SYNAPSE 9:3542 (1991)
An Arthropod NMDA Receptor CINDY PFEIFFER-LINN AND RAYMON M. GLANTZ Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251
KEY WORDS
EAA, Glutamate, Quisqualate, Crayfish, Invertebrate, Glycine, Potentiation, Kynurenate vision adaptation
ABSTRACT Identified crayfish visual interneurons respond to illumination with a compound EPSP of up to 40 mV. L-gultamate, quisqualate, and kainate mimic the depolarizing action of the natural transmitter. In reduced M g + , N-methyl-D-aspartate (NMDA) elicits a depolarization with a reversal potential (E,) = -60 mV. Ere, is independent of extracellular calcium but shifts to +4 mV if potassium conductances are blocked by intracellular CS+.The results suggest that NMDA may gate more than one class of ionic channel. The NMDA-elicited response is enhanced and prolonged by glycine, and kynurenate competitively blocks the action of glycine. The NMDA antagonist, D-AP7, selectively blocks the NMDA response while enhancing the EPSP. The actions of NMDA are consistent with a role in the neural mechanisms of visual adaptation. This is the first description of an NMDA receptor in an invertebrate. INTRODUCTION Glutamate receptors are well described in crustacean (Atwood, 1980) and insect (Pick, 1985) neuromuscular junctions, where they mediate a cation conductance and the excitatory synaptic potential. Although many of these receptors are particularly sensitive to the glutamate analog quisqualate (Atwood,1980; Stettmeier and Finger, 19831,they differ from their vertebrate counterparts with respect to antagonist binding and channel properties. Arthropod muscles also exhibit an assortment of extrajunctional receptors with different ligand affinities and ionic selectivity (e.g., chloride conductance, Cull-Candy and Usherwood, 1973; Hironaka, 1974). In arthropod neurons (e.g., the crab and lobster stomatogastric ganglion, Marder and PaupardinTritsch, 1978; Marder and Eisen, 1984) receptors for L-glutamate have been shown to mediate potassium and chloride conductances, in addition to a depolarizing response. The receptors in crab are insensitive to kainate, and the inhibitory responses in lobster are blocked by picrotoxin. In vertebrate central nervous system, glutamate appears to be the principal excitatory neurotransmitter and receptors for NMDA are widely distributed (Cotman and Monoghan, 1988). Considerable attention has focused on the pharmacological and other functional distinctions between NMDA and non-NMDA excitatory amino acid (EAA) receptors (Mayer and Westbrook, 1987) and on the role of the NMDA receptor in synaptic plasticity (Cotman and Monaghan, 1988; Gustafsson and Wigstrom, 1988) and central pattern generation (Alford and Williams, 1989). Although invertebrates collectively exhibit a remarkable diversity of glutamate 0 1991 WILEY-LISS, INC.
receptors, there is no known example of an invertebrate NMDA receptor (Mayer and Westbrook, 1987). The absence is noteworthy, because invertebrates provide some of the best examples of plasticity in the synaptic interactions among identified neurons (Byrne, 1987; Nguyen and Atwood, 1990). The issue is also important because the distribution and properties of transmitter receptors may hold important clues to the evolution of nervous systems (Hille, 1984; Lunt, 1986). In this report we describe some of the features of an NMDA receptor in a class of visual interneurons, the sustaining fibers (SF) of the crayfish optic lobe. The structure and functional properties of SFs were previously described (Wiersma and Yamaguchi, 1967; Kirk, Waldrop, and Glantz, 1983; Waldrop and Glantz; 1985). Here we show that the neuropile that contains the SF dendrites is a rich source of glutamatergic neurites and glutamate appears to mediate the visually elicited EPSP. Although the SF NMDA receptor differs in several regards from those in the vertebrates, it also shares some common features, such as suppression by M2+, dependence upon glycine, and sensitivity to both specific and nonspecific NMDA antagonists. Both pharmacological and reversal potential measurements distinguish the SF NMDA and non-NMDA receptors. METHODS Immunocytochemistry The distribution of glutamate in crayfish optic lobes was examined with a monoclonal antibody specific for
Received December 10,1990; accepted in revised form February 5,1991.
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C. PFEIFFER-LINN AND R.M. GLANTZ
carbodiimide-fixedglutamate (Madl, Larson, and Beitz, 1986)obtained from Immuno Nuclear (Stillwater, MN). Crayfish were cooled to 4°C and exsanguinated by exchanging blood for oxygenated saline for 2 hours. Sixteen eyestalks (from 8 animals) were processed. Eight of these were used in preliminary studies to establish the optimal antiserum concentration and exposure time. The optic lobe was excised and placed in Ca2+-freevan Harreveld's (1936) solution for 5 minutes at 4°C. Fixation and incubation procedures are slightly modified from Madl et al. (1986). The tissue was fixed with 5% carbodiimide in phosphate buffered saline (PBS), pH 7.2, for 15 minutes and postfixed with 5% glutaraldehyde in PBS for 2 hours. The fixed tissue was sectioned in a cryostat a t 50 pm, floated in culture slides, and incubated in 10% normal horse serum in 0.3% Triton X-100 and PBS for 1 hour. After two 15-minute washes in PBS, the sections were incubated in the primary antibody, diluted 1:1,000 in PBS and 0.3% Triton X-100, for 12 hours at 4°C. The sections were washed in PBS and incubated in biotinylated anti-mouse IgG antibody in 0.3%TritonX-100 and PBS for 1 hour at 23°C. Following 3 more PBS washes, the sections were incubated with avidin peroxidase reagent (Immuno Nuclear) for 1 hour, washed, and incubated in diaminobenzidine tetrachloride until the reaction product was visible (typically 5-20 minutes). The reaction was stopped by a wash in 0.9% NaCl, Tris buffer, pH 7.6. This procedure was replicated in 4 preparations. Two control procedures were used to assess nonspecific staining. In one (n = 2), the above procedure was repeated precisely as described, but the primary antiserum was deleted from the incubation protocol. In the second procedure (n = 2), the primary antiserum was preabsorbed in a solution of 5.0 mh4 gamma-l-glutamyl-L-glutamate for 1 hour prior to incubation with the tissue.
cells from electrodes containing 2.0 M CsCl with 0.5 nA of positive current for 5 minutes. Visual stimulation was performed with a system consisting of a 300-Watt quartz-iodide lamp, condenser lens, electromagnetic shutter, neutral density filter, and a 5-mm fiber-optic light guide, which conducted the illumination to the eye. The terminal of the light guide was held in a micromanipulator and positioned 5 cm from the eye. Pharmacological methods The agonists tested consisted of L-glutamate, (Ykainate (Sigma, St. Louis), quisqualate, and NMDA (Research Biochemicals, Natick, MA). All agonists were tested before and after replacing the saline in the eyestalk with a Ca2+-freesaline containing 20 mm CoC1,. The Co2+ reduced all synaptic potentials by 390% in 2-5 minutes. The antagonists tested included kynuD-AP7 renate, DNQX (6,7-dinitroquinoxaline-2,3-dione), (D-2-amino-7-phosphonoheptanoic acid) (Research Biochemicals), picrotoxin, and L-glutamate-gamma-methylester (GME) (Sigma). Agonists were pressure injected via a 5-pm tip glass pipette placed in the medullary neuropile. The injected volume (about 65 pl) was measured under oil in a compound microscope. Antagonists were applied to the surface of the desheathed ganglion in 1-2 pl volumes. Reversal potentials and membrane conductance were measured in current clamp with procedures described in Waldrop and Glantz (1985).
