Glutamate and GABA Activate Different Receptors and Clminus Conductances in Crab Peptide-Secretory Neurons

Shumin Duan and Ian M. Cooke

Békésy Laboratory of Neurobiology and Department of Zoology, University of Hawaii, Honolulu, Hawaii 96822


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Duan, Shumin and Ian M. Cooke. Glutamate and GABA Activate Different Receptors and Clminus Conductances in Crab Peptide-Secretory Neurons. J. Neurophysiol. 83: 31-37, 2000. Responses to rapid application of glutamic acid (Glu) and gamma -aminobutyric acid (GABA), 0.01-3 mM, were recorded by whole-cell patch clamp of cultured crab (Cardisoma carnifex) X-organ neurons. Responses peaked within 200 ms. Both Glu and GABA currents had reversal potentials that followed the Nernst Cl- potential when [Cl-]i was varied. A Boltzmann fit to the normalized, averaged dose-response curve for Glu indicated an EC50 of 0.15 mM and a Hill coefficient of 1.05. Rapid (t1/2 ~ 1 s) desensitization occurred during Glu but not GABA application that required >2 min for recovery. Desensitization was unaffected by concanavalin A or cyclothiazide. N-methyl-D-aspartate, alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, quisqualate, and kainate (to 1 mM) were ineffective, nor were Glu responses influenced by glycine (1 µM) or Mg2+ (0-26 mM). Glu effects were imitated by ibotenic acid (0.1 mM). The following support the conclusion that Glu and GABA act on different receptors: 1) responses sum; 2) desensitization to Glu or ibotenic acid did not diminish GABA responses; 3) the Cl--channel blockers picrotoxin and niflumic acid (0.5 mM) inhibited Glu responses by ~90 and 80% but GABA responses by ~50 and 20%; and 4) polyvinylpyrrolydone-25 (2 mM in normal crab saline) eliminated Glu responses but left GABA responses unaltered. Thus crab secretory neurons have separate receptors responsive to Glu and to GABA, both probably ionotropic, and mediating Cl- conductance increases. In its responses and pharmacology, this crustacean Glu receptor resembles Cl--permeable Glu receptors previously described in invertebrates and differs from cation-permeable Glu receptors of vertebrates and invertebrates.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Secretory neurons, defined as neurons whose axonal terminals are specialized for the release of hormones to the circulation, while often showing "spontaneous" electrical activity, are under synaptic and hormonal control from the CNS (see review by Cooke and Stuenkel 1985). The major neurosecretory system of crustaceans, the X-organ-sinus gland, is analogous to the vertebrate hypothalamic-neurohypophysial system in its physiological roles and cellular mechanisms (see reviews by Newcomb et al. 1988; Stuenkel and Cooke 1988). Recordings from the X-organ, the cluster of secretory neuronal somata, of crayfish (Procambarus clarkii) eyestalks showed inhibitory synaptic potentials, both spontaneously occurring and in response to stimulation of the optic peduncles (axonal tracts from the brain) (Iwasaki and Satow 1971).

Although there have been a number of reports pointing toward gamma -amino-butyric acid (GABA) as a synaptic transmitter of the X-organ-sinus gland system (e.g., Aréchiga et al. 1990; Fingerman 1985; Glantz et al. 1983; Nagano 1986), responsiveness to glutamate has not been reported, to our knowledge. An increase in conductance to Cl- in response to GABA has been shown in X-organ neurons of crayfish (P. clarkii) (García et al. 1994) and in cultured X-organ neurons of the crab used for this study (Cardisoma carnifex) (Rogers et al. 1997). Furthermore, acutely isolated secretory terminals of the crab sinus gland showed a Cl- conductance increase in response to GABA (Rogers et al. 1997).

