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The Journal of Neuroscience, September 15, 2002, 22(18):8010-8017
Kainate Receptor Subunits Underlying Presynaptic Regulation of
Transmitter Release in the Dorsal Horn
Geoffrey A.
Kerchner1,
Timothy J.
Wilding2,
James
E.
Huettner2, and
Min
Zhuo1
1 Washington University Pain Center and Departments of
Anesthesiology, Anatomy and Neurobiology, and Psychiatry, and
2 Department of Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
Presynaptic kainate (KA) receptors regulate synaptic transmission
at both excitatory and inhibitory synapses in the spinal cord dorsal
horn. Previous work has demonstrated pharmacological differences
between the KA receptors expressed by rat dorsal horn neurons and those
expressed by the primary afferent sensory neurons that innervate the
dorsal horn. Here, neurons isolated from mice deficient in the KA
receptor subunit were used to evaluate the contributions of glutamate
receptor subunit 5 (GluR5) and GluR6 to the presynaptic control of
transmitter release and to KA receptor-mediated whole-cell currents in
these two cell populations. Deletion of GluR6
produced a significant reduction in KA receptor-mediated current
density in dorsal horn neurons, whereas GluR5 deletion caused no change in current density but removed sensitivity to GluR5-selective antagonists. Presynaptic modulation of inhibitory transmission between dorsal horn neurons was preserved in cells from
either GluR5- or GluR6-deficient mice. In DRG neurons, in contrast,
GluR5 deletion abolished KA receptor function, whereas deletion of GluR6 had little effect on peak current density
but increased the rate and extent of desensitization. These results highlight fundamental differences in KA receptor physiology between the
two cell types and suggest possible strategies for the pharmacological modulation of nociception.
Key words:
kainate; glutamate receptor; presynaptic; GluR5; GluR6; spinal cord; dorsal horn; dorsal root ganglion; sensory
transmission
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INTRODUCTION |
Kainate (KA) receptors are
multisubunit ion channels that play an important role as postsynaptic
mediators of transmission at a variety of excitatory synapses in the
CNS. In addition, recent work indicates that presynaptic KA receptors
may serve to regulate transmitter release from both excitatory and
inhibitory nerve terminals (for review, see Chittajallu et al., 1999 ;
Frerking and Nicoll, 2000; Lerma et al., 2001). There are five
different subunits that can contribute to KA receptors (Hollmann and
Heinemann, 1994), including glutamate receptor subunit 5 (GluR5),
GluR6, and GluR7, which can form functional homomeric receptors, and KA1 and KA2, which combine in heteromeric receptors but do not form
functional ion channels on their own. Genetic deletions of GluR5 (Mulle
et al., 2000) and GluR6 (Mulle et al., 1998) have revealed important
and distinct roles for these subunits in synaptic transmission and
plasticity in the hippocampus (Bureau et al., 1999; Contractor et al.,
2000, 2001) and striatum (Chergui et al., 2000). Much less is known
about the roles of individual KA receptor subunits in other parts of
the nervous system.
In the spinal cord dorsal horn, presynaptic KA receptors regulate
transmission at both excitatory and inhibitory synapses (Huettner et
al., 2002). At excitatory primary afferent sensory synapses, KA
receptors expressed by a subset of DRG neurons are located on
presynaptic terminals (Hwang et al., 2001), where they regulate
glutamate release (Kerchner et al., 2001b). At inhibitory synapses
within the dorsal horn, presynaptic KA receptors, which respond to
glutamate released from dorsal root sensory fibers, regulate GABA and
glycine release by direct depolarization of interneuron terminals
(Kerchner et al., 2001a). In addition to these presynaptic receptors on
excitatory and inhibitory terminals, KA receptors also are found on the
postsynaptic membrane of neurons that respond to high-threshold dorsal
root fiber stimulation (Li et al., 1999).
