Glutamate is the Transmitter for N2v Retraction Phase Interneurons of the Lymnaea Feeding System

M. J. Brierley, M. S. Yeoman, and P. R. Benjamin

Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton, East Sussex BN1 9QG, United Kingdom

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Brierley, Matthew J., Mark S. Yeoman, and Paul R. Benjamin. Glutamate is the transmitter for the N2v retraction phase interneurons of the Lymnaea feeding system. J. Neurophysiol. 78: 3408-3414, 1997. Electrophysiological and pharmacological methods were used to examine the role of glutamate in mediating the excitatory and inhibitory responses produced by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network. The N2v right-arrow B3 motor neuron excitatory synaptic response could be mimicked by focal or bath application of L-glutamate at concentrations of >= 10-3 M. Quisqualate and alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were potent agonists for the B3 excitatory glutamate receptor (10-3 M), whereas kainate only produced very weak responses at the same concentration. This suggested that non-N-methyl-D-aspartate (NMDA), AMPA/quisqualate receptors were present on the B3 cell. The specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10-5 M) blocked 85% of the excitatory effects on the B3 cell produced by focal application of glutamate (10-3 M), confirming the presence of non-NMDA receptors. CNQX also blocked the major part of the excitatory postsynaptic potentials on the B3 cell produced by spontaneous or current-evoked bursts of spikes in the N2v cell. As with focal application of glutamate, a small delayed component remained that was CNQX insensitive. This provided direct evidence that glutamate acting via receptors of the non-NMDA, AMPA/quisqualate type were responsible for mediating the main N2v right-arrow B3 cell excitatory response. NMDA at 10-2 M also excited the B3 cell, but the effects were much more variable in size and absent in one-third of the 25 B3 cells tested. NMDA effects on B3 cells were not enhanced by bath application of glycine at 10-4 M or reduction of Mg2+ concentration in the saline to zero, suggesting the absence of typical NMDA receptors. The variability of the B3 cell responses to NMDA suggested these receptors were unlikely to be the main receptor type involved with N2v right-arrow B3 excitation. Quisqualate and AMPA at 10-3 M also mimicked N2v inhibitory effects on the B7 and B8 feeding motor neurons and the modulatory slow oscillator (SO) interneuron, providing further evidence for the role of AMPA/quisqualate receptors. Similar effects were seen with glutamate at the same concentration. However, CNQX could not block either glutamate or N2v inhibitory postsynaptic responses on the B7, B8, or SO cells, suggesting a different glutamate receptor subtype for inhibitory responses compared with those responsible for N2v right-arrow B3 excitation. We conclude that glutamate is a strong candidate transmitter for the N2v cells and that AMPA/quisquate receptors of different subtypes are likely to be responsible for the excitatory and inhibitory postsynaptic responses.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Pharmacological analysis of central pattern generator (CPG) circuits in vertebrates generating rhythmic motoractivity emphasized the importance of the amino acid, glutamate, as a neurotransmitter. For instance, in lower vertebrates such as the lamprey and the Xenopus tadpole, rhythmic motor activity in the spinal swim system can be initiated by glutamate-mimicking activity in descending glutamatergic reticulospinal interneurons (Grillner et al. 1994). In addition, glutamate is also important as a transmitter in excitatory interneurons that form part of the segmental pattern-generating network (Grillner et al. 1994; Roberts 1989). In both species, N-methyl-D-aspartate (NMDA)and non-NMDA receptors, alpha -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate are involved (Dale and Grillner 1986; Dale and Roberts 1984). The importance of glutamate as a transmitter in invertebrate rhythm generator networks is not as well established. The exception to this is the crustacean stomatogastric system where the importance of glutamate has been known for a considerable time (Cleland and Selverston 1995; Marder and Paupardin-Tritsch 1978). It generally is accepted that glutamate is a CNS and peripheral neurotransmitter in arthropods and mollusks (e.g., Gerschenfeld 1973; S.-Rózsa 1984; Shinozaki 1988; Walker 1986).