Glutamic acid (Glu) receptors producing an ionotropic Cl- conductance increase have been described in a number of arthropod and other invertebrate groups (see reviews by Cleland 1996; Cully et al. 1996a), although it is less clear whether such receptors are present in vertebrates. The coexistence of separate receptors for Glu- and GABA-mediated ionotropic Cl- conductance increases has been shown in several arthropod and molluscan preparations (see references in DISCUSSION). The observations reported here have been summarized in an abstract (Duan and Cooke 1997).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dissection and culturing

Experiments were performed on cultured X-organ somata that had been 2-3 days in culture. The procedures used to dissociate and culture X-organ neurons from the semiterrestrial tropical crab Cardisoma carnifex Herbst have been described in detail elsewhere (Cooke et al. 1989; Grau and Cooke 1992). Briefly, the X-organ with <1 mm of the axon tract was removed from the eye stalk of adult male crabs and agitated in the dark for 1.5 h in a Ca2+/Mg2+-free saline containing 0.1% trypsin (Gibco). A large volume of Ca2+/Mg2+-free saline was then added to retard enzymatic activity, and the cells were dissociated by gentle trituration in a 60 µl drop of culture medium on 35 mm Primaria dishes (Falcon 3801). The dishes were carefully flooded after allowing 1-2 h for the cells to adhere to the substratum. The culture medium consisted of Leibowitz L-15 (Gibco) diluted 1:1 with double-strength crab saline to which D-glucose (120 mM, Fisher), L-glutamine (2 mM, Sigma), and gentamicin (50 mg/ml, Gibco) were added. Cultures were maintained in humidified incubators (Billups-Rothenberg) in the dark at 22-24°C.

Cells whose regenerative outgrowth took the form of large, lamellipodial growth cones ("veilers," Grau and Cooke 1992), and were thus identifiable as containing crustacean hyperglycemic hormone (Keller et al. 1995), were chosen for this study. Before starting electrophysiological recording, the culture dish was rinsed three times and the medium replaced with filtered crab saline consisting of (in mM) 440 NaCl, 11 KCl, 13.3 CaCl2, 24 MgCl2, and 10 HEPES (pH 7.4) with NaOH. During experiments, the dish was constantly superfused with crab saline at a rate of ~0.2 ml/min. A self-priming siphon maintained a relatively constant fluid level. Somata were viewed using a Nikon Diaphot inverted microscope with a 40× objective and Hoffman modulation optics.

Drug application

The use of a miniature Y-tube (Murase et al. 1989) manipulated to within <25 µm of the soma permitted rapid (<100 ms) application of drug-containing saline to the neuron with minimal dilution or mixing during patch clamping. The solutions to be applied, held in reservoirs higher than the recording bath, were drawn through the arms of the Y, which are of larger diameter tubing (PE 50) than the stem (PE10), by gentle suction; some bath fluid was drawn through the stem to ensure that no agent was applied unintentionally. A solenoid pinch valve, controlled by a Grass S15 stimulator through which the arm on the suction side of the Y passes, was used to stop the suction for a selected duration permitting gravity flow of the material out of the Y while suction was blocked. Applications of agonists to neurons were separated by at least 3 min to avoid possible desensitization effects. When the effect of changed ionic conditions or the presence of inhibitors was to be tested, the neuron was pretreated by bath perfusion and application through the Y tube of the altered solution for 3-10 min before testing. Agonists, made up in the altered solution, were applied through the Y-tube while continuing perfusion with altered solution. Drugs and reagents were obtained from Sigma (St. Louis, MO).

