Institute of Neurobiology, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico 00901
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ABSTRACT |
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Krenz, Wulf D.,
Don Nguyen,
Nivia L. Pérez-Acevedo, and
Allen I. Selverston.
Group I, II, and III mGluR Compounds Affect Rhythm Generation in
the Gastric Circuit of the Crustacean Stomatogastric Ganglion.
J. Neurophysiol. 83: 1188-1201, 2000.
We
have studied the effects of group I, II, and III metabotropic glutamate
receptor (mGluR) agonists on rhythm generation by the gastric circuit
of the stomatogastric ganglion (STG) of the Caribbean spiny lobster
Panulirus argus. All mGluR agonists and some antagonists
we tested in this study had clear and distinct effects on gastric
rhythm generation when superfused over combined oscillating or blocked
silent STG preparations. A consistent difference between group I
agonists and group II and III agonists was that group I agonists acted
excitatory. The group I-specific agonists L-quisqualic acid
and (S)-3,5-dihydroxyphenylglycine, as well as the
nonspecific agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid
accelerated ongoing rhythms and could induce gastric rhythms in silent
preparations. The group II agonist
(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) and the group III
agonist L(+)-2-amino-4-phosphonobutyric acid
(L-AP4) slowed down or completely blocked ongoing gastric rhythms and were without detectable effect on silent preparations. The
action of L-CCG-I was blocked partially by the group-II-specific antagonist, (RS)-1-amino-5-phosphonoindan-1-carboxylic acid
[(RS)APICA], and the group-III-specific antagonist
(RS)--methyl-4-phosphonophenylglycine completely blocked the action
of L-AP4. Besides its antagonistic action, the
group-II-specific antagonist (RS)APICA had a remarkably strong apparent
inverse agonist action when applied alone on oscillating preparations.
The action of all drugs was dose dependent and reversible, although
recovery was not always complete. In our experiments, the effects of
none of the mGluR-specific agonists were antagonized or amplified by
the N-methyl-D-aspartate
(NMDA)-receptor-specific antagonist
D(
)-2-amino-5-phosphonopentanoic acid, excluding the contamination of responses to mGluR agonists by nonspecific
cross-reactivity with NMDA receptors. Picrotoxin did not prevent the
inhibitory action of L-CCG-I and L-AP4. We conclude that
mGluRs, probably similar to those belonging to groups I, II, and III
described in mammals, may play a role as modulators of gastric circuit
rhythm generation in vivo.
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INTRODUCTION |
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Work over the last decade has shown that in the
mammalian brain, glutamate not only acts on ionotropic glutamate
receptors (iGluR) but also on metabotropic glutamate receptors (mGluR). mGluRs are seven-transmembrane domain proteins. They are coupled via G
proteins to cyclic nucleotide and phosphoinositide intracellular messenger pathways and are capable of inducing a variety of cellular changes (Conn and Pin 1997; Pin and Duvoisin
1995
; Saugstad et al. 1995
). Depending on their
pre- or postsynaptic localization, they may modulate neurotransmitter
release (Cochilla and Alford 1998
) and/or intrinsic
membrane properties of neurons (Del Negro and Chandler
1998
; Lüthi et al. 1997
; Morisset
and Nagy 1996
; Russo et al. 1997
) in a time- and
activity-dependent way (Rodríguez-Moreno et al.
1998
). mGluRs have been shown to be involved in motor pattern generation (Krieger et al. 1998
). Three groups, I, II,
and III, have been identified for mammalian mGluRs. This classification is based on specific pharmacological profiles, amino acid sequence similarity, and distinct intracellular messenger coupling (Conn and Pin 1997
). The functional significance of their role in
fine tuning the electrical behavior of nerve cells and synapses, as well as the complexity of their dynamic interactions with other receptors and ion channels, is only beginning to emerge.
In invertebrate animals, glutamate receptors that are activated by
mGluR agonists have been identified in Aplysia neurons (Katz and Levitan 1993) and in leech glial cells
(Lohr and Deitmer 1997
). In the neuromuscular junction
(NMJ) of lobster and crayfish, where glutamate is the principal
excitatory neurotransmitter (Kravitz et al. 1970
;
Takeuchi and Takeuchi 1964
), presynaptic autoreceptors are known to modulate the release of glutamate and
-aminobutyric acid (GABA) (Miwa and Kawai 1986
; Miwa et al.
1990
, 1993
). Experiments done in lobster suggest that at least
some of these GluRs are coupled to G proteins (Miwa et al. 1987
,
1990
). In the crayfish NMJ, the nonspecific mGluR ligands
(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, agonist) and
(RS)-
-methyl-3-carboxymethylphenylglycine (MCPG, antagonist) both
modulate the quantum content of postsynaptic currents (Schramm
and Dudel 1997
). The available data suggest that mGluRs may be
as important for the function of the invertebrate CNS and peripheral
nervous system as they are in mammals and other vertebrates.