RESULTS Immunocytochemistry Glutamate is found in high concentrations in arthropod brains (McCaman, 1984). Immunocytochemical assays in crayfish optic lobe (Fig. 1)indicate high levels of anti-glutamate reactivity in the first optic neuromere, the lamina ganglionaris (LA), and the medulla externa (ME) and interna (MI) (second and third neuromeres of Physiological preparation the optic lobe, respectively). There is also substantial Adult crayfish of 8-10 cm length were prepared as in reactivity in the optic chiasm between the lamina and Pfeiffer and Glantz (1989). The crayfish were cooled, the medulla externa. Comparable staining in ME and exsanguinated as described above, and clamped in a MI was observed in each of the replicates, but the plexiglass chamber. The eyestalks were cemented to the reactivity in the lamina was variable. carapace with cyanoacrylate, and the optic lobe was Two control procedures suggest that the observed exposed by excising the cuticle on the distal eyestalk. staining pattern indicates the distribution of glutamate. The preparation was maintained a t 13°C in Van Harrev- In the first, the serum antibodies were incubated with eld's (1936)solution. Van Harreveld's solution contains the original antigen (glutamate-glutaryl-bovine serum 205 mM Na+,5.4 mM K+, 13.5 mM Ca2+,2.6 mMMg', albumin). An absence of staining indicated that the 243 mM C1-, and 2.4 mM NaHCO,. nonspecific reactivity of the serum for crayfish nerve Visual interneurons were impaled with 100 MO mi- tissue was extremely low and that the source of the cropipettes filled with 3.0 M potassium acetate. Signals reactivity is the antibody(s1to the glutamate conjugate. were amplified with a capacitance-compensated elec- An identical result was obtained when the antiserum trometer and stored on an FM tape recorder. Intracellu- was omitted from the initial incubation. lar current injection was performed through a bridge These data are consistent with the presence of high circuit, and electrodes were selected for fast response levels of glutamate in optic lobe neuropiles. This glutaand minimal polarization artifact. Cs+ was injected into mate could serve as a neurotransmitter or may be a
AN ARTHROPOD NMDA RECEPTOR
37
uniquely identified by their visual receptive fields (Wiersma and Yamaguchi, 1966) and spatially correlated dendritic geometries (Kirk, Waldrop, and Glantz, 1983). SF axons project to the brain, where they innervate ocular motoneurons and thus participate in optomotor reflexes (Glantz and Nudelman, 1988).The SFs respond to illumination with a tetrodotoxin-insensitive EPSP (Fig. 2A) of up to 40 mV. The EPSP is associated with an increase in input conductance (G,), as illustrated in Table I (Waldrop and Glantz, 1985). Glutamate (1.0-10 pM, Fig. 2B), quisqualate (.01 pM, Fig. 10, and kainate (1.0 pM) elicit approximately halfmaximal depolarizing responses that are unaffected by synaptic blockade with 20 mM CoC1, (Figs. 2C, 2D). The responses to the EAAs are associated with substantial increases in G, (Fig. 2D). L-glutamate, at 10 pM, increases G, by 70% (Table 1). EAA receptors with maximal sensitivity t o quisqualate have been described at the crayfish neuromuscular junction (Stettmeier and Finger, 1983). Ere, for responses elicited by glutamate were -23 k 3 mV (mean SD, n = 4) or about 43 mV above the resting potential (-66 mV; Fig. 3, open circles) and very similar to the extrapolated Ere, of the EPSP (Fig. 3, filled circles, Table 1). At 1.O mM, L-glutamate-gamma-methylester(GME) blocks the excitatoryjunction potential and the depolarizing action of glutamate a t the crayfish neuromuscular junction (Lowagie and Gerschenfeld, 1974). At 0.1 mM GME blocks the SF EPSP (Fig. 4A) and the response to 10 pM glutamate (Fig. 4B) and 1.0 pM kainate (not shown). A second antagonist, DNQX, which competitively blocks vertebrate non-NMDA receptors (Honore et al., 1988) was ineffective on both the EPSP and responses elicited by glutamate and quisqualate at antagonist concentrations of up t o 0.1 mM. NMDA, applied at resting potential in normal crayfish saline (with 2.6 mM Mg+),was without measurable effect in 8/8 cells tested (Fig. 5A, bottom trace). Depolarizations (with extrinsic current) of up to 20 mV failed to unmask an NMDA response in control saline. In M 2 + free saline, however, NMDA elicited a modest depolarization in 42/42 cells (Fig. 5A, middle trace). The NMDA response is associated with a 109% increase in G, (Fig. 5B, Table 1). The NMDA-elicited response is further enhanced (by 100-200%) and prolonged by the presence of 10 pM glycine (Fig. 5A, top trace). A comparable action of glycine on the NMDA-elicited membrane conductance change is shown in Figure 5C. At 10 pM, glycine more than doubles the NMDA-elicited increase in G, (Table 1).As observed in vertebrate neurons (Johnson and Ascher, 19871, this action of glycine is specific to the NMDA receptor. By itself, glycine produces no effect on the SF membrane potential or input conductance, and has no effect on the response to kainate (not shown). Glycine (1.0-100 pM) does, however, reduce the amplitude of the EPSP (by 50-70% at 100 pM) (Figs. 5D, 5E). If this glycine action is mediated by
*
Fig. 1. Immunocytochemical localization of glutamate in the crayfish optic lobe. Dark staining produced by indirect peroxidase method in the lamina ganglionaris (LA), the medulla externa (ME), and the medulla interna (MI). Scale (lower right) is 100 pm.
precursor for the synthesis of GABA. Previous studies have demonstrated that crayfish optic lobe contains high levels of GABA (Pfeiffer-Linn and Glantz, 1989). Furthermore, GABAergic inhibitory motoneurons in lobster contain about the same amount of glutamate as the presumed glutamatergic excitatory motoneurons (Otsuka, Kravitz, and Potter, 1967). Thus some or even all of the immunoreactivity shown in Figure 1 could reflect GABAergic neurons. Physiology and pharmacology Pharmacological studies were carried out with glutamate and several agonist analogs. The actions of the EAAs were examined in four classes of identified interneurons with dendrites in the medulla externa. The neurons include the SFs, amacrine cells, dimming fibers, and tangential cells. Each cell type has a distinct structure and visual response (Waldrop and Glantz, 1985; Wang-Bennett and Glantz, 1987; Pfeiffer and Glantz, 1989). Among these four neuron classes, only the SFs are responsive to glutamate. The SFs are the principal output cells of the medulla externa. The class consists of 14 neurons, which can be
C. PFEIFFER-LINN AND R.M. GIANT2
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B
A
B
Ar
D
C
Fig. 2. SF light response (A)and SF responses to 10 +M L-glutamate (B) and 10 nM quisqualate (C). At 10 nM, quisqualate reduces the SF input resistance from 16 to about 1.0 MR (D). In C and D, synaptic activity is blocked with 20 mM Co". Current pulses are -0.5 nA. The scale is 10 mV and 1.0 s.
-
-
Fig. 4. L-glutamate-gamma-methylester at 0.1 mM blocks the PSP and the glutamate-elicited responses (lowertraces) in A and B, respectively.
TABLE 1. Reuersal potentials and input conductance changes associated with the EPSP and responses elicited b y 10 p M L-glutamate and 10 p M NMDA EWl (mV) -19 k 9 i7)3 -23 f 3 (4) -60 f 5 (11) -60 f 9 (3) 4 f 8 (3)
Agonist EPSP L-Glutamate NMDA4 NMDA, Glycine NMDA, Cs'
AG' (% control)
121 f 51 (6) 70 f 55 (5) 109 28 (7) 285 f 64 (4) 27 f 20 (3)
+
5 mV (SD, n=35). 'Average membrane resting potential is -66 Siemens (n=23). 'Average resting input conductance is 8.9 & 2.6 X 3All results are mean +standard deviation and the number of separate determinations is indicated in parentheses. EPSP data from Waldrop and Glantz (1985). 4All NMDA measurements in Mg"+-free saline solution.
..
PSP
i , 0 GLUTAMATE
\
\
- 30mV -
B
E
A F
20
-- 10
Fig. 3. Sustaining fiber PSP and glutamate-elicited responses vs. membrane potential. Reversal potential of the PSP ( 0 ) and glutamate responses ( 0 )estimated by extrapolation from the least-square regressions.
the NMDA receptor, then it implies that NMDA-elicited currents contribute to the repolarization of the EPSP. This is observed when an NMDA pulse is superposed on the EPSP (Fig. 5F, arrow). The partial inhibition of the EPSP by glycine is consistent with the potentiation of the NMDA receptor and a relatively negative Ere, of the NMDA-elicited
Fig. 5. SF responses to NMDA (10 pM) and glycine. A Responses t o NMDA in control saline (bottom trace), in M$+-free saline (middle trace), and in 10 +M glycine in M$+-free saline (top trace). Each trace is the average of 5-10 responses. B,C: Input resistance changes in response to NMDAin 0 M$+ (B)and after the addition of 10 pM glycine (C). D: Control PSP. E PSP in 10 +M glycine. F: Responseto an NMDA pulse (arrow) superposed on a visually elicited EPSP. Scale (in panel F) is 10 mV and 1.0 s throughout.
response. When the SF membrane is hyperpolarized, the NMDA-elicited response increases dramatically with increases in membrane potential (Fig. 6, top, inset). Conversely, the response is inverted by membrane depolarizations of no more than 10-15 mV. The Ere, of the NMDA-elicited response (-60 2 5 mV, n = 11; Fig. 6 , O ) is about 6 mV above resting potential. As a consequence, the action of the NMDA receptor in a depolarized cell tends to move the membrane potential away from the glutamate E, toward -60 mV. Thus, the plateau phase of the sustaining fiber EPSP, which is generally about 20 mV above rest, may reflect the joint action of two EAA receptor classes that gate opposing currents.
AN ARTHROPOD NMDA RECEPTOR
w i20mV
-90 mV
-30
Fig. 6. Response amplitude vs. membrane potential of the NMDAelicited responses in Mg2+-freesaline ( 0 )and followingthe intracellular injection of Cs' (0).Continuous lines are least-square regressions. Insets (top) are NMDA-elicited responses in Mg2'-free saline and following intracellularinjection of Cs'. The scale is 10 mV and 1.0 s.