Electrophysiology

Voltage-clamp recordings were obtained in the whole-cell patch-clamp configuration using an EPC9 amplifier (Instrutech, Great Neck, NY). Data acquisition, storage, and analysis were performed using HEKA software (Instrutech) run on a Macintosh Centris 650. Signals were filtered with a corner frequency of 2.9 kHz. All experiments were recorded at room temperature (22-26°C). Pipettes used to obtain tight-seal whole-cell recordings were pulled from Kimax thin-walled glass capillaries (1.5-1.8 mm OD) on a vertical puller (TW 150F-4, David Kopf Instruments). Pipettes were coated with dental wax to reduce capacitance and were fire polished with a microforge (model MF-83, Narishige). Pipettes filled with the intracellular solution and immersed in the bath had resistances ranging from 1.5 to 5 MOmega , but were typically 1.5-3 MOmega . The intracellular solution, unless otherwise noted, was (in mM) 300 KCl, 10 NaCl, 5 Mg-ATP, 5 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, and 50 HEPES, pH adjusted to 7.4 with KOH. The extracellular solution was crab saline as above with the addition of 10 mM D-glucose. The tonicity was adjusted with sucrose to 1095-1100 mOsm for intracellular solutions and 1100 mOsm for extracellular solutions. After establishing an electrode seal and breaking in, neurons were generally held at -50 mV, a value close to the usual resting potential and therefore requiring very little current when recordings were not being made. Compensation for series resistance and capacitance was optimized.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Responses to glutamic acid

Close application of Glu (0.01-3 mM) to the soma elicited transient current from whole-cell patch-clamped crab peptidergic neurons. Application using a Y-tube (see METHODS) ensured rapid (<100 ms) flooding of the cell with agonist little diluted from that preloaded into the Y-tube. Figure 1A presents superimposed recordings of responses from one neuron to a series of concentrations of Glu. Responses consisted of inward current from a holding potential (Vh) of -50 mV using the standard high-Cl- pipette solution (calculated Nernst potential, ECl, is approximately -14 mV). Current reached a maximum within 200 ms and then immediately declined (t1/2 ~1 s for concentrations of 0.1 mM or more) during the continued application of Glu. Although not seen in the responses of Fig. 1A, in approximately half the neurons studied the transient peak subsided to a plateau having an amplitude of <= 20% of the peak (records from neurons having a plateau may be seen in Figs. 2A and 4). Any remaining Glu response relaxed rapidly (<100 ms) upon termination of the agonist application. The rapid commencement and relaxation of the current suggests that it represents the response of an ionotropic receptor to Glu. A dose-response curve for this neuron plotting the peak currents observed against the log of the Glu concentration applied is shown in Fig. 1B. Responses to seven applications of near-saturating concentrations of Glu (1-3 mM) were recorded from 4 neurons and showed peak amplitudes of ~12 nA [11.7 ± 0.54 nA (SE); n = 7]. The relatively consistent peak amplitudes observed, both for the same neuron and between neurons (noting that they represent an identifiable neuronal type; see METHODS), suggests that the Y-tube application technique provided sufficiently rapid and complete exposure of the cell to the agonist to minimize desensitization effects on the recording of peak amplitude.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. Responses under patch clamp of cultured crab X-organ neurons to glutamate (Glu). A: superimposed responses to a series of glutamate concentration clamps, as indicated. Note rapid desensitization during continued presence of Glu. B: plot of peak Glu current versus concentration of Glu (log scale) from A. C: average normalized peak current versus Glu concentration (2-5 observations per point). The line represents a nonlinear regression on the data using Inorm=1/[1 + (EC50/[Glu])n], with n, the Hill coefficient, and EC50, the Glu concentration giving half-maximal effect, being free parameters. The fit provided EC50 = 0.15 mM and n = 1.05. Responses of each cell were first normalized to its response to 0.1 mM, the normalized values were then averaged to obtain a maximum averaged number, and each value was then renormalized (Cleland and Selverston 1998).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Reversal potentials of responses to Glu and GABA correspond to the ECl. A: records to application of nonsaturating concentrations of Glu (left) or GABA (right) while holding the cultured neuron at the potentials indicated. Note that the currents reversed between -50 and -70 mV; the calculated Nernst potential for Cl- was -59 mV. B: plot of reversal potentials observed in experiments altering [Cl-]i, [Na+]o, or [K+]o (see legend); only changes of [Cl-] influenced the reversal potential of Glu responses.

