Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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Morishita, Wade and
Bradley E. Alger.
Evidence for Endogenous Excitatory Amino Acids as Mediators in
DSI of GABAAergic Transmission in Hippocampal CA1.
J. Neurophysiol. 82: 2556-2564, 1999.
Depolarization-induced suppression of inhibition (DSI) is a process
whereby brief ~1-s depolarization to the postsynaptic membrane of
hippocampal CA1 pyramidal cells results in a transient suppression of
GABAAergic synaptic transmission. DSI is triggered by a
postsynaptic rise in [Ca2+]in and yet is
expressed presynaptically, which implies that a retrograde signal is
involved. Recent evidence based on synthetic metabotropic glutamate
receptor (mGluR) agonists and antagonists suggested that group
I mGluRs take part in the expression of DSI and raised the
possibility that glutamate or a glutamate-like substance is the
retrograde messenger in hippocampal CA1. This hypothesis was tested,
and it was found that the endogenous amino acids
L-glutamate (L-Glu) and L-cysteine
sulfinic acid (L-CSA) suppressed
GABAA-receptor-mediated inhibitory postsynaptic currents (IPSCs) and occluded DSI, whereas L-homocysteic acid
(L-HCA) and L-homocysteine sulfinic acid
(L-HCSA) did not. Activation of metabotropic kainate
receptors with kainic acid (KA) reduced IPSCs; however, DSI was not
occluded. When iontophoretically applied, both L-Glu and
L-CSA produced a transient IPSC suppression similar in
magnitude and time course to that observed during DSI. Both DSI and the actions of the amino acids were antagonized by
(S)--methyl-4-carboxyphenylglycine ([S]-MCPG), indicating that the
effects of the endogenous agonists were produced through activation of
mGluRs. Blocking excitatory amino acid transport significantly
increased DSI and the suppression produced by L-Glu or
L-CSA without affecting the time constant of recovery from
the suppression. Similar to DSI, IPSC suppression by L-Glu
or L-CSA was blocked by N-ethylmaleimide
(NEM). Moreover, paired-pulse depression (PPD), which is unaltered
during DSI, is also not significantly affected by the amino acids.
Taken together, these results support the glutamate hypothesis of DSI
and argue that L-Glu or L-CSA are potential
retrograde messengers in CA1.
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INTRODUCTION |
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In hippocampal CA1 pyramidal cells (Alger
et al. 1996; Pitler and Alger 1992
, 1994
) and in
cerebellar Purkinje cells (Llano et al. 1991
;
Vincent et al. 1992
; Vincent and Marty
1993
), brief (~1-s) influx of Ca2+
through voltage-dependent Ca2+ channels produces
a transient suppression of GABAAergic inhibitory postsynaptic currents (IPSCs). This phenomenon, termed
depolarization-induced suppression of inhibition (DSI), appears to be
mediated by a retrograde message released after depolarization of the
pyramidal cell. This hypothesis is based on a great deal of recent
work. DSI is not accompanied by detectable changes in
GABAA receptor response properties, whether these
are assessed by iontophoretic GABA responses (Llano et al.
1991
; Pitler and Alger 1992
) or by spontaneous
(Llano et al. 1991
; Pitler and Alger
1994
), evoked (Alger et al. 1996
), or
Sr2+-induced (Morishita and Alger
1997
) miniature IPSCs. DSI is associated with a decrease in
GABA release as measured by the coefficient of variation (Alger
et al. 1996
), the method of failures (Alger et al.
1996
; Vincent et al. 1992
), or direct counting
of evoked asynchronous quanta in Sr2+
(Morishita and Alger 1997
). Evidently,
Ca2+ influx into a pyramidal cell causes a signal
to be sent to interneurons that prevents them from releasing GABA
efficiently for a time.