Important questions about mGluR function that remain difficult to
address in more complex mammalian preparations are under what
conditions are the different types of mGluRs activated and what are the
consequences of their activation for neuronal circuits. To determine
the consequences of mGluR activation for neuronal network function, we
have started to investigate mGluRs in a small defined, rhythmically
active circuit of ~30 neurons, the stomatogastric ganglion (STG) of
decapod crustaceans. All neurons of the STG, along with their synaptic
and electrical connections, are identified and can be studied at the
level of the whole network, as reduced subcircuits, or as isolated
cell-pairs or single neurons. Two main central pattern generator (CPG)
circuits have been described in the STG, the pyloric and the gastric
CPGs (Fig. 1), which produce the cyclic
motor patterns that drive the muscles of the foregut. The activities of
both are controlled by a large number of modulatory substances that
either are released into the STG by neurons projecting from the
commissural or esophageal ganglia or act via the bloodstream (Harris-Warrick et al. 1992; Marder 1987
;
Marder et al. 1995
).
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Glutamate is one of two major neurotransmitters in the STG
(Marder 1987). It is used by the motoneurons that
innervate intrinsic stomach muscles, i.e., muscles that originate and
insert on the stomach wall (Govind and Lingle 1987
).
Glutamate activates excitatory iGluRs at the NMJ of their target
muscles and inhibitory iGluRs at intraganglionic synapses that mediate
the mutual interactions between neurons (Cleland and Selverston
1995
, 1998
; Marder 1987
). Besides these
well-studied, fast ionotropic receptor-mediated synaptic events,
several slow postsynaptic potentials (Elson and Selverston
1995
) and presumably metabotropic slow responses to glutamate,
depolarizing and hyperpolarizing, have been described in crustacean STG
neurons (Marder and Paupardin-Tritsch 1978
; Tazaki and Chiba 1994
). The second major
neurotransmitter is acetylcholine (ACh) (Marder 1987
).
ACh within the STG also acts as a fast inhibitory transmitter between
neurons and in the periphery excites a subset of stomach muscles. In
addition, ACh is thought to be a powerful neuromodulator that activates
both the pyloric and the gastric CPG via muscarinic receptors. In this
study, we ask the questions, are mGluRs present in the gastric circuit
of the STG and does their activation modulate the gastric CPG?
To answer these questions, we examined several representative agonists from each of the three different groups for their role under three different conditions. In the simplest case, we blocked all or most of the modulatory inputs to the gastric mill so that no rhythm was present. The agonists then were perfused over the ganglion to see if this initiated a rhythm and what the characteristics of such rhythms were. To determine if the group-specific agonists can modulate ongoing rhythms, agonists were perfused over combined preparations (fully modulated by the commissural and esophageal ganglia) that were producing vigorous patterns. Reversible group-specific changes to the patterns then could be determined. Because bursting in blocked silent preparations also could be initiated by the application of the muscarinic agonist pilocarpine, the effects of the mGluR agonists on this very specific type of rhythm also was studied. A check on the specificity of some of the agonists was made using group-specific antagonists to try and block their effects. And to determine if glutamate is one of the modulators present in the mix normally supplied by upstream ganglia, the effects of antagonists only on normal combined rhythms also were measured.
In this study at the neuronal network level, we show that agonists for the three mammalian-defined groups I-III, as well as antagonists for group II and III, have strong, dose-dependent and reversible effects on CPG function. Our data indicate that in the gastric circuit of the decapod STG (Fig. 1), besides its well-studied role as the major fast inhibitory neurotransmitter, glutamate also may play a hitherto unrecognized yet important role as a neuromodulator.
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METHODS |
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Animals
Experiments were performed on STGs from 48 adult Caribbean spiny lobsters, Panulirus argus, of both sexes. Animals were provided by local fishermen and were kept in large aquaria at ambient temperature (24-25°C) before being used in experiments. Experiments were performed at the same ambient temperature.
Preparation
The combined stomatogastric nervous system (STNS) was dissected
as described previously (Mulloney and Selverston 1974)
and was pinned out in a silicone elastomer (Sylgard)-lined petri dish. The STG was desheathed. The stomatogastric nerve (stn) also
was desheathed over a length of 5-10 mm. The STG and the desheathed portion of the stn were isolated from the rest of the bath
volume in wells made from petroleum jelly (Vaseline) to enable separate superfusion. The volume of the STG well was ~500 µl and could be
exchanged rapidly.
Physiological and drug solutions
Dissection and experiments were done in standard Panulirus saline, which had the following composition (in mmol/l): 479.1 NaCl, 12.7 KCl, 13.7 CaCl2, 10 MgSO4, 3.9 Na2SO4, 5 HEPES, and 5 TES, pH 7.4. In most experiments, the STG was superfused continuously with Panulirus saline.
All mGluR drugs, ACPD, (S)-3,5-dihydroxyphenylglycine (DHPG),
(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I),
L(+)-2-amino-4-phosphonobutyric acid (L-AP4),
MCPG, (RS)--methylserine-O-phosphate (MSOP),
(RS)-
-methyl-4-phosphonophenylglycine (MPPG),
(RS)-1-amino-5-phosphonoindan-1-carboxylic acid [(RS)APICA], (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA),
(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG),
(RS)-
-methyl-4-tetrazolylphenylglycine (MTPG), as well as
D(
)-2-amino-5-phosphonopentanoic acid
(D-AP5), were purchased from Tocris Cookson (Bristol, UK).
They were dissolved as stock solutions in 0.1 N NaOH, except DHPG,
which was dissolved in H2O. L-Quisqualic acid was either from Tocris Cookson or from
Research Biochemicals International (Natick, MD). It was dissolved as
stock solution in H2O. Aliquots of stock
solutions were frozen and stored at
20°C for
1 mo. DHPG was
dissolved directly before being used because it showed rapid loss of
activity in solution. GluR compounds were added to the perfusate to
yield the desired concentration after adjustment of pH, if necessary.