An E, of -60 mV is unusual for an NMDA-activated conductance. NMDA receptors generally gate a nonspecific cation conductance with E, =: 0 mV and with a significant calcium permeability (Mayer and Westbrook, 1987). Since intracellular calcium can gate a calcium-activatedpotassium conductance (Meech, 1978), it is possible that the -60 mV Ere, is only secondarily associated with NMDA receptor activation (Nicol and Alger, 1981). Alternatively, it is possible that in SFs NMDA gates a mixture of inward and outward currents, as observed in glutamate responses of crab stomatogastric neurons (Marder and Paupardin-Tritsch, 1978)and locust neural somata (Giles and Usherwood, 1985). When SFs are injected with Cs+ to block potassium conductances (Gorman et al., 1982; Johnson and Ascher, 1987; Cull-Candy and Usowicz, 1987), Ere, of the NMDA-elicited response shifts from -60 to about + 4 mV (Table 1;Fig. 6,o). The difference in the slope of the two functions in Figure 6 also indicates that the Cs+
39
markedly reduces the magnitude of the NMDA-elicited change in input conductance (Table 1). The results indicate that NMDA can elicit a depolarizing response and (either directly or indirectly) a large outward current. The 4 mV E,, following Cs+ injection is consistent with an NMDA-elicited cation conductance. The outward current could be a secondarily activated potassium conductance or a directly elicited response in a subpopulation of glutamate receptors (Marder and Paupardin-Tritsch, 1978; Yarowsky and Carpenter, 1976; Atwood, 1980). To distinguish between these alternatives, the NMDA response was elicited in nominally Ca2+-freemedia in which Ca2+was replaced with Ba2+ or Co2+.None of these substitutions produced a significant shift in the -60 mV E,,,. This result implies that the -60 mV Ere, does not depend upon an inward calcium current and further suggests that the NMDA response may reflect a mixture of inward and outward currents, as observed in the glutamate responses of other arthropod neurons. Because Co2+ has a potent Mg+-like action on the vertebrate NMDA receptor (Ascher and Nowak, 1988), the persistence of the SF NMDA response in 20 mM Co2+ indicates another important difference between vertebrate and crayfish receptors. Because the vertebrate NMDA receptor is in part defined by its sensitivity t o a wide array of specific and nonspecific antagonists (Olverman and Watkins, 19891, we tested the crayfish receptor's sensitivity to a variety of blockers. GME, which blocked the SF EPSP and glutamate response at 0.1 mM, was ineffective on the NMDA-elicited response, even at 1.O mM. Picrotoxin blocks glutamate-elicited outward currents in lobster stomatogastric ganglion (Marder and Eisen, 19841, but had no effect on the NMDA-elicited response in SFs at 0.1 mM. Kynurenic acid is both a nonspecific EAA antagonist (Perkins and Stone, 1982; Ode11 and Christensen, 1989) and a competitive antagonist of the allosteric action of glycine on the NMDA receptor (Thomson et al., 1989). When 0.1 mM kynurenate is applied to SFs, it reduces the EPSP by 50430% (Fig. 7A, lower trace) and blocks the response to kainate (Fig. 7B, lower trace). Both of these effects are consistent with an antagonist action of kynurenate on the non-NMDA receptor. Kynurenate also blocks the response to NMDA (Fig. 7C, lower trace). In 10 p,M glycine and M$+-free saline, 0.1 mM kynurenate abolishes the NMDA response (Fig. 7D, lower trace), but the inhibition is reversed by 1 mM glycine (Fig. 7D, upper trace). Thus the actions of kynurenate in crayfish resemble its actions in vertebrates, i.e., a nonspecific antagonism to EAA receptors and a competitive block of glycine action an the NMDA receptor. D-AP7 is a potent and selective NMDA antagonist (Olverman and Watkins, 1989). In 3/3 tests, 10 pM
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C. PFEIFFER-LINN AND R.M. GLANTZ
8
C
A!”
D d P
*
Fig. 7. Kynurenate a t 0.1 mM (lower traces A-C) antagonizes the natural transmitter (A), the response to 10 pM glutamate (B), and NMDA in low Mg2+but in the absence of glycine (0.In 10 pM glycine kynurenate blocks the NMDAresponse (D, lower trace), but the block is overcome by elevating glycine to 1.0mM (D, upper trace).
Fig. 8. Actions of the NMDA antagonist D-AP7. At 10 pM, D-AP7 blocks the NMDA (10 pM) response (A, lower trace) and augments the visually elicited PSP (B,upper trace). The upper trace in A and the lower trace in B are control responses. Each trace is the average of 12 responses. All measurements are in Mg2+-freesaline. The scale is 1.4 mV and 0.4 s in A, and 6.0 mV and 75 ms in B.
D-AP7 abolished the response to 10 pM NMDA (Fig. 8A, tion with non-NMDA glutamate receptors (Jahr and lower trace), while it had no measurable effect on the Stevens, 1987). In several vertebrate neurons (Cotman response to kainate. In the same cells, D-AP7 potenti- et al., Dale, 1989),the non-NMDA receptor mediates the ated the visual response by 30-100% (Fig. 8B, upper initial, rapid phase of the EPSP, which is subsequently trace). This result implies that the activation of the modulated by the delayed action of the NMDA receptor. NMDA receptor tends to reduce the SF EPSP. The effect In crayfish SFs, the transient EPSP appears to be of D-AP7 is consistent with the effects of glycine on the mediated by the non-NMDA receptor. The actions of the SF EPSP (Fig. 5E) and the action of NMDA superposed NMDA receptor are consistent with a modulatory role, possibly related to neuronal mechanisms of light adapon the EPSP (Fig. 5F). tation. DISCUSSION An important property of the vertebrate NMDA reBoth pharmacological tests and reversal potential ceptor is that the M 2 + block of the ionic channel is measurements indicate that crayfish SFs possess at voltage dependent and the NMDA response exhibits a least two classes of EAA receptors. A non-NMDA recep- negative slope conductance between -60 and -30 mV tor mediates the initial phase of the visually elicited (Mayer and Westbrook, 1987). The crayfish NMDA EPSP, and it is highly sensitive to quisqualate. The response is completely blocked by normal levels of M 2 + , NMDA receptor(s1 appears to primarily mediate an and this block is insensitive to depolarizations of up to 20 mV. Another obvious difference between the crayfish outward current. The crayfish NMDA receptor exhibits some striking and vertebrate NMDA receptors is the apparent reversimilarities and important differences when compared sal potential. In this regard, the SF NMDA response to the vertebrate receptor. Both receptors are blocked by resembles the glutamate-elicited responses in crab and circulating levels of M$+ (Davies and Watkins, 1977) lobster stomatogastric ganglion (Marder and Pauparand by the specific antagonist D-AP7, and both have a din-Tritsch, 1978; Marder and Eisen, 1984) and the requirement for glycine, which is susceptible to compe- extrajunctional glutamate responses in arthropod mustition by kynurenate. In mammalian cerebrospinal cle (Hironaka, 1974; Atwood, 1980). The fact that the fluid, glycine concentrations are above 1.0 pM, which reversal potential is unaffected by deleting Caz+ from substantially exceeds the nM requirements of the the medium argues against an important role for a NMDA receptor (Ascher and Johnson, 1989) and masks Ca2+-activated potassium conductance. Following inthe glycine dependence in situ. Since the concentration tracellular Cs+injection, the E,, of the NMDA response of glycine in crayfish hemolymph is about 1.0 mM is more positive and resembles that in vertebrates. (Gardiner, 1972) and glycine potentiates NMDA action Taken together these results suggest that NMDA elicits at 1.0 pM, the same situation should obtain in crayfish. a mixture of inward and outward currents normally The in situ observations of glycine dependence were dominated by the latter. An insensitivity to Co2+further made possible by the exsanguination procedure that distinguishes the SF from vertebrate NMDA receptors replaces the hemolymph with Van Harreveld’s solution, (Ascher and Nowak, 19881,and an insensitivity to picrocomposed exclusively of inorganic ions. toxin distinguishes the SF receptor from the glutamateAn additional similarity between crayfish and ver- activated potassium conductances in the stomatogastric tebrate NMDA receptors is that they occur in conjunc- ganglion.