It is not yet known which KA receptor subunits underlie these distinct
synaptic functions in the dorsal horn. In previous work, a
pharmacological difference was identified between KA receptors on rat
DRG neurons, which were activated and potently desensitized by the
GluR5-preferring agonist
(RS)-2-amino-3-(3-hydroxy-5-tertbutylisoxazol-4-yl)propanoic acid (ATPA), and those on rat dorsal horn neurons, which were largely
insensitive to ATPA (Kerchner et al., 2001b; Wilding and Huettner,
2001). These results are consistent with the prevalence of GluR5 mRNA
in DRG but not dorsal horn neurons (Partin et al., 1993; Sato et al.,
1993; Tölle et al., 1993). However, the pharmacology of ATPA is
not definitive in this regard, because it also activates some
heteromeric KA receptors that do not contain the GluR5 subunit (Paternain et al., 2000). In addition, it remains unclear which subunits underlie KA responses in dorsal horn neurons. In this study,
we make use of mice deficient in the GluR5 and GluR6 KA receptor
subunits, as well as antagonists selective for the GluR5 subunit, to
test whether GluR5 and GluR6 are required for the assembly of
functional KA receptors in the dorsal horn.
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MATERIALS AND METHODS |
Mice. The protocols for handling animals were
approved by the Animal Studies Committee at Washington University.
GluR5 / and GluR6/ mice were obtained as
gifts from Stephen F. Heinemann (Salk Institute, San Diego, CA), and
wild-type mice were C57BL/6 × 129S6/SvEv hybrids purchased from
Taconic (Germantown, NY).
Primary neuronal culture. Dorsal horn neurons were taken
from postnatal mice killed by decapitation. The spinal cord was removed to a dish containing Earle's buffer, and the dorsal third of the cord
was dissected and incubated for 30-90 min at 30-35°C in oxygenated Earle's buffer containing papain (Huettner and Baughman, 1986; Wilding
and Huettner, 1997). Cells were dissociated mechanically in bovine
serum albumin and ovomucoid, both at 1 mg/ml, and plated onto 35 mm culture dishes coated with matrigel (Becton Dickinson, Bedford,
MA). For DRG/spinal cocultures, the DRGs were isolated, treated
as described above, and plated with dorsal horn neurons onto large
islands (~200 µm square), created by drawing a grid of agarose (1.5 mg/ml) on the bottom of 35 mm dishes, which were then sprayed with
collagen or a mixture of poly-DL-ornithine (0.2 mg/ml) and laminin (6 µg/ml). In some experiments, DRG soma were isolated and used for experiments within 24 hr (Wilding and Huettner, 1995). Long-term cultures were maintained at 37°C in a humidified, 5% CO2 incubator in Eagle's minimal essential
medium (supplemented with 20 mM glucose, 0.5 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml
streptomycin, and 4% rat serum) (nerve growth factor was added when
DRGs were plated), treated after 4 d in vitro with 10 µM cytosine
 -D-arabinofuranoside, and used for experiments in vitro between 7 and 35 d.
Electrophysiology. On the stage of an Axiovert
25 inverted microscope (Zeiss, Thornwood, NY), cultures were
bath-perfused with Tyrode's solution, containing (in
mM): 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 D-glucose, and 10 HEPES, pH 7.4, with NaOH. In
experiments testing NMDA receptor-mediated responses, a Tyrode's solution lacking MgCl2 was used. Rapid agonist
applications to characterize KA receptors were made from a
multibarreled pipette (Wilding and Huettner, 1997) fed by solution
reservoirs maintained under 8-10 psi of static air pressure. During
recordings of synaptic currents, neurons were under constant local
gravity-fed perfusion from a quartz glass pipette (inner diameter, 300 µm) connected to a manifold with <1 µl dead space (ALA Scientific
Instruments, Inc., Westbury, NY). The local perfusion solutions
consisted of Tyrode's solution plus various pharmacological agents.