Pilocarpine was from Sigma (St. Louis, MO). It was made up as a stock
solution at 10 mM in Panulirus saline and was added directly
to the perfusate to yield the desired concentration. Picrotoxin (PTX)
was from Sigma or Research Biochemicals International. It was dissolved
as a concentrated stock in ethanol and diluted into the experimental solution.
Drug application
All drugs were administered by bath application. mGluR agonists
were applied onto the STG for 10 min by superfusion in the Vaseline
well and were washed out for 30-60 min thereafter. Antagonists were
also superfused for 10 min always followed by a 60-min washout. For
experiments in which agonist action was to be blocked by a specific
antagonists, the antagonist was first preapplied alone for 10 min
followed by coapplication for 10 min of antagonist and agonist. Washout
was always 60 min in coapplication experiments. To test whether mGluR
agonist action might be due to cross-reactivity with
N-methyl-D-aspartate (NMDA) receptors as
described in mammals (Breakwell et al. 1997;
Contractor et al. 1998
; Wilsch et al. 1994
), we did control experiments where D-AP5, a
potent NMDA receptor antagonist was preapplied and subsequently
coapplied at 100-250 µM with the group I agonist
L-quisqualic acid (5-10 µM) and the group II agonist
L-CCG-I (50 µM) in the same protocol as used for mGluR antagonists.
PTX was used in an identical procedure at 10-20 µM together with
L-CCG-I (50 µM) and L-AP4 (100 µM) to test for
cross-reactivity with fast glutamate-gated chloride channels.
To block modulatory inputs from commissural and esophageal ganglia, the desheathed portion of the stn was superfused in its separate well with isotonic sucrose containing 0.1-1 µM TTX, thus blocking action potential conduction. If gastric rhythms had to be induced by pilocarpine, the muscarinic agonist was superfused in the STG well at concentrations of 35-150 µM.
Electrophysiological recording techniques and data processing
Extracellular recordings from motor nerves of the STNS were made
with stainless steel electrodes connected to Model 1700 differential AC
amplifiers (A-M Systems, Carlsborg, WA). Intracellular recordings from
STG neurons were made with glass micropipettes connected to Axoclamp 2B
(Axon Instruments, Foster City, CA) and Model 1600 (A-M Systems,
Carlsborg, WA) intracellular amplifiers. Micropipette resistances were
8-15 M when filled with 3 M KCl. Gastric neurons were identified by
correlating intracellular action potential recordings with the
extracellular ones from the identified motor nerves. Extra- and
intracellular recordings were digitized at 11 kHz with a PCM recording
adapter (Model 3000A, Vetter, Rebersburg, PA) and stored on video tape
for later analysis. Data were played back on a TA6000 chart writer
(Gould Instrument Systems, Valley View, OH) and processed using
Kaleidagraph (Macintosh version 3.08d) and Sigmastat (SPSS Science,
version 2.03) software. The significance of drug effects was determined
applying one-way ANOVA using Kruskal-Wallis analysis on ranks.
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RESULTS |
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All mGluR agonists we tried had clear effects that are detailed in the following text. By contrast, most mGluR antagonists we tried had no statistically significant effects on ongoing combined or pilocarpine-induced rhythms. They were also unable to block significantly the effect of agonists specific for their respective mGluR group. In the summary table, they are indicated as "no effect detected." Except for the group-I-specific antagonist AIDA (Fig. 4), no data are shown for these negative results. For those antagonists that had clear effects on ongoing rhythms, such as (RS)APICA, or were capable of at least partially blocking the effects of mGluR agonists, such as (RS)APICA and MPPG, experimental data are also shown in the following text.
In control experiments, we tested whether the potent NMDA receptor
antagonist D-AP5 interfered with mGluR agonists that were acting on the lobster gastric circuit as has been reported in mammalian
systems (Breakwell et al. 1997; Contractor et al.
1998
; Wilsch et al. 1994
). At a concentration of
100 µM, D-AP5 had no effect on the excitatory action of 5 and 10 µM L-quisqualic acid, and 250 µM was without
effect on the inhibitory action of 50 µM L-CCG-I (data not shown). At
both concentrations D-AP5 had no effect on gastric rhythms
when applied alone (data not shown). DL-AP5 and its
analogue D-AP7 have both been shown to block crustacean NMDA receptors (Parnas et al. 1996
; Pfeiffer-Linn
and Glantz 1991
)
Nonspecific agonist ACPD stimulates gastric rhythm generation
In the gastric circuit of the STG (Fig. 1.), ACPD accelerated endogenous as well as pilocarpine-induced rhythms in a concentration-dependent manner as shown in Fig. 2 for an endogenous gastric rhythm. This rhythm-accelerating effect was reproducible and could be observed at concentrations of 50 µM; however, the effect of ACPD was highly variable. In most of our experiments, the accelerating effect of ACPD decreased continuously during the 10-min superfusion period (Fig. 2E). In a total of eight experiments in which 50-100 µM ACPD was applied, ongoing oscillations were accelerated in all recorded motoneurons. In addition, in three experiments where the stn was blocked, oscillations were reversibly induced in silent [dorsal gastric (DG), gastric mill (GM), and median gastric (MG)] or in tonically firing [DG, lateral posterior gastric (LPG) and GM] motoneurons (data not shown).
