Békésy Laboratory of Neurobiology and Department of Zoology, University of Hawaii, Honolulu, Hawaii 96822
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ABSTRACT |
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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-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.
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INTRODUCTION |
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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
-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
).
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METHODS |
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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 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.
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RESULTS |
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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.
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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 -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.
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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.
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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%.
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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