Although the identity of this signal is not fully determined, recent
experiments on the cerebellum (Glitsch et al. 1996) and hippocampus (Morishita et al. 1998
) have implicated
activation of metabotropic glutamate receptors (mGluRs) in the DSI
process. The mGluR family consists of eight subtypes of
G-protein-coupled receptors: group I (mGluRs 1 and 5),
group II (mGluRs 2 and 3), and group III (mGluRs
4, 6, 7, 8), which can be distinguished by pharmacological and
biochemical criteria (Pin and Duvoisin 1995
). Support
for the metabotropic glutamate hypothesis of DSI includes the
observations based on the use of synthetic mGluR agonists. In the
cerebellum, the group II agonist DCG-IV mimics and occludes
DSI (Glitsch et al. 1996
), whereas in hippocampal CA1
the selective group I mGluR agonists quisqualate (at a low concentration) and DHPG have the same effects (Morishita et al. 1998
), and participation of group II or
III receptors can be ruled out. In CA1, the group
I and group II antagonist
(S)-
-methyl-4-carboxyphenylglycine (S)-MCPG reduced both the
synthetic agonist effects and DSI.
These findings support the hypothesis that glutamate or a
glutamate-like substance could be the retrograde signal in DSI. Nevertheless, the mGluR-glutamate hypothesis has not been tested using
endogenous amino acids in any preparation, and, except for the block of
DSI by MCPG in hippocampus, the hypothesis is based mainly on
similarity of action between synthetic mGluR agonists and DSI. We
examined the actions of L-glutamate (L-Glu) and
several endogenous amino acids on IPSCs and DSI. Although a number of neurotransmitter candidates exist in the CNS, we focused on the sulfur-containing amino acids, L-homocysteic acid
(L-HCA), L-homocysteine sulfinic acid
(L-HCSA), and L-cysteine sulfinic acid
(L-CSA), because they can activate group I
mGluRs (Kingston et al. 1998). We also examined the
possibility of a role for the metabotropic kainic acid (KA) receptor
(Rodriguez-Moreno and Lerman 1998
) in DSI. This KA
receptor is present on interneurons and, when activated, produces a
G-protein-mediated presynaptic suppression of IPSCs onto CA1 pyramidal
cells (Clarke et al. 1997
; Cossart et al.
1998
; Fisher and Alger 1984
; Frerking et
al. 1998
; Rodriguez-Moreno et al. 1997
). The
crucial prediction of the mGluR-glutamate hypothesis is that endogenous
excitatory amino acid agonists of mGluRs should meet criteria
established for the DSI messenger. The data indicate that
L-Glu or L-CSA are good
candidates for the retrograde messenger in DSI in CA1, but that
L-HCA and L-HCSA are not. A
role for KA receptors in DSI can be ruled out. Some of the results in
this study have appeared in abstract form (Morishita and Alger
1998
).
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METHODS |
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Preparation of slices
Transversely sectioned slices 400-µm thick were obtained from
the hippocampi of adult male Sprague-Dawley rats (125-250 g) as
previously described (Morishita and Alger 1997). The
slices were maintained at room temperature in a holding chamber at the interface of a physiological saline and humidified 95% -5%
CO2 mixture. After a minimum of 1 h of
incubation, a single slice was transferred to a submersion-type
recording chamber (Nicoll and Alger 1981
) where it was
perfused with saline (29-31°C) at a flow rate of 0.5-1 ml/min.
Solutions
The physiological saline comprised (in mM): 120 NaCl, 3 KCl, 1 NaH2PO4, 25 NaHCO3, 2.5 CaCl2, 2 MgSO4, and 10 glucose. In all experiments,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 40 or 100 µM) and either
(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801, 100 µM) or
D,L-2-amino-5-phosphonovaleric acid (APV, 100 µM) was added to the physiological saline to block ionotropic
glutamate-receptor-mediated responses. The noncompetitive -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist,
1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466, 50 µM) was also present in most
experiments. Patch electrodes (2-5 M
in the bath) were filled with
a solution containing (in mM) 100 CsMeSO3, 60 CsCl, 10 HEPES, 1 or 2 BAPTA, 1.0 or 0.2 CaCl2, 1 or 2 MgCl2, 4 MgATP, and 5 QX-314 (pH adjusted to
7.2 with CsOH).