Two recent reports suggested that glutamate receptors are important in the molluscan feeding system (Katz and Levitan 1993; Quinlan and Murphy 1991). This indicated that glutamate was likely to be a transmitter in neurons of the premotor interneuronal CPG network (Quinlan et al. 1995). The CPG feeding network is particularly well understood in the pond snail Lymnaea, and this offered the opportunity to investigate the possible presence of glutamatergic interneurons. This paper reports on the novel type of retraction phase interneuron, the N2v (N2 ventral), of which the plateauing properties and synaptic connections with motor neurons were described in Brierley et al. (1997a). We will show that its synaptic responses on feeding motor neurons (Brierley et al. 1997a) and an interneuron known as the slow oscillator (SO) (Elliott and Benjamin 1985; Rose and Benjamin 1981) appeared to be mediated by L-glutamate. Glutamate reproduced both excitatory and inhibitory postsynaptic effects on different postsynaptic neurons of the N2v cells as did the glutamate receptor agonists quisqualate and AMPA. NMDA produced occasional but inconsistent effects on N2v postsynaptic cells that were not enhanced by glycine or zero Mg2+. 6-cyano-7-nitroquinoxolaline-2,3-dione (CNQX) blocked an excitatory monosynaptic N2v right-arrow motor neuron effect, suggesting that the Lymnaea glutamate receptor responsible for the N2v excitatory response might be related to the vertebrate non-NMDA, AMPA/quisqualate type of ionotropic receptor. At least one other subtype of glutamate receptor must be responsible for the inhibitory effect of glutamate and AMPA on the N2v postsynaptic cells, which is CNQX insensitive.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

The methods were the same as those described in Brierley et al. (1997a) for the isolated CNS preparation. The "twisted preparation" was used to record simultaneously the ventrally located N2v cells and postsynaptic cells on the dorsal surface.

Cell types recorded

The large motor neurons B3, B7, and B8 were located visually (Benjamin and Rose 1979), and their identity confirmed by their characteristic firing patterns and synaptic inputs during spontaneous or SO-driven fictive feeding patterns. The SO is a single cell usually occurring between the B1 and B2 cells (Elliott and Benjamin 1985). The N2v cells are a single pair of small cells lying medially on the ventral surface of left and right buccal ganglia (Brierley et al. 1997a). Their high-frequency firing superimposed on a large depolarizing plateau potential was not seen in other ventrally located cells (Brierley et al. 1997a).

Pharmacology

A variety of glutamate agonists or antagonists were bath perfused across the isolated CNS preparation. Also focal application of agonists to the cell bodies of postsynaptic cells of the N2v cells was carried out using pressure pulses (usually of 1-s duration and <= 40 psi pressure) applied to a drug-filled micropipette. n values in the text refer to the number of cells tested. At least three applications of agonist were carried out for each cell. Normal N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (Benjamin and Winlow 1981) was applied continuously across the CNS during agonist application. A minimumperiod of 3 min was allowed between successive drug applications, sufficient to allow responses to return to control levels. High Mg2+/nominally zero Ca2+ was used to reduce spontaneous synaptic input to the target cell (composition in Yeoman et al. 1993) and to test for direct effects of agonists on target cells. It was possible that Mg2+ in normal saline (2 mM Mg2+) might be blocking NMDA-like glutamate receptors. To test this, saline in which the Mg2+ was replaced by Ca2+ ions (6 mM) was used. Glycine is known to be a coagonist for NMDA receptors (Johnson and Ascher 1987), and so it was used in Lymnaea in an attempt to amplify NMDA responses. 10-4 M glycine was first bath perfused for 10 min and then a glycine (10-4 M)/NMDA (10-3 M) mixture was applied focally to the surface of the B3 motor neurons via a pressure pipette and compared with NMDA pressure applied alone to the same cell. Glutamate agonists were: L-glutamate, kainate (Sigma-Aldrich, Dorset, UK), AMPA, quisqualate, ibotenate, NMDA (Tocris Cookson, Bristol, UK). Glutamate antagonists were CNQX, (Tocris Cookson), kyurinic acid, glutamate diethyl ester (GDEE), and DL-aminopimelic acid (Sigma-Aldrich).