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Group I mGluR agonists L-quisqualate and DHPG stimulate gastric rhythm generation
Of the five mGluR agonists that were tried on the STG, L-quisqualic acid had the strongest activating effect, in oscillating (Fig. 3B) and nonoscillating (Fig. 3D) preparations alike (n = 14). The action of L-quisqualate was observed in all gastric motoneurons starting at concentrations as low as 5 µM. When the gastric CPG was blocked, rhythmic bursting could be induced in silent and tonically firing motoneurons by superfusion of 25 µM L-quisqualate. Characteristically, the activation of the gastric CPG by L-quisqualate was transient and decreased or ceased completely over the 10-min application period.
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Figure 3A shows the results of an experiment where a vigorous gastric rhythm was induced by 35 µM pilocarpine. On application of 25 µM L-quisqualic acid, this rhythm gradually increased in frequency (Fig. 3B). Note in Fig. 3B that while the rhythm was accelerating, motoneuron AM (amn recording) also started firing during its interburst interval. This would gradually lead to tonic firing, sometimes with some gastric modulation remaining, as seen in Fig. 3C. Intracellular recordings revealed that the development of tonic firing was due to a buildup of a sustained tonic depolarization in the electrotonically coupled motoneurons AM and DG (shown for AM in Fig. 5). In parallel with the increased tonic firing of AM and DG, the gastric rhythm often became irregular and on extended application of L-quisqualic acid became slower than initially (Fig. 3C). This figure shows that the subthreshold membrane potential oscillations recorded in neuron LPG maintained an accelerated rhythm; however, due to the failure of almost every second subthreshold potential to produce a full amplitude depolarization, an overall slower burst rhythm than the control was produced. The slower gastric rhythm can also be seen from the MG recording.
In Fig. 3D the pilocarpine and the L-quisqualate were washed out and all rhythmic gastric activity ceased. Under these conditions, motoneurons AM (amn) and GM (aln) fired tonically, whereas LPG continued to receive some cyclic modulatory input that had neither a gastric nor a pyloric frequency. Application of 25 µM of L-quisqualic acid onto the nonoscillating gastric CPG resulted in a slow depolarization and a gradual increase in amplitude of subthreshold rhythmic depolarizations in MG until full-amplitude rhythmic depolarizations developed after ~40 s. The oscillations in MG were stable for 1 min at a frequency of ~0.09 Hz, which then accelerated during the following 4 min. After ~4 min, the high-amplitude oscillations ceased and also the weak bursts in amn and aln. Instead it can be seen that AM and the GMs were firing tonically at high frequencies. In Fig. 4A the development of cycle duration in a pilocarpine-induced gastric rhythm after application of 10 µM L-quisqualic acid and 200 µM of the group-I-specific antagonist AIDA is plotted against the cycle number. Although 10 µM L-quisqualate reduced the cycle duration by 26% (30 ± 13.03%, mean ± SD, n = 5), AIDA alone had no significant effect and also did not block the effect of L-quisqualic acid (Fig. 4B). Figure 5 shows for motoneuron AM the slow depolarization that was characteristic for the effect of L-quisqualic acid in the electrotonically coupled motoneurons AM and DG. In most experiments, it would lead to sustained tonic firing, which then was accompanied by a slowed gastric rhythm.
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DHPG had effects on the gastric CPG that were similar to L-quisqualate in that it reproducibly induced rhythmic discharges in silent gastric motoneurons when the stn was blocked. As with ACPD and L-quisqualate, the effect of DHPG (n = 4) was dose dependent; however, we did not observe the slow buildup of tonic firing in motoneurons AM and DG. Instead gastric rhythms were generated that appeared stable during the 10-min application period. In three experiments, these DHPG-induced rhythms were much slower in frequency (0.05-0.01 Hz) than combined gastric rhythms (~0.1 Hz). In one experiment, however, a stable rhythm was induced that looked indistinguishable from combined rhythms and had a normal gastric frequency of 0.08 Hz (Fig. 6B). Figure 6D shows the development of cycle duration over cycle number of gastric rhythms induced by different concentrations of DHPG in a silent preparation where the stn was blocked. Note the high variability at 25 µM DHPG, whereas at 50 µM, the cycle duration increased only slightly over the application period and at 100 µM, it was stable after a few cycles of adjustment.
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Group II agonist L-CCG-I inhibits gastric rhythm generation
The effect of L-CCG-I was always inhibitory (n = 16) on both endogenous and pilocarpine-induced gastric rhythms. When a preparation in which a vigorous gastric rhythm had been induced by application of pilocarpine was superfused with L-CCG-I, rhythmic activity was either slowed or halted entirely, depending on the concentration used. Concentrations of 50 µM stopped rhythmic and tonic activity in most experiments. Figure 7A shows an example of application of 10 µM L-CCG-I that resulted in complete block of oscillatory activity in AM after only four gastric cycles. The intracellular recording indicates that the neuron was held hyperpolarized with the ability to generate plateau potentials removed. As can be seen in Fig. 7B, some high-frequency, rhythmic input remained intact, and although action potential threshold was reached several times, no plateau potentials were triggered over the entire application period of 10 min, although it appears from the extracellular recordings in aln, lpgn, and mgn that each action potential in motoneuron AM was correlated with a single CPG cycle in the other neurons. Under these conditions, plateau potentials could not be induced by depolarizing current injection. The extracellular recording from lpgn shows that the motoneurons LPG also stopped bursting. The extracellular recording from aln shows that the GM motoneurons started to fire action potentials tonically but with a frequency that decreased slowly over the application period without ever being completely silenced. The extracellular recording from nerve mgn shows that bursting in motoneuron MG became irregular but did not stop completely. As can be seen from Fig. 7C, this effect of L-CCG-I was completely reversible.