When measuring KA-evoked currents, 300 µM KA
was used, except in experiments testing blockade by
(3S,4aR,6S,8aR)-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (LY382884) or
(3S,4aR,6R,8aR)-6-{2-[1(2)H-tetrazol-5-yl]ethyl}-decahydroisoquinoline-3-carboxylic acid (LY293558), in which case 50 µM KA was used.
Whole-cell recordings were established using heat-polished pipettes
pulled from filamented borosilicate capillary tubes (Warner Instruments, Hamden, CT) with a tip resistance of 3-8 M when filled
with a solution containing (in mM): 140 CsCH3SO3, 5 CsCl, 5 MgCl2, 10 EGTA, 10 HEPES, 5 Mg-ATP, and 1 Li-GTP,
pH 7.4, with CsOH. Neurons were typically held at 70 mV (for
measuring excitatory currents) or 0 mV (for inhibitory currents).
Neither series resistance compensation nor cell membrane capacitance
neutralization were routinely applied, but both were monitored
throughout experiments. Recorded currents were filtered at 2 kHz,
digitized at 10 kHz, and stored in a personal computer for display and
analysis with an Axopatch 200B amplifier, Digidata 1320 interface, and
the pClamp 8 software suite (Axon Instruments, Foster City, CA).
Extracellular stimulation of synaptic currents was achieved with the
S48 single-channel stimulator and SIU5 stimulus isolation unit (Grass
Instruments, Inc., West Warwick, RI) connected to a bipolar stimulating
electrode, constructed with two Ag/AgCl wires immersed in Tyrode's
solution within a  glass electrode, which was pulled and
heat-polished to a final tip diameter of ~10-20 µm. This stimulus
electrode was placed against the cell body of a neuron close to the
recorded cell. Experiments were included only when evoked postsynaptic
currents occurred at a fixed latency after stimulation. Typically,
synaptic stimulation was delivered every 15 sec, in the case of NMDA
receptor-mediated EPSCs, or every 5 sec, in the case of IPSCs.
Pharmacology. All experiments were conducted in
the presence of the AMPA receptor-selective antagonist
(±)-4-(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine (SYM2206) (100 µM) (Li et al., 1999; Wilding
and Huettner, 2001) to permit selective KA receptor activation. In
studies of inhibitory synaptic transmission,
DL-2-amino-5-phosphono-pentanoic acid (25 µM) was also present. In studies of
excitatory synaptic transmission, bicuculline (10 µM) and strychnine (1 µM) were also present. All compounds were
purchased from Sigma (St. Louis, MO), except ATPA and SYM2206 (Tocris
Cookson, Ellisville, MO) and LY382884 and LY293558, which were obtained
as gifts from Eli Lilly and Co. (Greenfield, IN).
Data analysis. Data are presented as means ± SEM. To
detect significant differences between two means, a paired t
test or signed rank test was used. For comparison of multiple groups, a
one-way ANOVA was performed with the Student-Newman-Keuls test for
post hoc comparison. Cumulative probability plots were
compared with the Kolmogorov-Smirnov test. In all cases,
p < 0.05 was considered significant. Time constants
for KA receptor desensitization were determined by fitting a sum of two
exponential functions plus a constant to the falling phase of evoked current.
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RESULTS |
KA receptors expressed by dorsal horn neurons
Previous physiological recordings have documented the expression
of functional KA receptors by nearly all rat dorsal horn neurons in
culture (Kerchner et al., 2001b; Wilding and Huettner, 2001) and by a
significant proportion of neurons in acute spinal cord slices (Li et
al., 1999; Kerchner et al., 2001a). However, anatomical studies of KA
receptor subunit distribution have not conclusively established the
composition of native KA receptors in this region (Tölle et al.,
1993; Petralia et al., 1994) (see Discussion). In previous work on rat
dorsal horn neurons (Kerchner et al., 2001b; Wilding and Huettner,
2001), we showed that whereas native KA receptors were activated
reliably by KA application, the GluR5-selective compound ATPA triggered
only small currents in a minority of cells and failed to cross
desensitize spinal receptors to activation by KA (Kerchner et al.,
2001b; Wilding and Huettner, 2001).