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Glutamate receptors on B3 motor neurons

The B3 neurons are a bilaterally symmetrical pair of rasp phase motor neurons of the feeding network (Benjamin and Rose 1979). They were chosen as a model cell for studying the excitatory effects of glutamate because they are easy to identify and reliably excited by the N2v cells via a monosynaptic chemical synapse (Brierley et al. 1997a). While the majority of the data presented used the B3 motor neurons as a target cell of the excitatory action of the N2v, a number of other N2v postsynaptic cells also were studied, particularly those that are inhibited by the N2vs. All probably are innervated monosynaptically by the N2v cells (Brierley et al. 1997a). The type of synaptic input they receive from the N2v cells is summarized in Fig. 1.


View larger version (14K):
[in this window]
[in a new window]
 
FIG. 1. Summary of monosynaptic connections (bullet , inhibitory; , excitatory) made by the rasp phase feeding interneuron, N2v.

Focal application of 10-2 M L-glutamate (Fig. 2A) strongly depolarized the B3 cells (n = 41). Usually a biphasic depolarizing effect was seen with a rapid initial phase followed by a larger amplitude long-duration phase lasting many seconds (1 and 2 in Fig. 2Bi). At 10-2 M, this second phase usually produced spikes (Fig. 2A). The threshold concentration was typically ~10-3 M in this cell type. This was the pipette concentration, and the actual concentration at the receptor was likely to be up to two orders of magnitude lower in the continuous saline flow system used here (Walden et al. 1988). Similar high concentrations of glutamate were required to produce depolarizing effects on other molluscan neurons in intact ganglia (Dale and Kandel 1993; Quinlan and Murphy 1991).


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 2. Focal application of L-glutamate and N-methyl-D-aspartate (NMDA) to the cell body of the B3 feeding motoneuron. A: glutamate produced much stronger depolarization response compared with NMDA at the same concentration. Bi: biphasic response (1 and 2) to 10-3 M glutamate. Bii: bath application of glycine for 10 min did not significantly increase the amplitude of the response of the B3 cell to focal application of glutamate compared with the control application of glutamate alone. Ci: some B3 cells showed a strong response to NMDA where 2 phases of the response (1 and 2) could more clearly be distinguished. Cii: bath application of glycine for 10 min did not enhance the amplitude of the glutamate response. Focal applications were for 1 s.

To classify the type of excitatory glutamate receptor present on the B3 cells in more detail, a variety of glutamate agonists were applied focally to the B3 cells. Initially the experiments were aimed at distinguishing between NMDA and non-NMDA-like receptors. When the effects of glutamate were compared with those of NMDA on the same cells (n = 25), it was found that glutamate produced a consistent depolarizing effect, whereas the effect of NMDA was much more variable. In some cells (n = 4), 10-2 M NMDA produced weak depolarizing effects compared with the much larger effects of glutamate at the same concentration (Fig. 2A). In other experiments, much larger effects of NMDA were seen on the B3 cells (Fig. 2Ci, n = 12). These effects were biphasic and consistently slower to reach peak amplitude than glutamate applied to the same cell (compare Fig. 2, Ci with Bi). This made it easier to distinguish between the two phases (1 and 2 in Fig. 2Ci) of the response with NMDA compared with glutamate. However, in about one-third of cells (n = 9), no effects of NMDA were seen at all. As spontaneous excitatory input due to N2v activity (Brierley et al. 1997a) was still present on the B3 cells in the same preparations, it seemed unlikely that the NMDA receptor could be main type of glutamate receptor responsible for the N2v synaptic input to the B3 motor neurons (confirmed by antagonist experiments, see further text).

Experiments were carried out to see if bath application of the glutamate coagonist glycine (10-5 M) could enhance the effects of focal application of NMDA or glutamate on the B3 cells (n = 10). These experiments showed no consistent increase in NMDA response neither in cells that had previously responded strongly to NMDA (n = 4, Fig. 2C, i and ii) nor in those cells that responded only weakly to NMDA applied alone (not shown). On the same cells, coapplication of glutamate (10-3 M) with glycine (10-5 M) also was tried, again without significantly enhancing the effect compared with glutamate applied alone (Fig. 2Bii). Neither did reducing saline magnesium concentration to zero reveal or enhance the effects of NMDA or glutamate (n = 4, not shown).

