Glutamate and GABA Activate Different Receptors and Cl⫺
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
INTRODUCTION
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
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toward ␥-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) (Garcı́a
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
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 ␮l 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 super-
0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society
31
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Duan, Shumin and Ian M. Cooke. Glutamate and GABA activate
different receptors and Cl⫺ conductances in crab peptide-secretory
neurons. J. Neurophysiol. 83: 31–37, 2000. Responses to rapid application of glutamic acid (Glu) and ␥-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, ␣-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid, quisqualate, and kainate
(to 1 mM) were ineffective, nor were Glu responses influenced by
glycine (1 ␮M) 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.
32
S. DUAN AND I. M. COOKE
fused 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
Electrophysiology
Voltage-clamp recordings were obtained in the whole-cell patchclamp 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 M⍀, but
were typically 1.5–3 M⍀. 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
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
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).
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 ⱕ20% 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
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The use of a miniature Y-tube (Murase et al. 1989) manipulated to
within ⬍25 ␮m 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).
CL⫺ CURRENT VIA DIFFERENT GLUTAMATE AND GABA RECEPTORS
33
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
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.
observed against the log of the Glu concentration applied is
shown in Fig. 1B. Responses to seven applications of nearsaturating 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.
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
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-Daspartate (NMDA) receptors, NMDA had no effect up to 1
mM, nor were responses affected by the presence or absence of
glycine (1 ␮M) 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.
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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 ␣-amino-3-hydroxy-5-methyl-4isoxazolepropionic 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).
34
S. DUAN AND I. M. COOKE
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.
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.
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.
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
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
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.
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).
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Glu and GABA responses represent conductance increases
to Cl⫺
CL⫺ CURRENT VIA DIFFERENT GLUTAMATE AND GABA RECEPTORS
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 voltageclamping 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%.
THERE IS A SELECTIVE INHIBITOR OF THE CRUSTACEAN GLU
RESPONSES THAT LEAVES GABA RESPONSES UNAFFECTED. Poly-
vinylpyrrolydone-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.
DISCUSSION
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 gastricmill 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) Xorgan neurons (Garcı́a 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 Garcı́a 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
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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).
35
36
S. DUAN AND I. M. COOKE
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.
Address for reprint requests: I. M. Cooke, Békésy Laboratory of Neurobiology, University of Hawaii, 1993 East-West Rd., Honolulu, HI 96822.
Received 6 July 1999; accepted in final form 3 September 1999.
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prove to be a general response. If so, it could prove to be a
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CL⫺ CURRENT VIA DIFFERENT GLUTAMATE AND GABA RECEPTORS
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