41
AN ARTHROPOD NMDA RECEPTOR
The failure of previous invertebrate studies to reveal NMDA receptors may reflect the particular sites tested. The crayfish NMDA receptors were found on particular interneurons and on their dendritic processes in central neuropile. Previous studies in lobster and locust were confined to the neuromuscular junction (Nistri and Constanti, 1979; Ushenvood and Machili, 19681, and those in the snail examined only neuronal cell body receptors (Parmentier and Case, 1972). In the leech (James et al., 19801, "IDA was bath applied but only the Retzius neurons were tested. Thus it is likely that further study of invertebrate neurons will reveal additional examples of NMDA receptors. The sustaining fiber non-NMDA receptor shares several properties with those a t the crayfish neuromuscular junction (Atwood, 1980). The receptor mediates an excitatory and presumably cationic conductance. It is particularly sensitive to quisqualate, and it is blocked by GME. On the other hand, the SF receptor is also sensitive to kainate, which is an antagonist a t crayfish junctional receptors but an agonist at the extrajunctional receptors (Onodera and Takeuchi, 1980). The SF agonist-elicited responses appear to be relatively slowmuch slower than the responses elicited by iontophoresis at the neuromuscular junction. This difference is probably a consequence of our agonist application procedure (pressure injection), which could produce a diffusion-limited activation in the dense neuropile. The SF EPSP is quite rapid and the rate of depolarization (up to 6 mV/ms) is comparable to that of the excitatory junction potential elicited by a "fast" motoneuron (10 mV/ ms, Atwood, 1980). A more significant difference between the SF and junctional EAA receptors may be the reversal potentials of the associated synaptic and agonist-elicited responses. At crustacean neuromuscular junctions, Ere, of the excitatory junction potential (e.j.p.) and glutamateelicited responses varies from 0 to +50 mV (Atwood, 1980). In SFs, E, of the EPSP and the glutamate elicited responses are about -20 mV (Fig. 3), as measured in current-clamped cells. This discrepancy could be due to a real difference in channel selectivity and/or transmembrane ion gradients, but it could also reflect the effects of a voltage-dependent potassium conductance. An overview of the results provides substantial but still incomplete evidence that glutamate (or an analog) is the excitatory transmitter to the SF. The immunocytochemistry suggests the presence of glutamate in the vicinity of the SF dendrites, but we have no evidence that visual stimulation promotes synaptic release of glutamate. The reversal potential of the glutamateelicited response is similar to that ofthe EPSP, and both are antagonized by GME and kynurenate. It is clear that the SF has an EAA receptor, since the responses to glutamate and all excitatory amino acid analogs persist following synaptic blockade with cobalt. As noted above, one possible role for the SF NMDA
receptor is a neuronal mechanism of light adaptation (Glantz, 1972). The SF response to a step increase in illumination intensity is a compound EPSP, consisting of a rapid transient phase of up to +40 mV and a steady-state plateau of about 20 mV above the resting potential. As a consequence of its relatively negative E,,,, the NMDA response could contribute an outward current, which would partially repolarize the SF during prolonged excitation. In summary, our results suggest that the SF compound EPSP is mediated by glutamate, which acts upon a non-NMDA receptor to elicit the initial transient depolarization and an NMDA receptor, which participates in the partial repolarization and plateau phase. The non-NMDA receptor is about 100 times more sensitive to quisqualate than to glutamate or kainate. It is blocked by GME and kynurenate but is insensitive to DNQX. The latter result indicates that the SF nonNMDA receptor is distinct from analogous receptors in vertebrates. The NMDA receptor mediates a mixture of inward and outward currents dominated by a Cs'sensitive conductance, with a reversal potential just positive to the resting potential. The NMDA response is blocked by M 2 + but is insensitive to extracellular Coz+ and Ca2+. These features distinguish the SF NMDA receptor from the vertebrate prototype. The SF NMDA response is potentiated by glycine, and kynurenate competitively blocks glycine action. D-AP7 completely suppresses the response to NMDA. In these aspects the SF receptor is similar to that of vertebrates. Since it is inferred that the depolarizing EPSP is principally mediated by the non-NMDA receptor, it follows that GME and kynurenate (as a nonspecific EAA antagonist) reduce or abolish the EPSP. Conversely, since D-AP7 blocks the NMDA-elicitedoutward current, it enhances the EPSP. Glycine potentiates the NMDAelicited current and thus reduces the EPSP. The principal deficiency in the above interpretation is that we have been unable to unambiguously document the action of the NMDA in saline of normal M$+ concentration. It is possible that the M 2 + block could be relieved at depolarizations beyond the reach of our current clamp system. Alternatively (but less likely) it is possible that in neuropile, the M 2 + concentration is less than that in hemolymph. Thus, although it is clear that the SF has a receptor for NMDA, its exact function cannot be established until the nature of the M 2 + block is clarified. ACKNOWLEDGMENTS Supported by N.S.F. grants BNS-8711141 and BNS9021216, and a Lodieska Stockbridge Vaughn Fellowship to C.P.-L. We thank Dr. Dan Johnston of Baylor Medical School and Dr. Michael Gustin of Rice for useful comments on an earlier draft of this manuscript.
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C. PFEIFFER-LINN AND R.M. GLANTZ
REFERENCES Alford, S., and Williams, T.L. (1985) Endogenous activation of glycine and NMDA receptors in lamprey spinal cord during fictive locomotion. J. Neurosci., 9:2792-2800. Ascher, P., and Nowak, L. (1988) The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J. Physiol. (Lond.),399:247-266. Atwood. H.L. (1980) Svnauses and neurotransmitters. In: Biolom of Crustacea, Vol. 3. HYL. Atwood and D.C. Sandeman, eds. Acadsmic Press, New York pp. 105-150. Byrne, J. (1987)Cellular analysis of associative learning. Physiol. Rev., 67:329-439. Cotman, C.W., Monaghan, D.T., and Ganong, A.H. (1988) Excitatory amino acid neurotransmission: NMDA receptors and Hebb-type .. snyaptic plasticity. Ann. Rev. Neurosci., ll:61:80. Cull-Candy, J.G., and Usowicz, M.M. (1987) Multiple-conductance channels activated by excitatory amino acids in cerebellar neurons. Nature, 325525428. Cull-Candy, S.G., and Usherwood, P.N.R. (1973) Two populations of glutamate receptors on locust muscle fibers. Nature New Biol., 246:62-64. Davies, J.D., and Watkins, J.C. (1977)Effects ofmagnesiumions on the responses of spinal neurones to excitatory amino acids and acetylcholine. Brain Res., 130:364-368. Gardiner, M.S. (1972) Biology of Invertebrates. McGraw Hill, New York, p. 397. Giles, D., and Usherwood, P. (1985) The effects of putative neurotrahsmitters on somata isolated from neurons of the locust central nervous system. Comp. Biochem. Physiol. 8OC:231-236. Glantz, R.M., and Nudelman, H.B. (1988) Interval coding and bandpass filtering a t oculomotor synapses in crayfish. J. Neurophysiol., 59:56-76. Gorman, A.L.F., Herman, A,, and Thomas, M.V. (1981) Intracellular calcium and the control of neuronal pacemaker activity. Fed. Proc., 40:2233-2239. Gustafsson, B., and Wigstrom, H. (1988) Physiological mechanisms underlying long-term potentiation. T.I.N.S., 11(4):156-162. Hille, B. (1984) Ionic Channels of Excitable Membranes. Sinaur, Sunderland, MA, pp. 382-383. Hironaka, T. (1974) Chloride related depolarisation of crayfish muscle membrane induced by L-glutamate. Nature, 248:251-253. Honore, T., Davies, S.N., Drejer, J. Fletcher, E.J., Jacobsen, P., Lodge, D., and Nielsen, F.E. (1988) Quihoxalinediones: Potent competitive non-NMDA glutamate receptor antagonists. Science, 241 :701-703. James, V.A., Walker, R.J., and Wheal, H.V. (1980) Structure-activity on a n excitatory recepter for glutamate on leach Retzius neurones. Br. J. Parmacol., 68:711-717. Johnson, J.W., and Ascher, P. (1987) Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature, 325529-531. Kirk, M.D., Waldrop, and B., Glantz, R.M. (1983) The crayfish sustaining fibers. I. Morphological representation of visual receptive fields in the second optic neuropile. J . Comp. Physiol., 150:419425. Lowagie, C., and Gershenfeld, H.M. (1974) Glutamate antagonists at a crayfish neuro-muscular junction. Nature, 248533-535. Lunt, G.G. (1986) Is the insect neuronal nicotinic acetylcholine receptor the ancestral acetylcholine receptor protein? T.I.N.S., 9:341-342. Madl, J.E., Larson, A.A., and Beitz, A.J. (1986) Monoclonal antibody specific for carbodiimide-fixed glutamate: Immunocytochemical localization in the rat CNS. J. Histochem. Cytochem., 34:317-326. Marder, E., and Paupardin-Tritsch, D. (1978) The pharmacological properties of some crustacean neuronal acetylcholine, y-aminobutyric acid and L-glutamate responses. J . Physiol., 280:213-236. Marder, E., and Eisen, J.S. (1984) Transmitter identification of pyloric neurons: Electrically coupled neurons use different transmitters. J . Neurophysiol., 51 3345-1361.