Figure 1C summarizes observations from seven neurons for which responses to three or more concentrations of Glu were obtained. The peak current amplitudes, normalized as described in the legend, are plotted against the log of the applied Glu concentration. The dose-response curve shows the usual S-shape, with a just-discernable effect at 0.01 mM and an asymptote indicating near-saturation at ~1 mM Glu. A good fit of the data to a Boltzmann function (Fig. 1C, continuous line; see legend) with the free parameters being the concentration for half-maximal effect (EC50) and the Hill coefficient (n) was obtained with EC50 = 0.15 mM and n = 1.05.

Desensitization of Glu responses

As mentioned in Responses to glutamic acid, the response to Glu during continued Glu perfusion declined with a t1/2 of ~1 s for concentrations of Glu of 0.1 mM or higher, but more slowly for lower concentrations. The rate of desensitization did not depend on the membrane holding potential, as may be seen in Fig. 2A. When desensitization had occurred, it could not be overcome by application of a high concentration of Glu (e.g., 1 mM), even though it was produced by application of a near-threshold concentration of Glu (0.01 mM),. In three experiments, the rate of recovery from Glu desensitization was examined by comparing the size of a second response to a brief (<5 s) pulse of 0.1 mM Glu with the first response. Recovery to 90% of the first response required ~140 s.

Exposure to the lectin concanavalin A (ConA) rapidly removes Glu desensitization of Aplysia neurons (Kehoe 1978) and vertebrate kainate-preferring Glu receptors (Partin et al. 1993; Wong and Mayer 1993). Thus the effect on Glu responses of pretreatment of crab neurons with ConA at 0.3 mg/ml for 10 min was examined. No differences in the rate of Glu desensitization were found (n = 3).

Cyclothiazide, another agent found to influence Glu desensitization of the vertebrate alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type Glu receptors, was tested at 0.1 mM for 5 min for effects on Glu responses of the crab neurons, but it showed no effects (n = 3).

Pharmacology of the neuronal Glu response

Several of the agents used to characterize vertebrate Glu receptors (Ozawa et al. 1998) were tested on the cultured crab peptidergic neurons. Of the agents diagnostic for N-methyl-D-aspartate (NMDA) receptors, NMDA had no effect up to 1 mM, nor were responses affected by the presence or absence of glycine (1 µM) or [Mg2+]o (0-26 mM). Of agents characterizing AMPA/kainate receptors, AMPA itself had no effect, nor was there any response to kainate or quisqualate (all tested up to 1 mM, n = 3 for each).

Glutamate-induced current is not mediated through a Na-dependent Glu transporter (Fairman et al. 1995; Robinson and Dowd 1997) because it was not affected by substitution of the bath saline with an Na+-free (choline-substituted) saline (Fig. 2B).

Ibotenic acid (IA) used as an agonist of ionotropic and metabotropic receptors (Conn and Pin 1997; Ozawa et al. 1998) proved to be an effective agonist (at 0.1 mM) on the crab neurons, producing inward current that showed rapid desensitization similar to the Glu responses (Fig. 3A). Further, after application of IA responses to Glu were attenuated and, similarly, after application of Glu IA responses were attenuated, indicating mutual cross-desensitization between Glu and IA. It will be seen from Fig. 3B that the neuron desensitized to Glu or IA responds to GABA as discussed in DESENSITIZATION TO GLU DOES NOT REDUCE GABA RESPONSES. The records shown in Fig. 3 are typical of observations from 5 neurons.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. A: imitation of Glu responses and cross- desensitization by ibotenic acid (IA). Records from the same neuron. Application of one agent after the other fails to elicit a response. B: application of Glu or IA does not inhibit response to GABA as compared with responses (not shown) recorded before Glu or IA application (records in B are from a different cell than A). Note much slower desensitization of responses to GABA than to Glu.