As shown in Figure 1, cultured dorsal
horn neurons from wild-type mice also express functional KA receptors.
To evaluate the contribution of GluR5 in wild-type cells, we tested the
sensitivity of KA-evoked currents to cross desensitization by ATPA or
inhibition by the GluR5-selective compounds LY382884 and LY293558
(Bleakman et al., 1996). Similar to results in rat dorsal horn cells,
ATPA (30-100 µM) evoked much smaller peak currents than
KA (50-300 µM) (Fig. 1D) and caused
little to no cross desensitization of receptors in spinal neurons from
wild-type mice (Fig. 1E). However, 10 µM LY382884 (Fig. 1A,D) and
LY293558 (data not shown) produced significant inhibition of KA-evoked
currents. The slow rise in current after agonist onset likely reflects
competitive displacement by KA of the antagonist, which was present
continuously. Because any receptors that were not affected by the
antagonist would be expected to contribute an instantaneous rise in
current at agonist onset, the appearance of little instantaneous inward
current in most recordings (Fig. 1A) suggests that
the majority of surface KA receptors were sensitive to the drug. In
experiments on wild-type neurons, the instantaneous current ranged from
0.5 to 53% of control peak current, with a mean of 18.6 ± 5.3%
(n = 12).
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Figure 1.
Whole-cell currents in dorsal horn neurons
from wild-type and knock-out mice. A, Superimposed
currents evoked by KA in the absence (thin line) and
presence (thick line) of 10 µM LY382884
(LY). Open bars indicate the
period of agonist exposure. B, KA-evoked current density
(measured by dividing peak current amplitude by whole-cell capacitance
for each cell) was similar in dorsal horn neurons from wild-type mice
(n = 41 cells) and GluR5/ mice
(n = 48) but was significantly smaller in cells
from GluR6/ mice (n = 115).
Although KA triggered inward currents (with negative amplitudes),
current densities are shown as absolute values in this figure and in
Figure 3. *Significantly different from wild type. C,
Cumulative probability as a function of current density illustrates the
prevalence of GluR6/ cells with little or no KA
receptor-mediated current. Curves for wild-type and
GluR5/ cells were not significantly different
(p = 0.524), whereas the curve for
GluR6/ cells was different from both
(p < 0.0005; Kolmogorov-Smirnov test). The
dotted curve shows cumulative probability data for the
subset of GluR6/ cells with current density >2.0
pA/pF. This curve was also significantly different from both the
GluR5/ and wild-type curves
(p < 0.0005). D, The
relative amplitude of peak currents elicited by KA in the continuous
presence of 10 µM LY382884 [10 µM LY293558
was used in some experiments (Bleakman et al., 1996)] or by 30 µM ATPA alone are compared with those elicited by KA
alone in wild-type, GluR5/, or
GluR6/ neurons (n = 6-20 cells
per observation). GluR5-selective antagonists blocked KA currents, and
ATPA alone triggered currents in neurons from wild-type and
GluR6/ but not GluR5/ mice.
*Significantly different from wild type. E, Cross
desensitization of peak current evoked by 300 µM KA
resulting from a 2 sec exposure to 100 µM ATPA. Agonists
were applied once per minute. Squares plot the peak
current evoked by ATPA; circles plot the current evoked
by KA in wild-type (n = 9),
GluR5/ (n = 5), and
GluR6/ (n = 11) cells.
Dotted line indicates 100% of normalized peak
current.
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Mice deficient in either GluR5 or GluR6 were used to evaluate further
the specific contributions of these subunits to KA receptors in dorsal
horn neurons. Cultured neurons isolated from GluR5/ mice
exhibited robust responses to KA (Fig. 1A), with
current densities similar to those recorded from wild-type neurons
(Fig. 1B,C). ATPA evoked little or no current (Fig.