Other drugs that were bath applied in this study were 1-1.5 mM
L-Glu, 1-1.5 mM L-CSA, 1 mM L-HCA,
1-1.5 mM L-HCSA , 10 µM KA, 2.5-3 mM (S)-MCPG, 250 µM
N-ethylmaleimide (NEM), 1 mM dihydrokainic acid (DHK), and 0.5 mM
L()-threo-3-hydroxyaspartic acid (THA). DHK, THA, and
(S)-MCPG were purchased from Tocris Cookson (Ballwin, MO). GYKI 52466, MK-801, and QX-314 were acquired from Research Biochemicals (Natick,
MA). All other drugs were obtained from Sigma (St. Louis, MO).
Electrophysiology
Whole cell voltage-clamp recordings were made from CA1 pyramidal
cells. The cells were clamped at 70 mV, and IPSCs were evoked by
stimulating stratum oriens with a concentric bipolar electrode (Rhodes
Electronics) at 0.33 Hz. Paired-pulse stimulation was achieved by
giving two identical stimulation pulses (repeated every 0.2 or 0.33 Hz)
to s. oriens separated by an interstimulus interval of 200 ms. Cells
were considered acceptable for an experiment if they had input
resistances >55 M
and exhibited stable holding currents. Series
resistance was compensated between 30 and 50% and continually
monitored throughout the experiment by observing the amplitude of the
capacitive current arising from a 5-mV, 50-ms hyperpolarizing voltage
step. Experiments were terminated if the series resistance was unstable
or exceeded 30 M
. The slight liquid-junction potential was not
corrected for.
DSI was induced by depolarizing the postsynaptic membrane to 0 or 10
mV for durations ranging between 0.5 and 3 s every 90 s.
Iontophoretic pipettes, filled with L-Glu (100 mM or 1 M,
pH 9) or L-CSA (50 or 100 mM, pH 9), were positioned in
stratum pyramidale in proximity (50-100 µm) to the recording
electrode. A 20-nA retaining current was applied to the iontophoretic
pipettes to minimize leakage of drugs, and iontophoretic applications
(performed with ejecting currents of 150-170 nA with durations ranging
between 0.5 and 4 s) were alternated with the DSI-inducing voltage step.
Current signals were filtered at 2 kHz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA), digitized at 10 kHz (DigiData 1200, Axon Instruments, Foster City, CA), and stored on a pentium-based personal computer (Dell Dimensions XPS M200). The signals were also acquired at 22 kHz with a 14-bit PCM digitizer (Neuro-Corder DR-484, Neuro Data Instruments) and stored on VHS videotape.
Data analysis
To quantify peak DSI the following formula was used
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(1) |
Nonlinear regression analysis was used to measure the time constant of
recovery from DSI. The time course of recovery was best fitted by the
single exponential equation
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(2) |
Off-line analysis was performed using pClamp 6 (Axon Instruments, Foster City, CA) and SigmaPlot 4 (SPSS, Chicago, IL) software. Statistical analysis of the data were performed using a Student's paired t-test (P < 0.05).
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RESULTS |
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We began by determining whether bath application of 1-1.5 mM L-Glu and L-CSA could mimic or occlude DSI. Typical experiments are shown in Fig. 1, A1 and B1. The dots represent the peak amplitudes of individual monosynaptic IPSCs that were evoked at 3-s intervals. DSI was elicited by a depolarizing voltage step at 90-s intervals (given at the points marked by the arrows). After each voltage step, the IPSC amplitudes were reduced and then recovered to control levels over the next minute. Note that both agents, when bath applied for ~7 min, strongly and reversibly suppressed the IPSCs (IPSC reduction in L-Glu, 53 ± 9.2%; in L-CSA, 69 ± 7.7%, Fig. 1, A3, n = 4 and B3, n = 5) and occluded DSI. DSI was 61 ± 5.6% in control and 36 ± 5.2% during L-Glu application and 46 ± 7.9% before and 20 ± 7.1% during L-CSA application (Fig. 1, A3, n = 4 and B3, n = 5, respectively). After washout of L-Glu or L-CSA, the IPSCs and DSI recovered to near control values. If, when DSI was reduced, the stimulation intensity was increased to elicit IPSCs comparable in amplitude with those in control (in L-Glu, n = 2, Fig. 1A2; in L-CSA n = 3, Fig. 1B2), DSI remained reduced, indicating that the actions of these agonists were on the DSI process and could not be accounted for by a simple reduction in IPSC size.