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Figure 7D shows an almost identical experiment as in Fig. 7A, however, at a higher concentration of 50 µM L-CCG-I. Again, gastric burst firing was rapidly blocked in motoneuron AM as shown in the extracellular recording in nerve amn. DG, recorded intracellularly, behaved essentially like AM shown in Fig. 7A. It remained hyperpolarized at an apparent resting potentials after only four cycles and thereafter remained silent for the 10-min application period. At 50 µM L-CCG-I, all recorded gastric cells were silenced as can be seen from the extracellular recordings of motoneurons GM, AM, and MG (Fig. 7E). The extracellular recording from nerve mgn also shows that the pyloric rhythm was inhibited by 50 µM L-CCG-I. The action of this higher concentration of L-CCG-I was fully reversible (Fig. 7F). Figure 8 shows the effect of 50 µM L-CCG-I on an endogenous gastric rhythm recorded in a combined preparation (Fig. 8A). As can be seen from Fig. 7B, rhythmic burst firing largely stopped in all gastric neurons recorded, although some subthreshold gastric modulation remained visible in LPG and AM continued to fire single action potentials or weak irregular bursts of few action potentials. The effect of L-CCG-I was fully reversible in combined preparations (Fig. 8C). The inhibitory action of L-CCG-I was not prevented by the chloride channel blocker PTX (Fig. 9) at concentrations between 10 and 20 µM (n = 3).
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The data presented in Figs. 7 and 8 (and Fig. 9 in PTX) are representative for the action of L-CCG-I on the gastric CPG in the majority of experiments. In a total of 16 experiments at concentrations of 10-100 µM, all gastric motoneurons were affected. However, the inhibitory action was always most immediate on the return-stroke phase motoneurons AM, DG, and LPG (Fig. 1), which were always the first to be affected and were silenced completely in the majority of experiments (AM: 68.8%, n = 16; DG: 85.7%, n = 14; LPG: 80%, n = 15). This particular sensitivity of AM, DG, and LPG to the inhibitory mGluR agonist L-CCG-I matched the also exceptional sensitivity of these motoneurons to the inverse action of the group II antagonist (RS)APICA (see following text and DISCUSSION). Although the powerstroke motoneurons MG, LG, and GM were also inhibited by L-CCG-I, in all experiments, they were less frequently completely silenced (MG: 11.1%, n = 9; LG 25%, n = 8; GM 50%, n = 14). Instead the powerstroke motoneurons continued irregular or tonic firing.
Antagonistic and inverse agonist action of the group-II-specific antagonist (RS)APICA
In both endogenous and pilocarpine-induced gastric rhythms the effect of the group-II-specific antagonist (RS)APICA was always opposite to the group II agonist L-CCG-I. Figure 10 shows an experiment with a pilocarpine-induced gastric rhythm (Fig. 10A). Note that in the presence of 50 µM L-CCG-I in pilocarpine (Fig. 10B), motoneurons LPG and AM were silenced although some subthreshold rhythmic activity remained visible in LPG. The latter was correlated with the continuing activity of the gastric CPG as seen from the mgn recording and also in aln. Note, however, that the GM motoneurons recorded in aln gradually switched to continuous firing. As described in the preceding text, the effect of L-CCG-I could be reversed completely by washing with saline (Fig. 10C). (RS)APICA (1 mM) partially blocked the action of 50 µM L-CCG-I when the two were coapplied (Fig. 10E). The recovery of oscillatory activity by the antagonistic effect of (RS)APICA was almost complete for the intracellularly recorded motoneuron LPG and a slightly irregular gastric rhythm was clearly discernible in all neurons except AM, which remained inhibited. In five different experiments, we never observed a complete block of the inhibitory action of 50 µM L-CCG-I by (RS)APICA when the antagonist was applied at a concentration of 1 mM.
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When superfused over the STG alone at a concentration of 1 mM (RS)APICA
had a particularly dramatic effect. As can be seen in Fig.
10D, the depolarizing phases in the return-stroke
motoneurons DG and LPG were remarkably prolonged (10 times the normal
cycle duration in 1 experiment), and the correspondingly prolonged
action potential bursts in the efferent nerve amn containing
the AM motor axon suggest the same for this motoneuron. In a separate
experiment where AM was recorded intracellularly, we confirmed that
this was indeed the case under identical conditions of drug application (data not shown). The effect of (RS)APICA on combined gastric rhythms
was principally identical to that on muscarinic agonist-induced rhythms, although less dramatic (Fig.
11). The prolongation of intracellular
depolarizations and axonal bursts generated by motoneurons AM, DG, and
LPG oscillating in an endogenous rhythm was about four times their
normal duration before (RS)APICA was applied for 10 min
(n = 3). Also, Fig. 11 shows clearly the augmented
inhibition of LG that resulted in low-frequency oscillation. As with
pilocarpine-induced rhythms (Fig. 10F), the effect of
(RS)APICA was fully reversible on washing with saline (Fig.