Glu and GABA responses represent conductance increases to Cl-

Recordings with bath or pipette salines having altered monovalent ion concentrations showed that the reversal potential for responses to both Glu and GABA closely tracked the calculated Nernst potential for Cl- but not for Na+ or K+ (Fig. 2). Note that the records shown in Fig. 2 were made with a pipette solution containing 50 mM Cl- rather than the usual 310 mM Cl-, thus giving an ECl of -59 mV (vs. -14 mV). The pipette, rather than bath, [Cl-] was changed to avoid the need for positive holding potentials and the errors that might result from using larger holding current. In contrast to the recordings of Fig. 1A, the Glu currents in Fig. 2 are seen to be outward at membrane potentials depolarized from the Vh of -50 mV, as typically seen for inhibitory postsynaptic events. As seen in the plot of the reversal potentials observed under various conditions against the pipette [Cl-] (Fig. 2B), changes made to bath Na+ or K+ concentration proved to have little effect on the responses, while the reversal potentials fall close to the line showing ECl. The conclusion that both Glu and GABA responses represent increases in conductance to Cl- is also supported by inhibitory effects of the Cl--channel blockers picrotoxin and niflumic acid, as detailed in the next section.

Evidence that Glu and GABA activate different receptors

RESPONSES TO GLU AND GABA SUM. If Glu and GABA acted on the same receptor, application of a saturating concentration of one would be expected to occlude response to the other. Figure 4 shows responses to a near-saturating concentration (3 mM) of Glu, GABA, and both combined. It will be seen that the response to the combined application was close to the sum of the response to each alone (in this example 97%). Given the somewhat slower time to peak of the GABA response, some desensitization of the Glu response may be expected to have occurred before the GABA response reached its peak.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Responses to Glu and GABA sum. Traces showing responses to near-saturating concentrations of Glu, GABA, and both are superimposed. The response when both are applied together is 97% of the sum of each alone.

DESENSITIZATION TO GLU DOES NOT REDUCE GABA RESPONSES. As mentioned in Pharmacology of the neuronal Glu response and illustrated in Fig. 3B, large responses to GABA were observable when applied immediately after Glu or IA that had desensitized the neuron to Glu. When compared with previously recorded GABA responses in the absence of previous Glu application, the responses after Glu proved to be undiminished (n = 5).

CL- CHANNEL BLOCKERS INHIBIT GLU AND GABA RESPONSES TO DIFFERENT EXTENTS. Picrotoxin was one of the first agents identified as an inhibitor of GABA-mediated responses and has more recently been shown to also inhibit other ligand-gated Cl- conductances (Cleland 1996; Etter et al. 1999; Yoon et al. 1993, 1998). Niflumic acid is an inhibitor of Cl- conductance used for control of endogenous Cl- currents during voltage-clamping of Xenopus oocytes (White and Aylwin 1990). Both of these agents inhibited responses of the crab neurons to Glu and GABA, but to different extents (Fig. 5). After pretreating neurons with the inhibitor at 0.5 mM by simultaneous bath and Y-tube perfusion for >3 min (see METHODS), Glu responses were inhibited ~90% by picrotoxin and 80% by niflumic acid. By contrast, GABA responses were inhibited by ~50 and 20%.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Glu and GABA responses are reduced to different extents by the Cl--conductance inhibitors niflumic acid and picrotoxin. A: inhibition by the presence of niflumic acid (0.5 mM) in the bath to focal application of Glu or GABA. Responses before and after adding niflumic acid are superimposed on the response in its presence. Note the much greater inhibition of the response to Glu than of that to GABA. B: inhibition by picrotoxin (0.5 mM) in the bath (superimposed traces as in A). Note that picrotoxin inhibits responses to both Glu and GABA more effectively than niflumic acid (A and B from the same neuron).