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Bath application of two other sulfur-containing amino acids that are
agonists at mGluRs (Kingston et al. 1998), 1 mM
L-HCA and 1-1.5 mM L-HCSA, produced effects
unlike those of L-Glu or L-CSA.
L-HCA reduced IPSCs by 95 ± 7.8%, but did not
occlude DSI, which was 67 ± 8.5% in control and 65 ± 2.9%
in L-HCA (n = 3). L-HCSA also reduced IPSCs by 88 ± 9.9%
without occluding DSI. L-HCSA actually increased
DSI as calculated from Eq. 1, from 37 ± 6.8% to
65 ± 3.1% (n = 3), although whether this
represents an enhancement of the DSI process or some other factor
cannot be determined from the present data. In any case, their failure to occlude DSI argues that neither L-HCA nor
L-HCSA is a strong candidate as the DSI
messenger, and we did not consider them further.
As previously reported (Cossart et al. 1998;
Fisher and Alger 1984
; Frerking et al.
1998
; Rodriguez-Moreno and Lerma 1998
; Rodriguez-Moreno et al. 1997
), KA (10 µM) also
suppressed IPSCs (IPSC reduction in KA, 49 ± 4.5%, Fig.
1C2, n = 5); however, unlike L-Glu and L-CSA, KA did not
occlude DSI (control DSI, 45 ± 3.4%; KA DSI, 46 ± 3.7%,
n = 5, Fig. 1, C1 and C2,
respectively). Additional evidence that activation of KA receptors does
not play a role in DSI can be seen in the effects of subsequent
application of 100 µM CNQX. At 100 µM, CNQX reversed the KA-induced
suppression of IPSCs. Concomitantly, as the IPSC amplitudes returned to
near control values, DSI also became more readily detectable (KA + CNQX DSI, 51 ± 3.9%). Had the KA receptor been involved in DSI, the IPSCs would have recovered, and DSI would have been blocked. The
recovery of IPSCs and the presence of DSI in 100 µM CNQX can be
contrasted with the effects of the mGluR antagonist MCPG, which reverses the effects of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] on IPSC amplitudes, and yet simultaneously reduces DSI (Fig. 3, cf. Morishita et al. 1998
).
The occlusion experiments shown in Fig. 1 suggested that
L-Glu or L-CSA could be candidates for the DSI
signal. We tested this hypothesis in more detail using iontophoresis.
Brief application of a candidate messenger (rather than the bath
application shown in Fig. 1) may be expected to mimic the time course
of the endogenous agent. In the case of a fast, ionotropic
neurotransmitter response, very rapid application of the suspected
transmitter very close to the receptors is required (e.g.,
Kuffler and Yoshikami 1975; Liu et al.
1999
). However, the requirements for speed and precise location
of application are less critical in the case of an mGluR-mediated action. Brief DCG-IV application to cerebellar cells produced IPSC
suppression with a time course similar to that of cerebellar DSI
(Glitsch et al. 1996
). The mGluRs are often located in
perisynaptic regions outside the immediate subsynaptic zone
(Lujan et al. 1996
) and therefore are probably not
subject to the same rapid exposure to synaptically released glutamate.