11C).
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Effect of group-III-specific mGluR agonist L-AP4 is similar to that of L-CCG-I
Mammalian group III mGluRs, which, like group II mGluRs, are
coupled to adenylylcyclase, are selectively activated by the agonist
L-AP4. In our experiments (n = 4), we found
that the action of L-AP4 on gastric rhythm generation was
always inhibitory as was L-CCG-I. However, the sensitivity of all
gastric cells was lower to L-AP4 than to L-CCG-I at
identical concentrations. Normally concentrations of 100 µM of the
group III agonist had to be administered to evoke effects comparable
with those observed with 50 µM of the group II agonist. Figure
12 shows an experiment in which 100 µM L-AP4 was superfused over an STG preparation
oscillating with a pilocarpine-induced rhythm (Fig. 12A).
The figure shows clearly that L-AP4 at a concentration of
100 µM blocked rhythm generation in all gastric neurons that we
recorded (Fig. 12B). The intracellularly recorded LPG neuron
remained silent at its apparent resting potential and the other
recorded cells fired action potentials tonically. The effect of
L-AP4 was reversible (Fig. 12C). The inhibitory
effect of L -AP4 was not prevented by 10-20 µM PTX (data
not shown).
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Effect of L-AP4 is blocked by group-III-specific antagonist MPPG
Figure 13 shows an experiment on a stable rhythm recorded in a combined preparation (Fig. 13A), where the inhibitory effect on the gastric CPG of 100 µM L-AP4 (Fig. 13B), was blocked by the group-III-specific antagonist MPPG at a concentration of 1 mM (Fig. 13C). Recordings were made extracellularly from motoneurons GM in nerve aln and from motoneuron LG recorded in nerve lgn. As in all experiments with antagonists, MPPG was first preapplied alone for 10 min before being coapplied with 100 µM L-AP4. Preapplication of the antagonist alone had no detectable effect on the gastric rhythm (data not shown). However, in the presence of 1 mM MPPG, the gastric rhythm was unaffected by 100 µM L-AP4 (Fig. 13C). As Fig. 13D shows, the effects of agonists and antagonists were reversible.
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DISCUSSION |
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The principal result of our study is that agonists and antagonists
that are specific for mGluR types I-III have clear and distinct
effects on the gastric CPG (summarized in Table
1). This allows us to put forward the
hypothesis that mGluRs of distinct groups are likely to be present in
the gastric circuit of the STG and could be important for normal CPG
function. Although never investigated before, the presence and
modulatory role of mGluRs in the decapod STG does not come
unexpectedly. Slow excitatory and inhibitory effects of
L-glutamate, when ejected onto STG neurons, have been
described in the past (Marder and Paupardin-Tritsch 1978; Tazaki and Chiba 1994
), suggesting
multiple mechanisms of action for glutamate. However, the level of
detail to which the electrophysiological pharmacology of mGluR agonists
in the gastric network resembles the action of these compounds in
mammals, as well as the potent effect of some of them on ongoing
rhythms, is remarkable and clearly suggests a role for glutamate as a
modulatory substance in the STG, acting on different types of mGluRs
expressed differentially in the STG. Similar data have been obtained
for the pyloric circuit of the lobster STG (N. L. Pérez-Acevedo, W. D. Krenz, Y. I. Arshavsky, and
A. I. Selverston, unpublished data).
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Can we conclude that lobster homologs of group I-III mGluR genes are
expressed in STG neurons? A definitive answer to this question will
have to await cloning of Panulirus mGluR genes so that
sequences and pharmacologies of individual recombinant receptors can be
determined and compared with mammalian receptors. This careful
consideration seems to be appropriate in the light of the sometimes
unusual pharmacology of invertebrate homologs of mammalian channels and
receptors (Benson 1988; Darlison 1992
; Jackel et al. 1994a
,b
). The only invertebrate mGluR gene
cloned thus far comes from Drosophila melanogaster. Amino
acid sequence and pharmacology of the recombinant receptor place it
closest to mammalian group II (Parmentier et al. 1996
).
Unfortunately, no in vivo pharmacology of Drosophila mGluRs
has yet been published, therefore we do not know whether native
Drosophila mGluRs show any similarity with group I, II, or
III pharmacology and whether intracellular messenger coupling is
conserved. Interestingly, first evaluations of the recently completed
Caenorhabditis elegans genome project indicate that this
simple nematode has at least four genes coding for mGluRs
(Bargmann 1998
). It is therefore reasonable to assume
that in more complex invertebrate animals mGluRs may be present in a
diversity as impressive as in vertebrates.
Specificity of the responses to mGluR compounds
Any study of the action of drugs on the electrophysiology of nerve cells has to fulfill a number of criteria to be considered meaningful for the characterization of a respective receptor: the drug should be effective at low concentrations to avoid nonspecific effects, the effect of the drug should be dose dependent, it should be at least partially reversible, and it should be antagonized by specific antagonists. If we take as a reference the data available from cloned and exogenously expressed mammalian mGluRs and those from the cloned Drosophila mGluR, then the effects of mGluR compounds we describe here for the gastric circuit of the Panulirus STG are specific and significant. Threshold doses for clearly identifiable effects of agonists for all three groups were at concentrations of between 5 and 50 µM. Considering that we measured effects at the network level in an intact ganglion where access of drugs to the receptor binding sites is likely to be not as immediate and rapid as for receptors expressed exogenously in cell lines or in Xenopus oocytes, these concentrations can be regarded as low. Also it is important to note that dose-response relationships obtained at the circuit level may be misleading because drugs applied to the STG act onto all susceptible elements of the circuit simultaneously, potentially leading to complex, concentration- and time-dependent interactions that may be difficult to interpret.