We assumed that the absence of a consistent response to NMDA indicated that a non-NMDA type of excitatory glutamate receptor was more likely to be responsible for the N2v-induced synaptic response on the B3 cells. Therefore, a more detailed analysis of non-NMDA agonists quisqualate and kainate was carried out by comparing their effects on the same cell. Quisqualate at 10-3 M pipette concentration produced a very strong excitatory effect (n = 9) and consistently depolarized the B3 cells more than glutamate at the same concentration (Fig. 3A). Kainate had weaker depolarizing effects than either glutamate or quisqualate but still produced a clear depolarization (Fig. 3A). The effects of glutamate and quisqualate persisted in high Mg2+, nominally zero Ca2+ saline (Fig. 3C, i and ii). This saline blocks chemical synapses (Brierley et al. 1997a) and was used to provide evidence that glutamate is acting directly on receptors on the B3 motor neuron.


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 3. Responses of the B3 motoneuron to the non-NMDA agonists quisqualate and kainate. A: focal application of quisqualate to the cell body produced larger depolarizing responses compared with L-glutamate or kainate applied at the same concentration. B: alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) but not ibotenate could reproduce the depolarizing responses. Ci: glutamate responses persisted in high Mg2+/zero calcium saline, which blocks chemical synapses. Cii: quisqualate responses similarly persisted in this altered saline. Both Ci and Cii suggest that the glutamate agonists are acting directly on the B3 receptors. Focal applications were for 1 s.

The depolarizing effect on the B3 cells evoked by quisqualate was analyzed further using two additional agonists that in vertebrates discriminate between the ionotropic (AMPA) and metabotropic (ibotenate) subtypes of receptors that quisqualate can activate.

AMPA depolarized the B3 cells, like glutamate (Fig. 3B) in a high Mg2+, nominally zero Ca2+ saline that was used to block synaptic transmission. Unlike AMPA, the non-NMDA metabotropic agonist, ibotenate, did not affect B3 (Fig. 3B) on the same cell (n = 3). However, ibotenate is not a well-established metabotropic agent in mollusks, and it is still possible that the slow responses to glutamate on Lymnaea motor neurons involve nonligand gated ion channels.

In summary, there appeared to be a non-NMDA, AMPA/quisqualate-sensitive receptor mediating the depolarizing effects on the B3 neurons. NMDA depolarized the B3 cells in some preparations, indicating the possible presence of NMDA receptors, but as these were not enhanced by the presence of glycine or by lowering the concentration of Mg2+ in the saline, they appear to be atypical in their pharmacological properties (Johnson and Ascher 1987; Nowak et al. 1984).


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 4. Both L-glutamate and quisqualate mimicked the inhibitory responses of the N2v cell on the B7 and B8 feeding motoneurons and the slow oscillator (SO) modulatory interneuron. A: bath application of 10-3 M glutamate generates hyperpolarization of the B7 and the opposite depolarizing response on the B3 cell. B: bath application of 10-4 quisquate excited the B3 cell and inhibited the B7 and B8 motoneurons. Ci: focal application of glutamate onto a SO cell body mimicked the spontaneous inhibiting synaptic inputs (star ) received by the SO. These are known to originate from the N2v central pattern generator interneurons. Horizontal bars are the duration of bath application of the agonists.


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 5. AMPA produces both inhibitory and excitatory responses on feeding neurons. A: AMPA mimicked the ability of L-glutamate to inhibit the spontaneous spike activity in the SO modulatory interneuron. As this experiment was carried out in a high Mg2+/zero Ca2+ saline, the responses were presumably due to receptors on the postsynaptic SO cell. B: opposite depolarizing responses were produced on the B3 motoneuron.

