18.79 s. In contrast, T1/2 for recovery of full-amplitude L-Glu
currents was ⬍500 ms (data not shown).
Dose-response data were obtained using concentrations of
D-Asp between 3 ␮M and 10 mM (Fig. 4B). Currents were
half-maximal at a concentration of ⬃42 ␮M D-Asp. The Hill
slope coefficient for the fitted curve was 0.67. Current amplitude was maximal at ⱖ1 mM D-Asp.
Ion permeability of D-Asp-activated channels. Each of the
major ions in solution was systematically replaced with a
larger, theoretically impermeant ion to determine which ions
contributed to whole cell D-Asp-induced currents. Significant
shifts in the reversal potential under these conditions indicate
channel permeation for that ion. Figures 5 and 6 summarize
these results.
Na⫹ in the ECS was replaced with NMDG, which resulted
in a significant negative shift in reversal potential of 47 mV
(P ⬍ 0.01, Student’s paired t-test; n ⫽ 7), indicating that Na⫹
Table 1. Activation and inactivation times and time constants for pressure-applied D-Asp and L-Glu
␶F
␶S
Spontaneous
Inactivation
(5 ms agonist)
2.9 ⫾ 0.9* (5)
6.2 ⫾ 2.5 (5)
12.7 ⫾ 9.9* (5)
38.1 ⫾ 12.3 (5)
401 ⫾ 223* (16)
657 ⫾ 462 (15)
Activation (100 ms agonist)
Time, ms
D-Asp
L-Glu
67.9 ⫾ 28.8* (22)
121 ⫾ 74.7 (21)
Steady-State
Inactivation
(3 s agonist)
497 ⫾ 314* (12)
595 ⫾ 399 (6)
Inactivation (100 ms agonist)
␶f
7.5 ⫾ 4.2* (5)
21.8 ⫾ 11.6 (5)
Values are means ⫾ SD (no. of neurons is given in parentheses). *P ⬍ 0.05, D-Asp vs. L-Glu for the indicated measure.
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␶s
80.4 ⫾ 46.6* (5)
171 ⫾ 49.4 (5)
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Fig. 3. Activation and inactivation of D-Asp
and L-Glu whole cell currents and desensitization of D-Asp currents. A: example D-Asp
(left) and L-Glu (right) currents in response
to brief (5 ms) and sustained (3 s) agonist
application. B: example time courses of activation and inactivation for D-Asp- and LGlu-induced currents (1 mM, 100 ms). The
data were fit using a double-exponential
Simplex/SSE algorithm (Axograph). Insets:
fast (␶F; solid lines) and slow (␶S; dashed
lines) activation time constants of D-Aspand L-Glu-induced currents. D-Asp: activation, ␶F ⫽ 4.2 ms and ␶S ⫽ 12.4 ms; inactivation, ␶F ⫽ 8.8 ms and ␶S ⫽ 151.2 ms.
L-Glu: activation, ␶F ⫽ 6.63 ms and ␶S ⫽
40.5 ms; inactivation, ␶F ⫽ 15.0 ms and ␶S ⫽
175 ms. C: superimposed currents in the
same BSC cell elicited in response to 2
applications of D-Asp (left) and L-Glu
(right). Applications 1 and 2 are separated by
10 s.
A DISTINCT EXCITATORY CURRENT FOR
D-Asp
IN APLYSIA
1633
Fig. 4. Dependence of whole cell D-Asp current amplitude on frequency of agonist application and on concentration. A: normalized
average (⫾SD) current amplitude in response
to a second application of D-Asp in a 2-pulse
protocol as a function of the increasing interval between application over 2- to 44-s intervals. Line is fitted to a single-exponential
equation (Robert and Howe 2003): It ⫽
[Imax ⫺ (Imax ⫺ I0)exp(⫺t/␶)] (n ⫽ 8; halftime for recovery ⫽ 13 s; ␶ ⫽ 18.79).
B: average (⫾SD) dose response for D-Asp. Line
is fitted to a Boltzmann equation (Verdoorn
and Dingledine 1988): I ⫽ Imax/[1 ⫹ (EC50/
[agonist])nH] (n ⫽ 6). See text for definitions.
Thus D-Asp activated a nonspecific cation channel that
excluded Ca2⫹.
Mg2⫹ was also replaced with NMDG to test for voltagedependent block or permeation through D-Asp channels (Fig.