In addition, the biochemical cascades downstream of activation of
metabotropic receptors should have a greater influence on the overall
duration of the response than on the receptor-binding steps. We
therefore iontophoresed L-Glu or L-CSA to CA1
cells while continuously evoking monosynaptic IPSCs. To compare the
agonist effects and DSI, we alternated iontophoretic applications with
DSI trials at 90-s intervals. Iontophoretic applications of
L-Glu (Fig. 2A)
and L-CSA (Fig. 2B) produced transient reductions in IPSCs. Similar maximal peak IPSC reductions induced with
DSI or amino acid iontophoresis recovered with very similar time
courses, as shown in the examples at the top and the group data in the
bottom graphs (L-Glu, n = 10;
L-CSA, n = 7).
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By alternating DSI trials with iontophoretic application of either
L-Glu or L-CSA, we found that the effects of
both agonists and DSI can be antagonized by (S)-MCPG (cf.
Morishita et al. 1998). (S)-MCPG (2.5-3 mM) caused a
significant, and comparable, decrease in the IPSC-suppressive effects
of both agonists and DSI. Control DSI and L-Glu reduction
of IPSCs were 54 ± 6.4% and 47 ± 3.1%, respectively. In
(S)-MCPG, DSI and the L-Glu suppression of IPSCs were
33 ± 6.3% and 31 ± 1.9%, respectively (Fig.
3A, n = 6).
Control DSI and L-CSA reduction of IPSCs were
47 ± 6.4% and 48 ± 3.4%, respectively. In (S)-MCPG, DSI
and L-CSA suppression of IPSCs were 23 ± 9.1% and 24 ± 6.7%, respectively (Fig. 3B,
n = 4). The data confirm that the agonists reduce IPSCs
by acting on mGluRs.
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If a glutamate-like agonist is the DSI signal, then DSI should be
affected by inhibition of glutamate uptake systems. Both L-Glu and L-CSA are substrates for glutamate
transporters, and we therefore examined the effects of a combination of
glutamate uptake inhibitors, DHK (1 mM) and THA (0.5 mM), on these
parameters, by means of alternating iontophoretic applications and DSI
induction. In these experiments the durations of DSI-inducing voltage
steps and iontophoretic applications were either 1 or 2 s. The
time constant of decay of DSI is independent of voltage-step duration (over a nearly 10-fold range of durations), provided a comparable degree of DSI is induced (Lenz and Alger 1999);
therefore, data from these experiments can be compared. Figure
4 shows the results of these experiments.
To maximize the chances of observing an effect of the uptake
inhibitors, in these experiments we adjusted conditions to obtain
minimal levels of IPSC suppression, by iontophoresis or DSI. Control
values of DSI and of IPSC suppression by L-Glu or
L-CSA (20-30%, rather than the 40-50% that are typical)
were similar. In the presence of uptake inhibitors, DSI and IPSC
suppression by L-Glu increased from 26 ± 8.8% to
39 ± 6.9% and from 20 ± 8.1% to 46 ± 10%,
respectively (Fig. 4A, n = 5). DSI and IPSC
suppression by L-CSA was also enhanced from
22 ± 5.3% to 37 ± 5.6% and from 25 ± 3.9% to
40 ± 3.7% (Fig. 4B, n = 7). These
increases were significant; P < 0.05. We also examined
the effects of the uptake inhibitors on the time constant of IPSC
suppression by DSI and by the amino acids. The graph in Fig.
4C shows averaged data for the time courses of IPSC
suppression and recovery in control and in the presence of the
glutamate transporter blockers. Although blocking glutamate uptake
produced greater IPSC suppression, it did not alter the time constant
of recovery itself. The total duration of DSI was, naturally, enhanced
because DSI was greater at every time point, as can be seen from the
dotted lines in the graphs of Fig. 4. DHK and THA slowed the time
course of fast ionotropic responses (cf. Tong and Jahr
1994
, Fig. 4C, inset), indicating that
they blocked glutamate transporters.