THERE IS A SELECTIVE INHIBITOR OF THE CRUSTACEAN GLU RESPONSES THAT LEAVES GABA RESPONSES UNAFFECTED. Polyvinylpyrrolydone-25 (PVP), which has been used in studies of the lobster stomatogastric ganglion (Cleland and Selverston 1995) as a membrane-stabilizing agent during application of salines deficient in divalent cations (Raditsch and Witzemann 1994), proved to be a selective blocker of the Glu response. With 2 mM PVP present in the bath, the responses to Glu were strongly inhibited or absent although those to GABA were unaltered (Fig. 6). PVP inhibition was rapidly reversible.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6. Polyvinylpyrrolidone-25 (PVP) inhibits responses to Glu but not GABA. A: superimposed traces showing responses to focal application of 0.1 mM Glu before the addition of PVP to the bath, in the presence of the indicated concentrations of PVP in the bath, and after the removal of PVP. Note that 2 mM PVP almost completely eliminates the response to Glu. B: superimposed responses to 0.2 mM GABA before PVP was added to the bath and with 2 mM PVP present in the bath (from a different neuron than A); the response is nearly unchanged. PVP was tested and inhibited the Glu response of this neuron (not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The observations reported here demonstrate responses to glutamate that result in a Cl- conductance increase of crab peptidergic neurons cultured from the X-organ-sinus gland system. These cells, identifiable by their broad lamellar growth cones, are known to produce hyperglycemic hormone (Keller et al. 1995). We further show that the Glu receptors are distinct from receptors to GABA that also produce a Cl- conductance increase. To our knowledge, this is the first report of these Glu receptors in a crustacean neurosecretory system. The characteristics of the responses and their pharmacology closely ally these receptors with those reported in a number of arthropod preparations, including the extrajunctional "H-receptors" of locust muscle (Cull-Candy 1976), crab stomatogastric ganglion neurons (Marder and Paupardin-Tritsch 1978), lobster gastric-mill muscle (Lingle and Marder 1981), and lobster stomatogastric ganglion neurons (Cleland and Selverston 1995, 1998; for a review see Cully et al. 1996a). In addition to being ionotropic receptors producing a Cl- conductance increase that is transient because of rapid desensitization, these receptors have several pharmacological characteristics in common. These include the ability of IA to imitate Glu effects and to cross-desensitize to Glu; failure to respond to the glutamate agonists of vertebrate CNS excitatory receptors such as NMDA, quisqualate, and kainate; and inhibition by picrotoxin and niflumic acid. Similar Cl--mediated responses to Glu have been described in molluscan neurons (e.g., Bolshakov et al. 1991; Ikemoto and Akaike 1988; Kehoe 1978; King and Carpenter 1989; Sawada et al. 1984). A receptor cloned from the nematode Caenorhabditis elegans (Cully et al. 1994) and one from Drosophila melanogaster (Cully et al. 1996b) expressed in frog oocytes also have similar pharmacological characteristics. While ConA effectively removes agonist desensitization of certain excitatory Glu responses of molluscan neurons, it did not influence the desensitization of Cl--mediated Glu responses in the molluscan neurons (Kehoe 1978) nor in the X-organ neurons (this study).

Iwasaki and Satow (1971) reported inhibitory postsynaptic potentials recorded from crayfish (Procambarus clarkii) X-organ neurons, their inhibition by picrotoxin, and their reversal potential near the resting potential. GABA was found to inhibit spontaneous electrical activity as recorded with KCl-filled electrodes intracellular to crab (C. carnifex) sinus gland terminals or X-organ somata when applied regionally to somata or terminals (Nagano 1986). Saturating concentrations of GABA moved the prevailing membrane potential toward a value of ~50 mV. Responses showing reversal at ECl have been reported in recordings from crayfish (Procambarus clarkii) X-organ neurons (García et al. 1994). Whether these responses should be considered excitatory as claimed requires evaluation in light of the previously mentioned observations and the reported selection in García et al. (1994) of only those neurons having initial resting potentials of -60 mV or more polarized. ECl was reported to be -45 mV, but it was not clear whether this was determined with KCl- or K acetate-filled electrodes. A patch clamp study of GABA responsiveness of the C. carnifex X-organ neurons in culture (Rogers et al. 1997) confirmed a Cl- conductance increase in response to applications to somata, but not neurites or growth cones. GABA responses were also obtained in patch clamp recordings from acutely isolated sinus gland terminals. The presence of receptors at the terminals, where there are no anatomically-distinguishable synapses (Weatherby 1981), suggests that GABA may act as a hormonal mediator. This possibility is supported by the presence of levels of GABA above the threshold for activating GABA responses in samples of C. carnifex hemolymph, and significantly higher than can be detected in neuronal or muscle tissue (R. Newcomb, B. Haylett, and I. Cooke, unpublished data).