1D) and had no effect on responses to KA (Fig.
1E). More importantly, KA receptor-mediated currents
in GluR5/ cells were completely insensitive to
GluR5-selective antagonists (Fig. 1A,D). In contrast,
dorsal horn neurons from GluR6/ mice exhibited
variable sensitivity to KA. In approximately one-half of the
GluR6/ neurons recorded, exposure to KA produced no
significant change in the holding current (<1-2 pA/pF); in most of
the remaining cells, KA-evoked currents were significantly smaller than
for wild-type and GluR5/ neurons (Fig.
1B,C). LY382884 blocked a greater proportion of
current in GluR6/ than in wild-type cells (Fig.
1A,D); instantaneous current in the presence of
LY382884 was 2.2 ± 0.9% (n = 8) of control peak
current for GluR6/ neurons. In addition, a greater
proportion of KA receptor-mediated current could be evoked by ATPA in
GluR6/ cells than in wild-type cells (Fig.
1D,E). The absolute density of ATPA-evoked current
was slightly, albeit not significantly, greater in our recordings from
20 GluR6/ cells than in 21 wild-type neurons (3.7 ± 0.9 pA/pF for GluR6/; 2.9 ± 0.6 pA/pF for wild
type). Moreover, exposure of GluR6/ cells to ATPA
produced a partial cross desensitization of currents evoked by KA, with
recovery occurring over the course of several minutes (Fig.
1E). Collectively, these results suggest that KA receptors in cultured murine dorsal horn neurons incorporate both the
GluR5 and GluR6 subunits. Although GluR5 deletion had no
effect on current density relative to wild type, GluR6
deletion reduced or eliminated functional KA receptor expression in
most cells, suggesting that GluR6 is more important than GluR5 for the
assembly of functional KA receptors in dorsal horn neurons.
KA receptor subunits underlying presynaptic regulation of
GABA/glycine release
Presynaptic KA receptors on rat dorsal horn interneuron terminals
trigger action potential-independent GABA and glycine release (Kerchner
et al., 2001a). We observed a similar effect in cells from wild-type
mice, in which KA (10 µM) elevated the frequency of
tetrodotoxin (TTX)-insensitive miniature IPSCs (mIPSCs) to 370 ± 60% of control (n = 8; p < 0.001). If subunit composition is the same for presynaptic KA
receptors as for receptors on the cell body, then our observation that
GluR5 deletion had little effect on whole-cell KA-evoked
current density (Fig. 1B,C) suggests that KA
application should affect inhibitory transmission similarly in
wild-type and GluR5/ cells. Indeed, exposure to KA (10 µM) triggered a comparable increase in mIPSC
frequency (Fig. 2A,B) in cultured GluR5/ dorsal horn neurons and in wild type.
ATPA (2 µM) was less effective than KA at
eliciting GABA/glycine release in wild-type cultures, and it showed no
activity in GluR5/ cells. (Fig.
2B).
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Figure 2.
Neither GluR5 nor GluR6 wholly accounts for
presynaptic KA receptors on dorsal horn interneurons. A,
B, The effects of 10 µM KA and 2 µM
ATPA on mIPSC frequency (recorded in the presence of 0.5 µM TTX) are compared in cultures of wild-type
(n = 8 KA recordings, 8 ATPA),
GluR5/ (n = 28 KA, 11 ATPA), and
GluR6/ (n = 20 KA, 5 ATPA)
dorsal horn neurons. A, Representative experiments for
GluR5/ (top) and
GluR6/ (bottom) cells.
B, Frequency (quantified during the first 4 sec of
agonist exposure) is normalized to the value in wild-type cultures in
the presence of KA (dotted line). *Significantly
different from the action of KA in wild-type cultures.