Mayer, M.L., and Westbrook, G.L. (1987) The physiology of excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol., 28:197-276. McCaman, M.W. (1984) Neurochemistry of invertebrates. In: Handbook of Neurochemistry, Vol. 7. A. Lajthe, ed. Plenum Press, New York, pp. 613-700. Meech, R.W. (1978) Calcium dependent potassium activation in nervous tissues. Ann, Rev. Biophys. Bioeng., 73-18, Nguyen, P.V., and Atwood, H.L. (1990) Expression of long-term adaptation of snyaptic transmission requires a critical period of protein synthesis. J. Neurosci., 10:1099-1109. Nicol, R.A., and Alger, B.E. (1981) Synaptic excitation may activate a calcium-dependent potassium conductance in hippocampal pyramidal cells. Science, 212:957-959. Nistri, A,, and Constanti, A. (1979) Pharmacological characterization of different types of GABA and glutamate receptors in vertebrates and invertebrates. Prog. Neurobiol., 13:117-235. Odell, T.J., and Christensen, B.N. (1989) A voltage-clamp study of isolated stingray horizontal cell non-MNDA excitatory amino acid receptors. J . Neurophysiol., 61:162-172. Onodera, K., and Tskeuchi, A. (1980) Distribution and pharmacological properties of synaptic and extrasynaptic glutamate receptors on crayfish muscle. J. Physiol. (Lond.), 306:233-250. Otsuka, M., Kravitz, E.A., and Potter, D.D. (1967) Physiological and chemical architecture of a lobster ganglion with particular reference to GABA and glutamate. J . Neurophysiol., 30:725-752. Parmentier, J., and Case, J. (1972) Structure activity relationships of amino acid receptor sites on a n identifiable cell body in the brain of the land snai1Helz.x aspersa. Comp. Biochem. Physiol., 43A.511-518. Perkins, M.N., and Stone, T.W. (1984) A study of kynurenic acid and excitatory amino acids in the rat hippocampus. J. Physiol. (Lond.), 357:118. Pfeiffer, C., and Glantz, R.M. (1989) Cholinergic synapses and the organization of contrast detection in crayfish optic lobe. J . Neurosci., 9:1872-1882. Pfeiffer-Linn, C., and Glantz, R.M. (1989) Acetylcholine and GABA mediate opposing actions on neuronal chloride channels in crayfish. Science, 245 :1249-1 25 1. Piek, T. (1985) Neurotransmission and neuromodulation of skeletal muscle. In: Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 1. G.A. Kerkut and L.I. Gilbert, eds. Pergamon, New York, pp. 55-118. Stettmeier, H., and Finger, W. (1983) Excitatory postsynaptic channels operated by quisqualate in crayfish muscle. Pfiugeri Ar'ch., 397:237242. Thomson, A.M., Walker, V.E., and Flynn, D.M. (1989) Glycine enhances NMDA-receptor mediated synaptic potentials in neocortical slices. Nature, 338:422424. Usherwood, P.N.R., and Machili, P. (1969) Pharmacological properties of excitatory neuromuscular synapses in the locust. J. Exp. Biol., 49:341-361. Van Harreveld, A. (1936) A physiological solution for fresh water Exp. Biol. Med., 34:428-432. crustaceans. Proc. SOC. Waldrop, B., and Glantz, R.M. (1985) Synaptic mechanisms of a tonic EPSP in crustacean visual interneurons:Analysis and simulation. J. Neurophysiol., 54:636-650. WangBennett, L., and Glantz, R.M. (1987) The functional organization of the crayfish lamina ganglionaris. 11. Large field spiking and nonspiking cells. J. Comp. Physiol., 161:147-160. Wiersma, C.A.G., and Yamaguchi, T. (1966) The neuronal components of the optic nerve of the crayfish as studied by single unit analysis. J. Comp. Neurol., 128:333-358. Womack, M.D., MacDermott, A.B., and Jessell, T.M. (1988) Sensory transmitters regulate intracellular calcium in dorsal horn neurons. Nature, 334:351-353. Yarowsky, P.J., and Carpenter, D.O. (1976)Aspartate: Distinct receptors on Aplysia neurons. Science, 192:807-809.
An Arthropod NMDA Receptor CINDY PFEIFFER-LINN AND RAYMON M. GLANTZ Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251
KEY WORDS
EAA, Glutamate, Quisqualate, Crayfish, Invertebrate, Glycine, Potentiation, Kynurenate vision adaptation
ABSTRACT Identified crayfish visual interneurons respond to illumination with a compound EPSP of up to 40 mV. L-gultamate, quisqualate, and kainate mimic the depolarizing action of the natural transmitter. In reduced M g + , N-methyl-D-aspartate (NMDA) elicits a depolarization with a reversal potential (E,) = -60 mV. Ere, is independent of extracellular calcium but shifts to +4 mV if potassium conductances are blocked by intracellular CS+.The results suggest that NMDA may gate more than one class of ionic channel. The NMDA-elicited response is enhanced and prolonged by glycine, and kynurenate competitively blocks the action of glycine. The NMDA antagonist, D-AP7, selectively blocks the NMDA response while enhancing the EPSP. The actions of NMDA are consistent with a role in the neural mechanisms of visual adaptation. This is the first description of an NMDA receptor in an invertebrate. INTRODUCTION Glutamate receptors are well described in crustacean (Atwood, 1980) and insect (Pick, 1985) neuromuscular junctions, where they mediate a cation conductance and the excitatory synaptic potential. Although many of these receptors are particularly sensitive to the glutamate analog quisqualate (Atwood,1980; Stettmeier and Finger, 19831,they differ from their vertebrate counterparts with respect to antagonist binding and channel properties. Arthropod muscles also exhibit an assortment of extrajunctional receptors with different ligand affinities and ionic selectivity (e.g., chloride conductance, Cull-Candy and Usherwood, 1973; Hironaka, 1974). In arthropod neurons (e.g., the crab and lobster stomatogastric ganglion, Marder and PaupardinTritsch, 1978; Marder and Eisen, 1984) receptors for L-glutamate have been shown to mediate potassium and chloride conductances, in addition to a depolarizing response. The receptors in crab are insensitive to kainate, and the inhibitory responses in lobster are blocked by picrotoxin. In vertebrate central nervous system, glutamate appears to be the principal excitatory neurotransmitter and receptors for NMDA are widely distributed (Cotman and Monoghan, 1988). Considerable attention has focused on the pharmacological and other functional distinctions between NMDA and non-NMDA excitatory amino acid (EAA) receptors (Mayer and Westbrook, 1987) and on the role of the NMDA receptor in synaptic plasticity (Cotman and Monaghan, 1988; Gustafsson and Wigstrom, 1988) and central pattern generation (Alford and Williams, 1989). Although invertebrates collectively exhibit a remarkable diversity of glutamate 0 1991 WILEY-LISS, INC.
receptors, there is no known example of an invertebrate NMDA receptor (Mayer and Westbrook, 1987). The absence is noteworthy, because invertebrates provide some of the best examples of plasticity in the synaptic interactions among identified neurons (Byrne, 1987; Nguyen and Atwood, 1990). The issue is also important because the distribution and properties of transmitter receptors may hold important clues to the evolution of nervous systems (Hille, 1984; Lunt, 1986). In this report we describe some of the features of an NMDA receptor in a class of visual interneurons, the sustaining fibers (SF) of the crayfish optic lobe. The structure and functional properties of SFs were previously described (Wiersma and Yamaguchi, 1967; Kirk, Waldrop, and Glantz, 1983; Waldrop and Glantz; 1985). Here we show that the neuropile that contains the SF dendrites is a rich source of glutamatergic neurites and glutamate appears to mediate the visually elicited EPSP. Although the SF NMDA receptor differs in several regards from those in the vertebrates, it also shares some common features, such as suppression by M2+, dependence upon glycine, and sensitivity to both specific and nonspecific NMDA antagonists. Both pharmacological and reversal potential measurements distinguish the SF NMDA and non-NMDA receptors. METHODS Immunocytochemistry The distribution of glutamate in crayfish optic lobes was examined with a monoclonal antibody specific for
Received December 10,1990; accepted in revised form February 5,1991.