5B). Replacing external Mg2⫹ with NMDG did not cause a
significant shift in reversal potential (n ⫽ 10). Removing Mg2⫹
caused an increase in current amplitude at voltages ranging
from ⫺60 to ⫺15 mV (at ⫺60 mV, amplitude was increased
306 ⫾ 228%; at ⫺45 mV, 217 ⫾ 193%; at ⫺30 mV, 189 ⫾
159%; and at ⫺15 mV, 263 ⫾ 180%; P ⬍ 0.05, Student’s
paired t-test; n ⫽ 10). Current amplitudes at positive voltages
in Mg2⫹-free conditions were not significantly different from
those in ASW that contained 55 mM Mg2⫹. Thus D-Aspactivated currents appeared to possess voltage-dependent block
by Mg2⫹ similar to that of NMDA receptor channels but lacked
the Ca2⫹ permeability of NMDA channels.
Fig. 5. Average I-V relationships in D-Asp currents with external cation replacement and higher resolution view of reversal potentials (insets). A: significant
reversal potential shift of ⫺47 mV after replacement of external Na⫹ with N-methyl-D-glucamine (NMDG) (P ⬍ 0.05; n ⫽ 7). B: absence of shift in reversal
potential shift after replacement of external Mg2⫹ with NMDG (n ⫽ 10). C: absence of shift in reversal potential after replacement of external Ca2⫹ with NMDG
(n ⫽ 12). D: absence of shift in reversal potential after replacement of external Ca2⫹ and Mg2⫹ (n ⫽ 6). ASW, artificial seawater.
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was permeant (Fig. 5A). K⫹ in the intracellular solution was
also replaced with NMDG and caused a positive shift in
reversal potential of 34 mV (Fig. 6A; P ⬍ 0.01, Student’s
paired t-test; n ⫽ 7), indicating that K⫹ was permeant.
External Ca2⫹ was replaced in the ECS with NMDG, in
addition to the addition of 1 mM EGTA to the ECS to chelate
any residual Ca2⫹ (Fig. 5C). Replacing Ca2⫹ did not result
in a shift in reversal potential from currents in Ca2⫹-containing
ASW (n ⫽ 12). Because this apparent absence of a reversal
potential shift may have been due to the small amount of Ca2⫹
being excluded (ASW contains 10 mM), Ca2⫹ permeability
was further investigated by replacing external Mg2⫹ with
additional Ca2⫹. Addition of 55 mM extra Ca2⫹ did not cause
a shift in reversal potential (Fig. 5D; n ⫽ 6). The absence of
a shift when Ca2⫹ was either excluded or amended in the
ECS indicated D-Asp channels were not permeable to Ca2⫹.
1634
A DISTINCT EXCITATORY CURRENT FOR
D-Asp
IN APLYSIA
Fig. 6. I-V relationships of D-Asp currents with K⫹ and Cl⫺ replacement and higher resolution view of reversal potentials (insets). A: significant reversal potential
shift of 34 mV after replacement of internal K⫹ with NMDG (P ⬍ 0.05; n ⫽ 7). B: I-V relationships before and after replacement of external NaCl with
Na-gluconate (n ⫽ 9).
DISCUSSION
The excitatory Na⫹ and K⫹ current activated by D-Asp in
BSC neurons had a Hill coefficient for agonist binding near 1,
indicating independent binding of D-Asp to its target receptors.
These results are consistent with those observed for D-Asp at
NMDAR and non-NMDARs expressed in Xenopus oocytes
(Verdoorn and Dingledine 1988) and for L-Glu channels at
higher agonist concentrations (Patneau and Mayer 1990). Similarities between the whole cell currents induced by D-Asp and
L-Glu in BSC cells were their activation ranges, current amplitudes, relatively slow activation and inactivation, and desensitization. These features are most similar to the kinetics of
NMDAR channels (Dingledine et al. 1999; Paoletti 2011), the
purported site of action of D-Asp (Huang et al. 2005; Kiskin et
al. 1990; Olverman et al. 1988). Yet in contrast to L-Glu
currents in Aplysia, D-Asp currents had faster kinetics, an
outward rectification of the I-V relationship, and a prolonged
desensitization on repeated applications of agonist, requiring
⬃40 s for current amplitude to fully recover. L-Glu currents
recovered from desensitization quickly and potentiated as currents were elicited multiple times, characteristic of known
L-Glu channels (Heckmann and Dudel 1997; Vicini et al. 1998;
Wilding and Huettner 1997).