 Significantly different from baseline frequency in the
absence of KA or ATPA. C, D, A 3 µM
concentration of KA reduced eIPSC amplitude similarly in wild-type
(n = 4), GluR5/
(n = 4), and GluR6/
(n = 14) dorsal horn neurons. KA action was reduced
by the GABAB receptor antagonist CGP55845
(CGP; 10 µM). Traces from representative
recordings (C) also document an increase in the
paired-pulse ratio between baseline (dashed line) and KA
(solid line) conditions. D, Values
normalized to the degree of KA-induced suppression of eIPSC amplitude
in wild-type cultures (dotted line). *Significantly
different from KA action in wild-type cultures.
Significantly different from baseline responses in the
absence of KA.
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In contrast, GluR6 deletion, which resulted in lower
KA-evoked current densities (see above), might be expected to hinder KA-induced GABA/glycine release (Fig. 1B,C). In some
recordings from GluR6/ neurons, KA produced no change in
mIPSC frequency. On average, the effect of KA was reduced by nearly
70% in GluR6/ cells relative to wild type. Consistent
with the observation that ATPA evoked a larger proportion of KA
receptor-mediated current in GluR6/ neurons than in
wild-type cells (Fig. 1D), ATPA also stimulated a
greater increase in GABA/glycine release in GluR6/ cultures than in wild type (Fig. 2A,B). The larger
effect of ATPA versus KA in GluR6/ cultures may reflect
activation of a greater proportion of receptors by 2 µM ATPA than by 10 µM
KA, consistent with the 20-fold lower EC50 of
ATPA compared with KA at native receptors on DRG neurons (0.6 vs 12 µM, respectively) (Clarke et al., 1997).
Presynaptic KA receptors also mediate a reduction in action
potential-evoked inhibitory transmission between rat dorsal horn neurons, in a mechanism involving GABAB receptor
activation (Kerchner et al., 2001a). KA (3 µM) depressed
the amplitude of IPSCs evoked by extracellular stimulation [evoked
IPSCs (eIPSCs)] between mouse dorsal horn neurons to 66 ± 4% of
control (n = 4; p = 0.029), an effect
that was significantly reduced by the GABAB receptor antagonist
(2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845) (Fig. 2D). As expected, this
action of KA was not affected by GluR5 deletion (Fig.
2C,D); however, surprisingly, this action was also preserved
in GluR6/ cultures (Fig. 2C,D). The ability
of the GluR6 deletion to reduce KA action on mIPSCs but not
eIPSCs may suggest that a subtle increase in ongoing GABA/glycine release was sufficient to cause GABAB
autoreceptor activation (Kerchner et al., 2001a). Thus, neither GluR5
nor GluR6 wholly accounts for presynaptic KA receptors on mouse dorsal
horn inhibitory neurons.
KA receptors expressed by DRG neurons
Although there is evidence for the expression of all five KA
receptor subunits in DRGs (Partin et al., 1993; Petralia et al., 1994),
both Northern blot analysis (Partin et al., 1993) and pharmacology data
(Swanson et al., 1998; Kerchner et al., 2001b; Wilding and Huettner,
2001) point to the predominance of GluR5. In addition, the report
describing the initial generation of GluR5/ mice (Mulle
et al., 2000) found a lack of KA receptor-mediated currents in 17 freshly dissociated DRG neurons from these mice, suggesting that the
expression of GluR5 was essential for the production of functional
receptors by DRG cells. As shown in Figure
3, our results confirm the observations
of Mulle et al. (2000): in only 2 of 28 GluR5/ DRG neurons did
rapid exposure to KA produce a detectable current, and the currents in
both of those cells were small (<55 pA). In contrast, KA elicited
currents in the majority of wild-type (47 of 62) and
GluR6/ (39 of 60) neurons tested. As observed previously
in rat DRG cells (Kerchner et al., 2001b; Wilding and Huettner, 2001),
brief exposure to ATPA caused profound cross desensitization of
currents evoked by KA in both wild-type and GluR6/
neurons (Fig. 3C). However, the currents evoked by KA in
GluR6/ cells unexpectedly showed more rapid and more
complete desensitization than in cells from wild-type mice (Fig.