36
C. PFEIFFER-LINN AND R.M. GLANTZ
carbodiimide-fixedglutamate (Madl, Larson, and Beitz, 1986)obtained from Immuno Nuclear (Stillwater, MN). Crayfish were cooled to 4°C and exsanguinated by exchanging blood for oxygenated saline for 2 hours. Sixteen eyestalks (from 8 animals) were processed. Eight of these were used in preliminary studies to establish the optimal antiserum concentration and exposure time. The optic lobe was excised and placed in Ca2+-freevan Harreveld's (1936) solution for 5 minutes at 4°C. Fixation and incubation procedures are slightly modified from Madl et al. (1986). The tissue was fixed with 5% carbodiimide in phosphate buffered saline (PBS), pH 7.2, for 15 minutes and postfixed with 5% glutaraldehyde in PBS for 2 hours. The fixed tissue was sectioned in a cryostat a t 50 pm, floated in culture slides, and incubated in 10% normal horse serum in 0.3% Triton X-100 and PBS for 1 hour. After two 15-minute washes in PBS, the sections were incubated in the primary antibody, diluted 1:1,000 in PBS and 0.3% Triton X-100, for 12 hours at 4°C. The sections were washed in PBS and incubated in biotinylated anti-mouse IgG antibody in 0.3%TritonX-100 and PBS for 1 hour at 23°C. Following 3 more PBS washes, the sections were incubated with avidin peroxidase reagent (Immuno Nuclear) for 1 hour, washed, and incubated in diaminobenzidine tetrachloride until the reaction product was visible (typically 5-20 minutes). The reaction was stopped by a wash in 0.9% NaCl, Tris buffer, pH 7.6. This procedure was replicated in 4 preparations. Two control procedures were used to assess nonspecific staining. In one (n = 2), the above procedure was repeated precisely as described, but the primary antiserum was deleted from the incubation protocol. In the second procedure (n = 2), the primary antiserum was preabsorbed in a solution of 5.0 mh4 gamma-l-glutamyl-L-glutamate for 1 hour prior to incubation with the tissue.
cells from electrodes containing 2.0 M CsCl with 0.5 nA of positive current for 5 minutes. Visual stimulation was performed with a system consisting of a 300-Watt quartz-iodide lamp, condenser lens, electromagnetic shutter, neutral density filter, and a 5-mm fiber-optic light guide, which conducted the illumination to the eye. The terminal of the light guide was held in a micromanipulator and positioned 5 cm from the eye. Pharmacological methods The agonists tested consisted of L-glutamate, (Ykainate (Sigma, St. Louis), quisqualate, and NMDA (Research Biochemicals, Natick, MA). All agonists were tested before and after replacing the saline in the eyestalk with a Ca2+-freesaline containing 20 mm CoC1,. The Co2+ reduced all synaptic potentials by 390% in 2-5 minutes. The antagonists tested included kynuD-AP7 renate, DNQX (6,7-dinitroquinoxaline-2,3-dione), (D-2-amino-7-phosphonoheptanoic acid) (Research Biochemicals), picrotoxin, and L-glutamate-gamma-methylester (GME) (Sigma). Agonists were pressure injected via a 5-pm tip glass pipette placed in the medullary neuropile. The injected volume (about 65 pl) was measured under oil in a compound microscope. Antagonists were applied to the surface of the desheathed ganglion in 1-2 pl volumes. Reversal potentials and membrane conductance were measured in current clamp with procedures described in Waldrop and Glantz (1985).
RESULTS Immunocytochemistry Glutamate is found in high concentrations in arthropod brains (McCaman, 1984). Immunocytochemical assays in crayfish optic lobe (Fig. 1)indicate high levels of anti-glutamate reactivity in the first optic neuromere, the lamina ganglionaris (LA), and the medulla externa (ME) and interna (MI) (second and third neuromeres of Physiological preparation the optic lobe, respectively). There is also substantial Adult crayfish of 8-10 cm length were prepared as in reactivity in the optic chiasm between the lamina and Pfeiffer and Glantz (1989). The crayfish were cooled, the medulla externa. Comparable staining in ME and exsanguinated as described above, and clamped in a MI was observed in each of the replicates, but the plexiglass chamber. The eyestalks were cemented to the reactivity in the lamina was variable. carapace with cyanoacrylate, and the optic lobe was Two control procedures suggest that the observed exposed by excising the cuticle on the distal eyestalk. staining pattern indicates the distribution of glutamate. The preparation was maintained a t 13°C in Van Harrev- In the first, the serum antibodies were incubated with eld's (1936)solution. Van Harreveld's solution contains the original antigen (glutamate-glutaryl-bovine serum 205 mM Na+,5.4 mM K+, 13.5 mM Ca2+,2.6 mMMg', albumin). An absence of staining indicated that the 243 mM C1-, and 2.4 mM NaHCO,. nonspecific reactivity of the serum for crayfish nerve Visual interneurons were impaled with 100 MO mi- tissue was extremely low and that the source of the cropipettes filled with 3.0 M potassium acetate. Signals reactivity is the antibody(s1to the glutamate conjugate. were amplified with a capacitance-compensated elec- An identical result was obtained when the antiserum trometer and stored on an FM tape recorder. Intracellu- was omitted from the initial incubation. lar current injection was performed through a bridge These data are consistent with the presence of high circuit, and electrodes were selected for fast response levels of glutamate in optic lobe neuropiles. This glutaand minimal polarization artifact. Cs+ was injected into mate could serve as a neurotransmitter or may be a
AN ARTHROPOD NMDA RECEPTOR
37
uniquely identified by their visual receptive fields (Wiersma and Yamaguchi, 1966) and spatially correlated dendritic geometries (Kirk, Waldrop, and Glantz, 1983). SF axons project to the brain, where they innervate ocular motoneurons and thus participate in optomotor reflexes (Glantz and Nudelman, 1988).The SFs respond to illumination with a tetrodotoxin-insensitive EPSP (Fig. 2A) of up to 40 mV. The EPSP is associated with an increase in input conductance (G,), as illustrated in Table I (Waldrop and Glantz, 1985). Glutamate (1.0-10 pM, Fig. 2B), quisqualate (.01 pM, Fig. 10, and kainate (1.0 pM) elicit approximately halfmaximal depolarizing responses that are unaffected by synaptic blockade with 20 mM CoC1, (Figs. 2C, 2D). The responses to the EAAs are associated with substantial increases in G, (Fig. 2D). L-glutamate, at 10 pM, increases G, by 70% (Table 1). EAA receptors with maximal sensitivity t o quisqualate have been described at the crayfish neuromuscular junction (Stettmeier and Finger, 1983). Ere, for responses elicited by glutamate were -23 k 3 mV (mean SD, n = 4) or about 43 mV above the resting potential (-66 mV; Fig. 3, open circles) and very similar to the extrapolated Ere, of the EPSP (Fig. 3, filled circles, Table 1). At 1.O mM, L-glutamate-gamma-methylester(GME) blocks the excitatoryjunction potential and the depolarizing action of glutamate a t the crayfish neuromuscular junction (Lowagie and Gerschenfeld, 1974). At 0.1 mM GME blocks the SF EPSP (Fig. 4A) and the response to 10 pM glutamate (Fig. 4B) and 1.0 pM kainate (not shown). A second antagonist, DNQX, which competitively blocks vertebrate non-NMDA receptors (Honore et al., 1988) was ineffective on both the EPSP and responses elicited by glutamate and quisqualate at antagonist concentrations of up t o 0.1 mM. NMDA, applied at resting potential in normal crayfish saline (with 2.6 mM Mg+),was without measurable effect in 8/8 cells tested (Fig. 5A, bottom trace). Depolarizations (with extrinsic current) of up to 20 mV failed to unmask an NMDA response in control saline. In M 2 + free saline, however, NMDA elicited a modest depolarization in 42/42 cells (Fig. 5A, middle trace). The NMDA response is associated with a 109% increase in G, (Fig. 5B, Table 1). The NMDA-elicited response is further enhanced (by 100-200%) and prolonged by the presence of 10 pM glycine (Fig. 5A, top trace). A comparable action of glycine on the NMDA-elicited membrane conductance change is shown in Figure 5C. At 10 pM, glycine more than doubles the NMDA-elicited increase in G, (Table 1).As observed in vertebrate neurons (Johnson and Ascher, 19871, this action of glycine is specific to the NMDA receptor. By itself, glycine produces no effect on the SF membrane potential or input conductance, and has no effect on the response to kainate (not shown). Glycine (1.0-100 pM) does, however, reduce the amplitude of the EPSP (by 50-70% at 100 pM) (Figs. 5D, 5E). If this glycine action is mediated by
*
Fig. 1. Immunocytochemical localization of glutamate in the crayfish optic lobe. Dark staining produced by indirect peroxidase method in the lamina ganglionaris (LA), the medulla externa (ME), and the medulla interna (MI). Scale (lower right) is 100 pm.
precursor for the synthesis of GABA. Previous studies have demonstrated that crayfish optic lobe contains high levels of GABA (Pfeiffer-Linn and Glantz, 1989). Furthermore, GABAergic inhibitory motoneurons in lobster contain about the same amount of glutamate as the presumed glutamatergic excitatory motoneurons (Otsuka, Kravitz, and Potter, 1967). Thus some or even all of the immunoreactivity shown in Figure 1 could reflect GABAergic neurons. Physiology and pharmacology Pharmacological studies were carried out with glutamate and several agonist analogs. The actions of the EAAs were examined in four classes of identified interneurons with dendrites in the medulla externa. The neurons include the SFs, amacrine cells, dimming fibers, and tangential cells. Each cell type has a distinct structure and visual response (Waldrop and Glantz, 1985; Wang-Bennett and Glantz, 1987; Pfeiffer and Glantz, 1989). Among these four neuron classes, only the SFs are responsive to glutamate. The SFs are the principal output cells of the medulla externa. The class consists of 14 neurons, which can be
C. PFEIFFER-LINN AND R.M. GIANT2
38
B
A
B
Ar
D
C
Fig. 2. SF light response (A)and SF responses to 10 +M L-glutamate (B) and 10 nM quisqualate (C). At 10 nM, quisqualate reduces the SF input resistance from 16 to about 1.0 MR (D). In C and D, synaptic activity is blocked with 20 mM Co". Current pulses are -0.5 nA. The scale is 10 mV and 1.0 s.