Quisqualate/AMPA responses on other N2v postsynaptic cells

Quisqualate and AMPA both mimic the depolarizing effects that N2v cells have on B3 neurons (Fig. 2), and this was initial evidence that glutamate may be the N2v excitatory transmitter. Further evidence would be provided for this hypothesis if glutamate or its agonists (quisqualate and/or AMPA) also could mimic inhibitory effects on other types of motoneurons, the B7 and B8 cells and the SO modulatory interneuron, other postsynaptic cells of the N2vs (Fig. 1). This was shown to be true in Fig. 4, where bath application of glutamate (Fig. 4A) or quisqualate (Fig. 4B) both produced inhibition (B7 and B8) and the opposite excitatory effect on the B3 cell on buccal motor neurons recorded at the same time (n = 3, for this combination of cells). The other cell tested, the SO, also was inhibited by focal application of both glutamate (Fig. 4Ci) and quisqualate (Fig. 4Cii), and this mimicked spontaneous N2 inputs occurring on the same cell (Fig. 4C). Brierley et al. (1997b) showed that this spontaneous hyperpolarizing input originated from the N2v cells. AMPA also consistently produced an effect on the SO that, again, was inhibitory, mimicking the N2v input to this cell type (Fig. 5A). These experiments were performed in high Mg2+/nominally zero Ca2+ saline, indicating that the recorded effects were due to the activation of glutamate receptors on the SO. In comparing the effects of AMPA and glutamate on the SO and B3 in the same preparation (Fig. 5, A and B), it can be seen that both agonists produce the same response on any particular cell type (hyperpolarizing on the SO, depolarizing on the B3).


View larger version (10K):
[in this window]
[in a new window]
 
FIG. 6. Bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) progressively blocks the N2v-induced depolarizing response on the B3 postsynaptic cell but leaves a small delayed response. CNQX was applied continuously from the beginning of the record.


View larger version (11K):
[in this window]
[in a new window]
 
FIG. 7. A 10-min bath perfusion with 10-5 M CNQX partially, but never completely, blocks the response to focal application of 10-3 glutamate. Response returns on washing for 15 min.

Blocking of N2v spontaneous and evoked excitatory inputs on B3 motor neurons by CNQX

Although mimicking N2v inhibitory or excitatory effects on appropriate postsynaptic cells is consistent with glutamate being the N2v transmitter, more conclusive evidence would come from blocking the synaptic effect of the N2v cells on their target neurons using a specific glutamate receptor antagonist. This has been successful, but only in the case of the excitatory N2v right-arrow B3 response on the B3 motor neuron (n = 4). An example of this is shown in Fig. 6, where excitatory postsynaptic potentials (EPSPs) and spike activity in the B3 cell, evoked by spontaneous bursts of N2v spikes, were blocked progressively by the bath application of the non-NMDA antagonist, CNQX. Eighty-five percent of the compound EPSP was blocked after a 45-s perfusion with 10-5 M CNQX. This was partially reversible after a 30-min wash in normal saline (not shown). A variety of other broad spectrum glutamate antagonists (GDEE, kyurinic acid, aminopimelic acid) were ineffective in blocking the excitatory effect of glutamate in these spontaneously active preparations (not shown).

Although CNQX blocked the excitatory postsynaptic effects of the N2v, it had no effect on the inhibitory effects on other postsynaptic cells. Long-term perfusion of CNQX [or other antagonists, 2-amino-5-phosphonobutyric acid (AP5), GDEE] did not block the inhibitory effect of the B8 cell or other identified neurons normally inhibited by the N2vs, suggesting that a different receptor subtype is involved (not shown).

Although CNQX is a well-known non-NMDA blocker, it was necessary to show that it could block the excitatory effects of glutamate on the specific system used here. This would confirm that blocking the N2v excitatory inputs to the B3 with CNQX was blocking glutamate receptors. The experiment shown in Fig. 7 showed that bath perfusion of 10-5 M CNQX reversibly blocked the majority of the depolarizing effects of focally applied 10-3 M glutamate (n = 2). A small delayed component of depolarization remained that was CNQX insensitive. The same concentration of CNQX then was used to block the depolarizing response on the B3 produced by current induced bursts of N2v spikes. It was important to carry out this type of experiment because the excitatory inputs on B3 of the type shown in Fig. 6A could have been due to simultaneous input to both N2v and B3 from a completely different interneuron. The experiment shown in Fig. 8 and other similar ones showed that a 10-min perfusion substantially reduced the amplitude of the B3 depolarizing postsynaptic potential (n = 6), without completely eliminating it. It recovered after washing for 30 min in normal HEPES-buffered saline. Care has to be taken in interpreting this result because of the difficulty in activating the same number of spikes in control and experimental traces. It should be noted that in the experiment like the one shown in Fig. 8, where N2v activity was evoked artificially by current injection, a two-component EPSP occurred, seen best after the wash (arrowed). Both of these components were shown previously to be due to direct effects of the N2v cells (Brierley et al. 1997a) and could be evoked by glutamate application (Fig. 2Bi). As with glutamate, a late component of the N2v-induced depolarization remained after CNQX perfusion, again indicating a CNQX-insensitive component of the synaptic potentials (see also Fig. 6).