The receptors producing a Cl- conductance increase to Glu have been found to be distinct from those responding to GABA in most of the arthropod preparations (Cleland and Selverston 1998; Lingle and Marder 1981; Marder and Paupardin-Tritsch 1978). Evidence for a shared receptor producing a Cl- conductance increase was obtained in single-channel recordings from crayfish (Austropotomobius torrentium) claw muscles (Franke et al. 1986). Unlike the lobster stomatogastric ganglion neuron Glu receptor (Cleland and Selverston 1995), quisqualate was an effective agonist of the muscle Glu receptor.

Studies of Cl--mediated responses to Glu and GABA of molluscan neurons provide a less clear picture, as there appears to be a number of types of receptors present together in single neurons that are responsive to Glu and/or GABA (e.g., Kehoe 1978; Oyama et al. 1990). Several studies provide observations supporting the existence of different receptors (e.g., Ikemoto and Akaike 1988; King and Carpenter 1987, 1989; Oyama et al. 1990). It has been suggested that there are receptors to Glu or GABA which share a channel producing Cl- conductance increases in molluscan neurons (King and Carpenter 1989).

For the crab X-organ neurons, the evidence that different receptors are responsible for Cl- conductance responses to GABA and Glu is compelling: the presence of desensitization to Glu but not to GABA, lack of cross-desensitization, summation of responses to Glu and GABA, different responsiveness to picrotoxin and niflumic acid, and identification of a selective agonist (ibotenic acid) and antagonist (PVP) acting on the Glu but not the GABA receptor.

In their study of what seems likely to be a homologous Glu receptor of lobster stomatogastric neurons, Cleland and Selverston (1995) used PVP as a membrane stabilizing agent in nominally Ca2+-free bath saline and left the Glu response unchanged. It remains to be seen whether the selective inhibition of the Cl--mediated Glu response by PVP (in normal saline) that has been observed in crab X-organ neurons will prove to be a general response. If so, it could prove to be a useful pharmacological tool for separating the responses of Glu receptors mediating ionotropic Cl-conductance increases from other responses.

We conclude that there are two types of receptors in the crab X-organ-sinus gland system responding to different transmitters (or hormones) to increase Cl- conductance. This is an effect that would be expected to stabilize the membrane potential near the prevailing resting level because there is no evidence for active regulation of internal Cl- in these neurons. The presence of two inhibitory systems suggests that activity of the neurosecretory cells is carefully regulated by the CNS.


    ACKNOWLEDGMENTS

We thank J. W. Labenia for preparing primary cultures of X-organ neurons and for unfailing technical assistance; we also thank Dr. Marc Rogers for helpful discussion.

This work was supported by the Cades Fund and by National Institute of Neurological Disorders and Stroke Grant NS-15453.

Present address of S. Duan: Institute of Neuroscience, Chinese Academy of Sciences, 320 Yue-yang Rd., Shanghai, PR China.


    FOOTNOTES

Address for reprint requests: I. M. Cooke, Békésy Laboratory of Neurobiology, University of Hawaii, 1993 East-West Rd., Honolulu, HI 96822.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 July 1999; accepted in final form 3 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society