-
-
Fig. 4. L-glutamate-gamma-methylester at 0.1 mM blocks the PSP and the glutamate-elicited responses (lowertraces) in A and B, respectively.
TABLE 1. Reuersal potentials and input conductance changes associated with the EPSP and responses elicited b y 10 p M L-glutamate and 10 p M NMDA EWl (mV) -19 k 9 i7)3 -23 f 3 (4) -60 f 5 (11) -60 f 9 (3) 4 f 8 (3)
Agonist EPSP L-Glutamate NMDA4 NMDA, Glycine NMDA, Cs'
AG' (% control)
121 f 51 (6) 70 f 55 (5) 109 28 (7) 285 f 64 (4) 27 f 20 (3)
+
5 mV (SD, n=35). 'Average membrane resting potential is -66 Siemens (n=23). 'Average resting input conductance is 8.9 & 2.6 X 3All results are mean +standard deviation and the number of separate determinations is indicated in parentheses. EPSP data from Waldrop and Glantz (1985). 4All NMDA measurements in Mg"+-free saline solution.
..
PSP
i , 0 GLUTAMATE
\
\
- 30mV -
B
E
A F
20
-- 10
Fig. 3. Sustaining fiber PSP and glutamate-elicited responses vs. membrane potential. Reversal potential of the PSP ( 0 ) and glutamate responses ( 0 )estimated by extrapolation from the least-square regressions.
the NMDA receptor, then it implies that NMDA-elicited currents contribute to the repolarization of the EPSP. This is observed when an NMDA pulse is superposed on the EPSP (Fig. 5F, arrow). The partial inhibition of the EPSP by glycine is consistent with the potentiation of the NMDA receptor and a relatively negative Ere, of the NMDA-elicited
Fig. 5. SF responses to NMDA (10 pM) and glycine. A Responses t o NMDA in control saline (bottom trace), in M$+-free saline (middle trace), and in 10 +M glycine in M$+-free saline (top trace). Each trace is the average of 5-10 responses. B,C: Input resistance changes in response to NMDAin 0 M$+ (B)and after the addition of 10 pM glycine (C). D: Control PSP. E PSP in 10 +M glycine. F: Responseto an NMDA pulse (arrow) superposed on a visually elicited EPSP. Scale (in panel F) is 10 mV and 1.0 s throughout.
response. When the SF membrane is hyperpolarized, the NMDA-elicited response increases dramatically with increases in membrane potential (Fig. 6, top, inset). Conversely, the response is inverted by membrane depolarizations of no more than 10-15 mV. The Ere, of the NMDA-elicited response (-60 2 5 mV, n = 11; Fig. 6 , O ) is about 6 mV above resting potential. As a consequence, the action of the NMDA receptor in a depolarized cell tends to move the membrane potential away from the glutamate E, toward -60 mV. Thus, the plateau phase of the sustaining fiber EPSP, which is generally about 20 mV above rest, may reflect the joint action of two EAA receptor classes that gate opposing currents.
AN ARTHROPOD NMDA RECEPTOR
w i20mV
-90 mV
-30
Fig. 6. Response amplitude vs. membrane potential of the NMDAelicited responses in Mg2+-freesaline ( 0 )and followingthe intracellular injection of Cs' (0).Continuous lines are least-square regressions. Insets (top) are NMDA-elicited responses in Mg2'-free saline and following intracellularinjection of Cs'. The scale is 10 mV and 1.0 s.
An E, of -60 mV is unusual for an NMDA-activated conductance. NMDA receptors generally gate a nonspecific cation conductance with E, =: 0 mV and with a significant calcium permeability (Mayer and Westbrook, 1987). Since intracellular calcium can gate a calcium-activatedpotassium conductance (Meech, 1978), it is possible that the -60 mV Ere, is only secondarily associated with NMDA receptor activation (Nicol and Alger, 1981). Alternatively, it is possible that in SFs NMDA gates a mixture of inward and outward currents, as observed in glutamate responses of crab stomatogastric neurons (Marder and Paupardin-Tritsch, 1978)and locust neural somata (Giles and Usherwood, 1985). When SFs are injected with Cs+ to block potassium conductances (Gorman et al., 1982; Johnson and Ascher, 1987; Cull-Candy and Usowicz, 1987), Ere, of the NMDA-elicited response shifts from -60 to about + 4 mV (Table 1;Fig. 6,o). The difference in the slope of the two functions in Figure 6 also indicates that the Cs+
39
markedly reduces the magnitude of the NMDA-elicited change in input conductance (Table 1). The results indicate that NMDA can elicit a depolarizing response and (either directly or indirectly) a large outward current. The 4 mV E,, following Cs+ injection is consistent with an NMDA-elicited cation conductance. The outward current could be a secondarily activated potassium conductance or a directly elicited response in a subpopulation of glutamate receptors (Marder and Paupardin-Tritsch, 1978; Yarowsky and Carpenter, 1976; Atwood, 1980). To distinguish between these alternatives, the NMDA response was elicited in nominally Ca2+-freemedia in which Ca2+was replaced with Ba2+ or Co2+.None of these substitutions produced a significant shift in the -60 mV E,,,. This result implies that the -60 mV Ere, does not depend upon an inward calcium current and further suggests that the NMDA response may reflect a mixture of inward and outward currents, as observed in the glutamate responses of other arthropod neurons. Because Co2+ has a potent Mg+-like action on the vertebrate NMDA receptor (Ascher and Nowak, 1988), the persistence of the SF NMDA response in 20 mM Co2+ indicates another important difference between vertebrate and crayfish receptors. Because the vertebrate NMDA receptor is in part defined by its sensitivity t o a wide array of specific and nonspecific antagonists (Olverman and Watkins, 19891, we tested the crayfish receptor's sensitivity to a variety of blockers. GME, which blocked the SF EPSP and glutamate response at 0.1 mM, was ineffective on the NMDA-elicited response, even at 1.O mM. Picrotoxin blocks glutamate-elicited outward currents in lobster stomatogastric ganglion (Marder and Eisen, 19841, but had no effect on the NMDA-elicited response in SFs at 0.1 mM. Kynurenic acid is both a nonspecific EAA antagonist (Perkins and Stone, 1982; Ode11 and Christensen, 1989) and a competitive antagonist of the allosteric action of glycine on the NMDA receptor (Thomson et al., 1989). When 0.1 mM kynurenate is applied to SFs, it reduces the EPSP by 50430% (Fig. 7A, lower trace) and blocks the response to kainate (Fig. 7B, lower trace). Both of these effects are consistent with an antagonist action of kynurenate on the non-NMDA receptor. Kynurenate also blocks the response to NMDA (Fig. 7C, lower trace). In 10 p,M glycine and M$+-free saline, 0.1 mM kynurenate abolishes the NMDA response (Fig. 7D, lower trace), but the inhibition is reversed by 1 mM glycine (Fig. 7D, upper trace). Thus the actions of kynurenate in crayfish resemble its actions in vertebrates, i.e., a nonspecific antagonism to EAA receptors and a competitive block of glycine action an the NMDA receptor. D-AP7 is a potent and selective NMDA antagonist (Olverman and Watkins, 1989). In 3/3 tests, 10 pM
40
C. PFEIFFER-LINN AND R.M. GLANTZ
8
C
A!”
D d P
*
Fig. 7. Kynurenate a t 0.1 mM (lower traces A-C) antagonizes the natural transmitter (A), the response to 10 pM glutamate (B), and NMDA in low Mg2+but in the absence of glycine (0.In 10 pM glycine kynurenate blocks the NMDAresponse (D, lower trace), but the block is overcome by elevating glycine to 1.0mM (D, upper trace).
Fig. 8. Actions of the NMDA antagonist D-AP7. At 10 pM, D-AP7 blocks the NMDA (10 pM) response (A, lower trace) and augments the visually elicited PSP (B,upper trace). The upper trace in A and the lower trace in B are control responses. Each trace is the average of 12 responses. All measurements are in Mg2+-freesaline. The scale is 1.4 mV and 0.4 s in A, and 6.0 mV and 75 ms in B.