View larger version (17K):
[in this window]
[in a new window]
 
FIG. 8. A 10-min bath perfusion of CNQX partially blocks the neuronally induced depolarizing response on the B3 motoneuron. Bursts of N2v spikes were induced by current injection through the (unbalanced) recording electrode. Washing for 15 min did not completely recover the response, and this allowed the 2 depolarizing components of the neuronal response (1 and 2) to be seen clearly.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The objective of the work described in this paper was to obtain evidence for glutamate as a transmitter in an identified type of interneuron, the N2v cells, of the Lymnaea feeding system. The N2v excites some feeding motor neurons (B3) and inhibits others (B7 and B8) (Brierley et al. 1997a), and so a pharmacological characterization of the glutamate receptors was carried out on these postsynaptic cells to see if glutamate agonists mimicked both types of postsynaptic effects of N2v neuronal stimulation. Chemical synapses were blocked routinely with high Mg2+, nominally zero Ca2+ and compared with normal saline to eliminate possible indirect effects of applied agonists on cells other than the N2v postsynaptic cells. Focal application of glutamate to the surface of these postsynaptic cells was successful in reproducing the pattern of inhibitory and excitatory responses in these different motor neurons and also the inhibitory response on the modulatory interneuron SO, also thought to be monosynaptically innervated by the N2v cells (Brierley et al. 1997a).

More potent than glutamate itself was quisqualate. Importantly, this glutamate agonist also produced the appropriate inhibitory and excitatory effects on the neurons, postsynaptic to the N2vs. The excitatory response was unusual because the most recent reports describing the effects of quisqualate on molluscan neurons report that the responses are usually inhibitory, mediated by increases in chloride or potassium conductances (e.g., Bolshakov et al. 1991; Katz and Levitan 1993). On the other hand, Walker (1976) showed that quisqualate could produce both inhibitory and excitatory effects on Helix neurons, and it is well known that quisqualate activates excitatory glutamate receptors on insect and crustacean muscles (e.g., Brundell et al. 1991; Shinozaki 1988). On feeding motor neurons of the related snail, Helisoma, quisqualate again mimicked inhibitory synaptic inputs, but unlike Lymnaea, kainate was more potent in producing the excitatory responses in the same snail (Quinlan and Murphy 1991). Although kainate was excitatory on Lymnaea neurons, it was much less potent than quisqualate in reproducing the excitatory responses of N2v cells on B3 motor neurons. These variable potencies of quisqualate/kainate are typical of the diversity seen in glutamate receptors in the vertebrate CNS.

AMPA is an agonist of ionotropic glutamate receptors of much greater specificity than quisqualate (Lodge and Collingridge 1990), and in Lymnaea it could reproduce both excitatory and inhibitory responses on N2v postsynaptic cells. We were surprised that it could produce both excitatory and inhibitory effects on different cells, given that it is only excitatory in vertebrate systems. The metabotropic glutamate agonist had no effect on B3, whereas AMPA had the expected depolarizing effect on the same cell (Fig. 3B). This suggested that glutamate effects on the B3 cell were restricted to ionotropic mechanisms, but it would still surprise us if part of the effects of glutamate was not due to an additional metabotropic mechanism because the second phase of the response continues for >= 1 min (Fig. 2A).