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DSI has a number of properties that can be used to screen candidate DSI
messengers. For instance, DSI and the (1S,3R)-ACPD-induced suppression
of IPSCs are blocked by 250 µM NEM, before NEM markedly suppresses
the IPSCs themselves (Morishita et al. 1997). We now show (Fig. 5) that NEM also blocked the
effects of L-Glu and L-CSA on IPSCs (IPSC
reduction by L-Glu and L-CSA, 54 ± 9.4%
and 43 ± 3.1%, respectively; IPSC reduction by L-Glu
and L-CSA in NEM, 9 ± 3.8% and 4 ± 2.8%,
respectively, Fig. 5, A and B, n = 3).
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An interesting property of DSI is that, although it represents a
presynaptic inhibitory process, it is unlike many forms of conventional
presynaptic inhibition in that it does not alter paired-pulse
responsiveness (Alger et al. 1996; Morishita et
al. 1998
). Paired-pulse depression (PPD) is a prominent feature
of monosynaptic IPSCs in the hippocampus (Davies et al.
1990
). The second response to a pair of stimuli administered
200 ms after the first is typically suppressed relative to the first
response, a reduction commonly expressed by the paired-pulse ratio as
the amplitude of IPSC2 divided by that of
IPSC1. DSI reduced IPSC amplitudes without altering PPD;
both responses were decreased in parallel. L-Glu and
L-CSA also reduced IPSCs without significantly altering the
paired-pulse ratio. In control solution the PPD ratio was 0.83 ± 0.01; in L-Glu it was 0.81 ± 0.01, Fig.
6, A1 and
A2, n = 5; in control solution the
PPD ratio was 0.84 ± 0.9; and in L-CSA it was
0.86 ± 1.8 (Fig. 6, B1 and B2, n =
7).
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DISCUSSION |
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Although mGluRs have been implicated in DSI in both cerebellum and
hippocampus, endogenous amino acids have not been tested. Our data
indicate that both L-Glu and another excitatory amino acid
neurotransmitter candidate, L-CSA, meet criteria that
should distinguish the DSI messenger. They mimicked and occluded DSI, their effects were blocked by NEM and (S)-MCPG, and they did not alter
PPD. Other very similar amino acids, L-HCA and
L-HCSA, did not occlude DSI. KA serves as a very important
control in this case because, although it is a glutamate agonist and
antagonizes evoked IPSCs (Clarke et al. 1997;
Cossart et al. 1998
; Fisher and Alger
1984
; Frerking et al. 1998
;
Rodriguez-Moreno et al. 1997
) at a presynaptic site
linked to a G-protein (Rodriguez-Moreno and Lerma 1998
),
a role for the KA receptor in DSI could be ruled out. It was possible
to reverse the effects of KA on IPSCs without altering DSI. Conversely,
the mGluR antagonist (S)-MCPG antagonized the effects of
L-Glu and L-CSA on IPSCs, as well as DSI,
consistent with a role for mGluR agonists in DSI. At this point we
cannot distinguish between L-Glu and L-CSA, and
both must be considered equally good candidate retrograde messengers
for DSI.
Glutamate is by far the best established excitatory amino acid
neurotransmitter; however, other endogenous amino acid candidates exist. L-CSA is an especially interesting one that fulfills
most of the commonly accepted criteria for transmitter identification, including Ca2+-dependent release. Besides acting
on ionotropic (Conn et al. 1994) and metabotropic
(Boss and Boaten 1995
) glutamate receptors, L-CSA also acts on an excitatory amino acid receptor that
is unique in catalyzing breakdown of membrane phospholipids through the activation of phospholipase D (PLD) (Boss et al. 1994
).
The PLD-coupled receptor is probably not involved in DSI because it is
insensitive to glutamate and MCPG, whereas it is activated by
L-AP3 (Conn et al. 1994
). As shown in the
present report, DSI is very effectively mimicked by glutamate, is
blocked by MCPG and, as previously noted, is unaffected by
L-AP3 (Morishita et al. 1998
).