D-AP7 abolished the response to 10 pM NMDA (Fig. 8A, tion with non-NMDA glutamate receptors (Jahr and lower trace), while it had no measurable effect on the Stevens, 1987). In several vertebrate neurons (Cotman response to kainate. In the same cells, D-AP7 potenti- et al., Dale, 1989),the non-NMDA receptor mediates the ated the visual response by 30-100% (Fig. 8B, upper initial, rapid phase of the EPSP, which is subsequently trace). This result implies that the activation of the modulated by the delayed action of the NMDA receptor. NMDA receptor tends to reduce the SF EPSP. The effect In crayfish SFs, the transient EPSP appears to be of D-AP7 is consistent with the effects of glycine on the mediated by the non-NMDA receptor. The actions of the SF EPSP (Fig. 5E) and the action of NMDA superposed NMDA receptor are consistent with a modulatory role, possibly related to neuronal mechanisms of light adapon the EPSP (Fig. 5F). tation. DISCUSSION An important property of the vertebrate NMDA reBoth pharmacological tests and reversal potential ceptor is that the M 2 + block of the ionic channel is measurements indicate that crayfish SFs possess at voltage dependent and the NMDA response exhibits a least two classes of EAA receptors. A non-NMDA recep- negative slope conductance between -60 and -30 mV tor mediates the initial phase of the visually elicited (Mayer and Westbrook, 1987). The crayfish NMDA EPSP, and it is highly sensitive to quisqualate. The response is completely blocked by normal levels of M 2 + , NMDA receptor(s1 appears to primarily mediate an and this block is insensitive to depolarizations of up to 20 mV. Another obvious difference between the crayfish outward current. The crayfish NMDA receptor exhibits some striking and vertebrate NMDA receptors is the apparent reversimilarities and important differences when compared sal potential. In this regard, the SF NMDA response to the vertebrate receptor. Both receptors are blocked by resembles the glutamate-elicited responses in crab and circulating levels of M$+ (Davies and Watkins, 1977) lobster stomatogastric ganglion (Marder and Pauparand by the specific antagonist D-AP7, and both have a din-Tritsch, 1978; Marder and Eisen, 1984) and the requirement for glycine, which is susceptible to compe- extrajunctional glutamate responses in arthropod mustition by kynurenate. In mammalian cerebrospinal cle (Hironaka, 1974; Atwood, 1980). The fact that the fluid, glycine concentrations are above 1.0 pM, which reversal potential is unaffected by deleting Caz+ from substantially exceeds the nM requirements of the the medium argues against an important role for a NMDA receptor (Ascher and Johnson, 1989) and masks Ca2+-activated potassium conductance. Following inthe glycine dependence in situ. Since the concentration tracellular Cs+injection, the E,, of the NMDA response of glycine in crayfish hemolymph is about 1.0 mM is more positive and resembles that in vertebrates. (Gardiner, 1972) and glycine potentiates NMDA action Taken together these results suggest that NMDA elicits at 1.0 pM, the same situation should obtain in crayfish. a mixture of inward and outward currents normally The in situ observations of glycine dependence were dominated by the latter. An insensitivity to Co2+further made possible by the exsanguination procedure that distinguishes the SF from vertebrate NMDA receptors replaces the hemolymph with Van Harreveld’s solution, (Ascher and Nowak, 19881,and an insensitivity to picrocomposed exclusively of inorganic ions. toxin distinguishes the SF receptor from the glutamateAn additional similarity between crayfish and ver- activated potassium conductances in the stomatogastric tebrate NMDA receptors is that they occur in conjunc- ganglion.
41
AN ARTHROPOD NMDA RECEPTOR
The failure of previous invertebrate studies to reveal NMDA receptors may reflect the particular sites tested. The crayfish NMDA receptors were found on particular interneurons and on their dendritic processes in central neuropile. Previous studies in lobster and locust were confined to the neuromuscular junction (Nistri and Constanti, 1979; Ushenvood and Machili, 19681, and those in the snail examined only neuronal cell body receptors (Parmentier and Case, 1972). In the leech (James et al., 19801, "IDA was bath applied but only the Retzius neurons were tested. Thus it is likely that further study of invertebrate neurons will reveal additional examples of NMDA receptors. The sustaining fiber non-NMDA receptor shares several properties with those a t the crayfish neuromuscular junction (Atwood, 1980). The receptor mediates an excitatory and presumably cationic conductance. It is particularly sensitive to quisqualate, and it is blocked by GME. On the other hand, the SF receptor is also sensitive to kainate, which is an antagonist a t crayfish junctional receptors but an agonist at the extrajunctional receptors (Onodera and Takeuchi, 1980). The SF agonist-elicited responses appear to be relatively slowmuch slower than the responses elicited by iontophoresis at the neuromuscular junction. This difference is probably a consequence of our agonist application procedure (pressure injection), which could produce a diffusion-limited activation in the dense neuropile. The SF EPSP is quite rapid and the rate of depolarization (up to 6 mV/ms) is comparable to that of the excitatory junction potential elicited by a "fast" motoneuron (10 mV/ ms, Atwood, 1980). A more significant difference between the SF and junctional EAA receptors may be the reversal potentials of the associated synaptic and agonist-elicited responses. At crustacean neuromuscular junctions, Ere, of the excitatory junction potential (e.j.p.) and glutamateelicited responses varies from 0 to +50 mV (Atwood, 1980). In SFs, E, of the EPSP and the glutamate elicited responses are about -20 mV (Fig. 3), as measured in current-clamped cells. This discrepancy could be due to a real difference in channel selectivity and/or transmembrane ion gradients, but it could also reflect the effects of a voltage-dependent potassium conductance. An overview of the results provides substantial but still incomplete evidence that glutamate (or an analog) is the excitatory transmitter to the SF. The immunocytochemistry suggests the presence of glutamate in the vicinity of the SF dendrites, but we have no evidence that visual stimulation promotes synaptic release of glutamate. The reversal potential of the glutamateelicited response is similar to that ofthe EPSP, and both are antagonized by GME and kynurenate. It is clear that the SF has an EAA receptor, since the responses to glutamate and all excitatory amino acid analogs persist following synaptic blockade with cobalt. As noted above, one possible role for the SF NMDA
receptor is a neuronal mechanism of light adaptation (Glantz, 1972). The SF response to a step increase in illumination intensity is a compound EPSP, consisting of a rapid transient phase of up to +40 mV and a steady-state plateau of about 20 mV above the resting potential. As a consequence of its relatively negative E,,,, the NMDA response could contribute an outward current, which would partially repolarize the SF during prolonged excitation. In summary, our results suggest that the SF compound EPSP is mediated by glutamate, which acts upon a non-NMDA receptor to elicit the initial transient depolarization and an NMDA receptor, which participates in the partial repolarization and plateau phase. The non-NMDA receptor is about 100 times more sensitive to quisqualate than to glutamate or kainate. It is blocked by GME and kynurenate but is insensitive to DNQX. The latter result indicates that the SF nonNMDA receptor is distinct from analogous receptors in vertebrates. The NMDA receptor mediates a mixture of inward and outward currents dominated by a Cs'sensitive conductance, with a reversal potential just positive to the resting potential. The NMDA response is blocked by M 2 + but is insensitive to extracellular Coz+ and Ca2+. These features distinguish the SF NMDA receptor from the vertebrate prototype. The SF NMDA response is potentiated by glycine, and kynurenate competitively blocks glycine action. D-AP7 completely suppresses the response to NMDA. In these aspects the SF receptor is similar to that of vertebrates. Since it is inferred that the depolarizing EPSP is principally mediated by the non-NMDA receptor, it follows that GME and kynurenate (as a nonspecific EAA antagonist) reduce or abolish the EPSP. Conversely, since D-AP7 blocks the NMDA-elicitedoutward current, it enhances the EPSP. Glycine potentiates the NMDAelicited current and thus reduces the EPSP. The principal deficiency in the above interpretation is that we have been unable to unambiguously document the action of the NMDA in saline of normal M$+ concentration. It is possible that the M 2 + block could be relieved at depolarizations beyond the reach of our current clamp system. Alternatively (but less likely) it is possible that in neuropile, the M 2 + concentration is less than that in hemolymph. Thus, although it is clear that the SF has a receptor for NMDA, its exact function cannot be established until the nature of the M 2 + block is clarified. ACKNOWLEDGMENTS Supported by N.S.F. grants BNS-8711141 and BNS9021216, and a Lodieska Stockbridge Vaughn Fellowship to C.P.-L. We thank Dr. Dan Johnston of Baylor Medical School and Dr. Michael Gustin of Rice for useful comments on an earlier draft of this manuscript.
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C. PFEIFFER-LINN AND R.M. GLANTZ
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