A number of studies have shown that mGluR compounds have significant
nonspecific cross-reactive effects on native and exogenously expressed
NMDA receptors (Breakwell et al. 1997; Contractor
et al. 1998
; Wilsch et al. 1994
). Therefore
although responses to NMDA have never been described in STG neurons, we
tested whether responses to mGluR compounds were antagonized by
D-AP5, a widely used potent NMDA receptor antagonist. Our
negative results further strengthen the case for specific
mGluR-mediated modulation within the lobster STG. Similarly, PTX, a
chloride channel blocker frequently used to block fast synaptic
inhibition within the STG (Bidaut 1980
) failed to block
the inhibitory action of L-CCG-I and L-AP4. This indicates
that there is no significant interaction between mGluR agonists and the
fast synaptic glutamate-gated chloride channels.
In our investigation, antagonists always required much higher doses
than agonists for clear effects. Concentrations of 1 mM were needed to
block the action of agonists at least partially, and blocks were only
obtained with (RS)APICA and MPPG. This finding is reminiscent of the
effect of mGluR antagonists in many mammalian preparations where
IC50 values for antagonists are frequently high
(Conn and Pin 1997), mandating high concentrations for
complete blockade of agonist responses. Again the observation that
invertebrate receptors may have significantly different pharmacologies
than their vertebrate counterparts (Benson 1988
;
Darlison 1992
; Jackel et al. 1994a
,b
)
offers the most likely interpretation for the inability of most
antagonists to block mGluR agonists.
Intracellular messenger coupling
It is known that cyclic AMP is involved as an intracellular
messenger in modulation of at least some STG neurons by biogenic amines
(Flamm et al. 1987; Hempel et al. 1996
).
Also application of the muscarinic agonist pilocarpine induces a rise
in intracellular cyclic AMP in the pyloric pacemaker neuron AB
(Hempel et al. 1996
). No such information is available
for responses to glutamate in the STG; however, a G-protein-coupled
activation cascade has been suggested for presynaptic GluRs at the
crustacean NMJ (Michel et al. 1991
; Miwa et al.
1987
, 1990
). mGluRs of type II and III are coupled to adenylyl
cyclase and reduce cyclic AMP synthesis in mammalian systems. In the
STG, modulatory compounds that are capable of inducing CPG activation,
such as biogenic amines and muscarinic agonists, increase cyclic AMP
production in pyloric neurons and the gastric neurons GM. Thus it
appears reasonable to hypothesize that group II and III agonists that
reduce or block gastric CPG activity, known from other systems to
reduce cyclic AMP synthesis, are probably also inhibiting cyclic AMP
synthesis in the STG. The particularly strong inverse agonist action on pilocarpine induced gastric rhythms would be in accordance with this
notion. Clearly more research is needed to elucidate this possibility.
In mammals it has been demonstrated in several systems that activation
of mGluRs, especially by group I agonists and the nonspecific ligand
ACPD, results in release of calcium from intracellular stores
(Geiling and Schild 1996; Linden et al.
1994
; Netzeband et al. 1997
). The same was found
in leech glial cells (Lohr and Deitmer 1997
). Caffeine,
known to release calcium from the endoplasmatic reticulum in neurons,
induces rhythmic discharges of the pyloric CPG in crayfish.
(Hermann 1981
). Because group I agonists also induce
rhythmic discharges in the gastric and pyloric networks (N. L. Pérez-Azevedo, W. D. Krenz, Y. I. Arshavsky, and A. I. Selverston, unpublished data), it could be that in the
crustacean STG type I mGluRs also may act via an increase in
intracellular calcium concentration by release from intracellular
stores. Again, more research is needed to address this question.
Functional implications of mGluRs in the STG
Studies at the network level do not give information about target cells and real dose-dependence relationships. However, the differential effects of group-specific compounds on rhythm generation suggest that different types of mGluRs may be present in the STG, specific activation of the different types of mGluRs leads to differential, sometimes opposite actions on the gastric rhythm, and in particular the group-II-specific drugs show apparent preference for action on the return-stroke motoneurons AM, DG, and LPG (Figs. 7-11) Again, we cannot claim that the apparent preference of group-II-specific mGluR agonists and antagonists is the result of the expression of group II mGluRs in the membranes of AM, DG, and LPG or whether they are under modulatory control of other neurons that express these mGluRs. However, it can be inferred from our data that group II mGluRs could play a key role in the regulation of rhythm generation in this functional subgroup of neurons (Fig. 1). Our results make them key candidates for cellular studies.
In our experiments, the group-II-specific antagonist (RS)APICA had a
very strong effect on ongoing endogenous and pilocarpine-induced rhythms (Figs. 10 and 11). It is possible, that this effect of
(RS)APICA is due to its peculiar inverse agonist action as described
earlier in recombinant mammalian receptors (Ma et al.