The broad-spectrum mGluR antagonist (S)-MCPG is more potent on mGluR1
than on mGluR5 and, even at mGluR5, is much more potent when
(1S,3R)-ACPD is the agonist than when L-Glu is
(Brabet et al. 1995; Joly et al. 1995
;
Littman and Robinson 1994
). L-CSA, similar
to L-Glu, is a full agonist in eliciting phosphoinositide turnover through activation of the group I human
mGluR1
and mGluR5
when these receptors are expressed in the
Syrian hamster tumor cell line AV12-664 (Kingston et al.
1998
). Actions at these receptors are likely to be relevant for
DSI. Synthetic agonists at group I
receptors
(1S,3R)-ACPD, quisqualate at a low concentration and (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I; high
concentration)
mimic and occlude DSI, whereas the group
II agonists DCG-IV and L-CCG-I (low concentration)
have no effect on IPSCs in CA1 (Morishita et al. 1998
;
cf. Gereau and Conn 1995
). Of the group I
receptors, mGluR5 is the more likely candidate for mediating DSI in
CA1, because mGluR1 receptors are confined to a subgroup of
interneurons in s. oriens, whereas mGluR5s are densely distributed
throughout CA1 (Shigemoto et al. 1997
). The mGluR
antagonist (S)-4-carboxyphenylglycine (4CPG), much more potent in
blocking mGluR1 than mGluR5 (Brabet et al. 1995
;
Joly et al. 1995
), had no effect on DSI or the reduction in IPSCs caused by 1S,3R-ACPD (Morishita et al. 1998
).
Blocking the effects of L-Glu and L-CSA
required high concentrations of MCPG, as did the antagonism of DSI.
Thus, these data continue to point to an endogenous glutamate-like
amino acid and mGluR5 as mediators of DSI.
The glutamate transport blockers DHK and THA enhanced DSI and the
effects of L-Glu and L-CSA. This adds to the
number of close similarities between the actions of mGluR agonists and
DSI. The specificity of DHK and THA provides a direct link between
glutamate agonists and DSI. Although it is taken up by the glutamate
transporter, L-CSA is not an ideal substrate and in fact
can reduce glutamate uptake, giving rise to concerns that some of its
effects could be caused by competitive heteroexchange (Kingston
et al. 1998) for glutamate. If this were to explain the actions
of L-CSA in our experiments it would strengthen the
candidacy of glutamate as the retrograde signal in DSI. Although it is
difficult to rule out this possibility completely, we think that it is
not likely to be responsible for the effects of L-CSA in
our experiments. The suppressive effect of L-CSA on IPSCs
should have been occluded in the presence of the uptake blockers if it
had been secondary to block of glutamate uptake. On the contrary,
L-CSA produced a greater effect in the presence of DHK and THA.
The absence of effect of uptake inhibitors on the time constant of IPSC
suppression and DSI is consistent with the interpretation that the
availability of the ligand at the receptor is not limiting for the
duration of metabotropic transmitter actions, which are largely
functions of downstream effector steps. Inhibiting glutamate transporters may be equivalent to increasing the concentration of the
ligand (Fitzsimonds and Dichter 1996), which could
explain the enhancement of IPSC suppression by DSI and the mGluR agonists.
If L-Glu or L-CSA is the retrograde messenger
in DSI, then an important challenge will be to elucidate its mechanism
of release from the pyramidal cells. DSI is caused by Ca2+
influx into the somatodendritic regions of these cells, and yet no
presynaptic specializations or concentrations of synaptic vesicles have
been found in these regions. A recent study has shown that DSI has a
Ca2+ dependence that in many ways more closely resembles
that of hormonal, or neuropeptide, slow release than it does fast
neurotransmitter release (Lenz and Alger 1999). It will
be interesting to determine whether DSI involves a conventional release process.
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ACKNOWLEDGMENTS |
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We thank laboratory members N. Varma, Y. Wang, and D.-S. Wei for comments on a draft of the manuscript. We also thank E. Elizabeth for expert typing and editorial assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-30219 and NS-22010 to B. E. Alger.
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FOOTNOTES |
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Address for reprint requests: B. E. Alger, Dept. of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 April 1999; accepted in final form 29 July 1999.
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REFERENCES |
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