One of the most conspicuous characteristics distinguishing
NMDAR channels from non-NMDAR channels is a high Ca2⫹
permeability (Mayer and Westbrook 1987), not observed presently with D-Asp, and voltage-sensitive Mg2⫹ block (Ascher
and Nowak 1988; Dale and Kandel 1993), which we did
observe. Since Aplysia has both NMDA-like receptors and
AMPA-like receptors free of constitutive block by Mg2⫹ (Li et
al. 2005), D-Asp may act at these receptors and/or at novel
receptors.
The assertion that D-Asp activates non-L-Glu channels is
supported by the observation that approximately one-quarter of
BSC cells responded to both L-Glu and D-Asp, whereas another
one-quarter responded only to D-Asp and not to L-Glu. Although previous reports have demonstrated separate actions of
J Neurophysiol • VOL
the L-isoform of aspartate and L-Glu in Aplysia (Yarowsky and
Carpenter 1976), D- and L-Asp appear to act independently in
BSC neurons (Carlson and Fieber, in press). These data reflect
previous results from our laboratory in pleural ganglia (Fieber
et al. 2010) but contrast with earlier findings that D-Asp is an
alternate agonist of L-Glu channels in vertebrate brain (Kiskin
et al. 1990; Olverman et al. 1988). Our results are consistent
with findings of Errico et al. (in press) in mouse hippocampal
neurons, in which D-Asp activated NMDA receptors but generated excitatory postsynaptic potentials persisting under conditions of NMDAR block. Correspondingly, we demonstrated
that D-Asp elicits currents in Aplysia BSC cells that lacked
AMPAR and NMDAR currents (Carlson and Fieber, in press).
It is possible that the current responses observed in this study
represent a mixed population of receptor channels, with D-Asp
activating a combination of NMDAR channels and another
non-L-Glu channel. This could confound interpretation of results involving D-Asp-induced currents, since some of the
channel ion-conducting characteristics observed in this study
could be attributed to L-Glu receptor channels. The reported
observations, however, were consistent with activation of a
population of receptor channels distinct from, at least, L-Gluactivated NMDARs. The finding that D-Asp current responses
are modulated by similar mechanisms to AMPAR responses
(Carlson and Fieber, in press) suggests that D-Asp activation of
a putative D-Asp receptor may substitute for L-Glu activation of
AMPARs in some Aplysia neurons. Pharmacological antagonists targeting different L-Glu receptor subtypes may be useful
in elucidating the identity of channels activated by both L-Glu
and D-Asp and is an area of emphasis. Additional characterization of the endogenous neurotransmitter role of D-Asp will
benefit from the use of synaptic preparations, as well as
molecular characterization of the receptors activated by D-Asp.
The modulatory actions of D-Asp in previous studies (Brown
et al. 2007; Dale and Kandel 1993; Gong et al. 2005) might be
explained in terms of high-affinity binding of D-Asp to its
receptor, slowing recovery from desensitization (Zhang et al.
2006). D-Asp binding may provide an endogenous mechanism
of synaptic modulation if D-Asp binds with high affinity to
L-Glu receptors but does not activate them.
Furthermore, the long desensitization time of D-Asp currents
may be important in the physiology of the channel, representing an endogenous mechanism for protection from excitotoxicity (Trussel and Fischbach 1989). Cross-activation of D-Asp
with L-Glu receptors in mammalian systems may be protective
in this respect, and lower levels of free D-Asp in patients with
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External Cl⫺ was replaced to determine whether this anion
contributed to whole cell D-Asp-induced currents (Fig. 6B).
NaCl, the major ionic constituent of the ECS, was replaced
with Na-gluconate. After correction for a 17-mV junction
potential generated by the switch to gluconate, the reversal
potential was not significantly different from that in ASW,
suggesting that Cl⫺ does not permeate D-Asp-activated
channels.
A DISTINCT EXCITATORY CURRENT FOR
Alzheimer’s disease would support this hypothesis (D’Aniello
et al. 1998).
Our results demonstrate features of the channels activated by
D-Asp in the nervous system of Aplysia. D-Asp may have
multiple sites of action, similar to L-Glu, and may overlap with
L-Glu receptors. Several of our recent results, including some
described in this report, however, allude to an undescribed
receptor.
ACKNOWLEDGMENTS
We gratefully acknowledge assistance from the staff of the University of
Miami Aplysia Resource.
Present address of S. L. Carlson: Bowles Center for Alcohol Studies, The
University of North Carolina at Chapel Hill, Campus Box 7178, Thurston
Bowles Building, Chapel Hill, NC 27599-7178.
This work was funded by National Institutes of Health Grant P40RR01029,
the Korein Foundation, and a University of Miami Fellowship to S. L. Carlson.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the
author(s).
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