1997). An alternative explanation would be that, during
rhythmic behavior, the antagonist is blocking a population of
group-II-like mGluRs. This would imply that during endogenous as well
as pilocarpine-induced gastric rhythms, glutamate release may be
involved in the regulation of membrane conductances that determine the
oscillatory behavior of the gastric CPG. According to this model, group
II mGluRs would be acting in a feedback loop regulating the duration of
plateau potentials. Our experimental data do not allow us to
distinguish between the two possibilities. Evidently the two mechanisms
of action may well be acting synergistically to produce the strong effect of (RS)APICA.
One of the interesting properties of mGluRs is the fact that some of
the subtypes show significant desensitization (Guérineau et al. 1997; Herrero et al. 1994
) on sustained
presence of glutamate and specific ligands as studied in detail in
recombinant mGluRs (Gereau and Heinemann 1998
). Recent
data from mammals provide evidence that receptor desensitization may
play a role in dynamic switching of presynaptic mGluR function from
facilitation to depression of neurotransmitter release at mammalian
hippocampal synapses (Herrero et al. 1998
;
Rodríguez-Moreno et al. 1998
). In our
experiments, the effects of group I agonists showed transient
characteristics in most experiments. Although we cannot exclude the
possibility of desensitizing receptors, alternative explanations that
implicate changing mutual inhibition of neurons by sequential
activation of different signaling pathways in the continued presence of
group-I-specific mGluR ligands are equally possible. Experiments with
individual isolated neurons have to be performed to clarify these questions.
Possible sources for the modulatory action of glutamate
Two possible sources for glutamate acting as a neuromodulator
within the STG may be considered. These may be acting either in
isolation or in combination and in a time- and/or activity-dependent manner. The first possibility would refer to extrinsic modulation (Katz 1995). Neurons in the higher ganglia or in the
brain may project into the STG where they release
L-glutamate exclusively or as a cotransmitter. Examples of
extrinsic modulation are well documented in the STG of decapod
crustacea. Examples include the gastropyloric muscle receptor GPR
(Katz and Harris-Warrick 1989
; Katz et al.
1989
; Kiehn and Harris-Warrick 1992
) and the
modulatory neuron APM, which has its cell body in the esophageal
ganglion (Dickinson et al. 1988
; Nagy et al.
1988
). In this scenario, the targets for glutamate could be STG
motoneurons directly or nerve endings that release a neuromodulator
other than glutamate, which in turn would excite or inhibit target
cells. Presynaptic inhibition has indeed been shown in the crab STG to
be involved in motor pattern selection (Bartos and Nusbaum
1997
). In addition, positive or negative feedback via
presynaptic autoreceptors is possible as described in vertebrates
(Cochilla and Alford 1998
) and also suggested for the
crustacean neuromuscular junction (Miwa et al. 1993
;
Parnas et al. 1994
, 1996
; Schramm and Dudel 1997
). At present we cannot distinguish between these different possibilities.
A second alternative is intrinsic modulation (Katz
1995), where L-glutamate from the fast inhibitory
glutamatergic synapses within the ganglion would diffuse away from the
synaptic cleft and reach para- or extrasynaptic metabotropic receptors,
which again may be localized on the pre- or postsynaptic side. If
located presynaptically, they could enhance or suppress transmitter
release (Schramm and Dudel 1997
). As has been suggested
from work in vertebrates, the degree of diffusion of glutamate to
mGluRs remote from the subsynaptic membrane may depend on the activity
level of the synapse (Min et al. 1998
). If located
postsynaptically, they could act on various postsynaptic currents or
modulate intrinsic electrical membrane properties leading to
excitation, oscillation, or inhibition. Intrinsic modulation by
glutamate is particularly interesting because much of the
neurotransmitter release in the STG is graded (Graubard et al.
1983
; Raper 1979
). Therefore the effective
output of glutamate into the synaptic cleft, and as a consequence
diffusion to more remote synaptic sites or presynaptic receptors, might be under dynamic control of oscillatory cycles. Evidently extrinsic and
intrinsic modulation are not mutually exclusive but instead may very
well act in concert.
We clearly have shown that mGluR-specific compounds have important activating and inactivating effects on motor patterns generated by the gastric circuit of the lobster STG. Now we have to elucidate the molecular identity of the metabotropic receptors involved, their localization in specific identified motoneurons of the STG, their intracellular messenger coupling, their evolutionary relationship with mammalian mGluRs, and the conditions under which they are activated in the intact isolated STNS and further in the intact animal. The latter parameter is of particular importance when one wants to understand the modulatory role of glutamate in the STG.
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ACKNOWLEDGMENTS |
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We thank Drs. D. Baro, Y. Arshavsky, R. Levi, and F. Nagy for critically reading the manuscript. Also, the remarks of two anonymous reviewers have greatly improved the manuscript. We thank S. Ramirez for expert technical assistance. Part of this work has appeared in the form of abstracts.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-09322. N. L. Pérez-Acevedo was supported by the minority fellowship program of the American Psychological Association, Grant T32MH18882.
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FOOTNOTES |
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Address for reprint requests: W. D. Krenz, Institute of Neurobiology, Medical Sciences Campus, University of Puerto Rico, 201 Blvd. Del Valle, San Juan, PR 00901.
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 23 April 1999; accepted in final form 19 October 1999.
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