We have obtained no conclusive evidence that NMDA-like glutamate receptors are responsible for N2v effects on feeding motor neurons or interneurons on Lymnaea buccal cells, although we cannot completely rule out the possibility that they mediate part of the response. Focal application of NMDA produced excitatory effects on B3 buccal motor neurons, but responses were inconsistent in different preparations and, in preliminary experiments, we could not block the occurrence of N2v-mediated EPSPS using the specific NMDA antagonist AP5 to confirm their role in synaptic transmission.

We tried to enhance NMDA effects with glycine applied as a cotransmitter. This produced no reliable effect, suggesting that the NMDA responses, when present, were glycine insensitive. In a second type of experiment, we reduced saline concentration of Mg2+ to zero to remove the possible Mg2+ blocking effects of NMDA-gated ion channels reported in vertebrate NMDA receptors (Nowak et al. 1984). Again this was ineffective in revealing NMDA responses. Moroz et al. (1993) recently have shown that other NMDA-induced currents in Lymnaea light yellow cells are also Mg2+ insensitive. However in Aplysia, Dale and Kandel (1993) showed that reducing saline Mg2+ levels to 5 mM, from its normal level in the bathing medium of 55 mM (seawater), removed the Mg2+-induced inhibition of a glutamate-induced inward current responsible for depolarization; so at least some glutamate receptors in mollusks can be blocked by Mg2+. The lack of Mg2+ block would be an unusual pharmacological property for NMDA receptors in vertebrates, suggesting that the Lymnaea NMDA receptors differ significantly from their vertebrate counterparts.

We can conclude reasonably that the main synaptic responses in feeding neurons in Lymnaea are due to non-NMDA (presumably AMPA/quisqualate) receptors, because CNQX, a specific non-NMDA glutamate receptor antagonist, effectively blocked most of the excitatory response on the B3 motor neuron type due to N2v activation. This occurred with spontaneous patterns of synaptic input due to N2v bursting (as described by Quinlan and Murphy 1991 in Helisoma) but, more importantly, when bursts of spikes were induced artificially in N2v cells by current injection. This is the best evidence we have that the N2vs are glutamatergic. However, we cannot conclude that only one receptor is involved because CNQX did not completely block the excitatory responses to N2v stimulation (B3 cells) and had no effect on the inhibitory ones recorded on other cells (SO cells).

Molecular analysis of Lymnaea glutamate receptors has discovered a glutamate receptor subunit type with homology (43-47%) to the GluR1-GluR4 rat glutamate receptor subunits (Hutton et al. 1991). The preferred agonist to these receptor subunits in vertebrates was AMPA, and this may be significant in relation to the present AMPA-sensitive glutamate receptors in Lymnaea feeding motor neurons, although so far it has not been possible to obtain in vitro expression in Xenopus oocytes (Darlison et al. 1994). A further cDNA clone (Lym-eGluR2) has been isolated (#21) that shows strong sequence homology to mammalian kainate and domoate sensitive glutamate receptors (Stühmer et al. 1996). Expression of the mRNA in Xenopus oocytes showed that it had a different pharmacology to the ones described in the present paper. Kainate, ibotenate were both agonists for Lym-eGluR2 as was AMPA, although they were not as effective as glutamate. Quisqualate was not an agonist. CNQX was an antagonist to Lym-eGluR2 similar to the AMPA/quisqualate receptors present on B3 but AP5 also had antagonist effects. Significantly, Lym-eGlu2R is expressed on different buccal motoneurons to the ones innervated by the N2v. These are the B4CL cells (Stühmer et al. 1996) known to be innervated by the N2d cells (Brierley et al. 1997a) rather than the N2vs. This suggests that native kainate-like receptors, perhaps similar to those described by Quinlan and Murphy (1991) in the related snail, Helisoma, also exist in Lymnaea and these may be the targets of a second type of N2 cell, the N2d.

    ACKNOWLEDGEMENTS

  We thank A. Bacon for typing the manuscript. M. Darlison and T. Stühmer made useful criticisms of the manuscript.

  This work was supported by a graduate studentship and research grant from the United Kingdom Biotechnology and Biological Research Council.

    FOOTNOTES

   Present address of M. J. Brierley: Dept. of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

  Address for reprint requests: P. R. Benjamin, School of Biological Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 9QG, UK.

  Received 29 October 1997; accepted in final form 8 August 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society