3A,C,D). The ratio of peak to steady-state current (peak/ss)
was significantly greater for GluR6/ cells than for wild-type cells (Fig. 3E, peak/ss). In addition,
the initial time constant for current desensitization (see Materials
and Methods) was shorter in GluR6/ cells (Fig.
3E, Tau 1), and the relative contribution by the
faster exponential was greater for GluR6/ cells than for
wild type (Fig. 3E, Amp 1). Thus, our results
support a requirement for GluR5 in the assembly of functional KA
receptors in DRG cells and identify a potential role for GluR6 in
modulating receptor kinetics.
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Figure 3.
GluR5 is required for functional KA receptor
expression in DRG neurons. A, Whole-cell currents evoked
by 300 µM KA in freshly dissociated DRG neurons from
wild-type, GluR5/, and GluR6/
mice. Open bars indicate the periods of agonist
exposure. Smooth curves are the best fits of the sum of
two exponentials plus a constant. B, Peak current
density was significantly reduced in cells from
GluR5/ mice (n = 28) relative to
wild-type (w.t.) (n = 62) and
GluR6/ (n = 60) mice.
*Significantly different from wild type. C, Superimposed
traces show currents evoked by 300 µM KA before
(pre) and after (post)
exposure to 500 nM ATPA for 2 sec. The percentage of
steady-state cross desensitization by ATPA is plotted for five
wild-type and six GluR6/ cells. D,
Best fits of the sum of two exponentials plus a constant for 10 wild-type and 10 GluR6/ cells are shown normalized
to the same initial peak. Dashed line indicates zero current
level. E, Plots compare the ratio of peak to
steady-state current for wild-type (n = 27) and
GluR6/ (n = 36) cells, as well
as parameters from the best fits to the time course of desensitization
(n = 16 wild type and 26 GluR6/), including the amplitude of the first
exponential (Amp 1) and the time constants of the first
(Tau 1) and second (Tau 2) exponential
functions. *Significantly different from wild type.
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KA receptor subunits underlying presynaptic regulation of DRG to
spinal transmission
Activation of presynaptic KA receptors on DRG cells reduces
glutamatergic transmission onto dorsal horn target neurons in rats
(Kerchner et al., 2001b). KA application had a similar effect in
DRG/spinal neuron cocultures from wild-type mice (Fig.
4). KA (10 µM) reduced the
amplitude of NMDA receptor-mediated EPSCs evoked in dorsal horn
neurons by extracellular stimulation directed at DRG cell bodies to
52 ± 2% of the control value (n = 4;
p = 0.029). Based on the results for agonist-evoked
currents in subunit-deficient mice (Fig. 3), we anticipated that
GluR5 but not GluR6 deletion would disrupt the
presynaptic regulation of DRG to spinal transmission by KA. To our
surprise, KA action was reduced to a similar extent, but not
eliminated, by the deletion of either subunit. To explain the action of
KA in GluR5/ cocultures, we initially speculated that
GluR6-containing somatodendritic receptors on dorsal horn neurons might
shunt postsynaptic current, thereby reducing EPSC amplitude (Frerking
et al., 1999); however, at 10 µM, KA caused no
significant change in input resistance in GluR5/ dorsal
horn neurons (88 ± 11% of control; n = 5;
p = 0.31) (Kerchner et al., 2001b).
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Figure 4.
Roles of GluR5 and GluR6 in presynaptic KA
receptor-mediated inhibition of dorsal horn excitatory transmission.
A, Traces from representative experiments illustrate
EPSCs in the absence (bottom traces) and presence
(top traces) of 10 µM KA in cocultures
comprised of neurons with the indicated genotypes. EPSCs were evoked in
dorsal horn neurons (DHNs) by an extracellular
stimulating electrode placed against the cell body of a nearby DRG
cell. B, The effects of KA (10 µM) and
ATPA (2 µM) on EPSC amplitude in DRG/spinal cocultures
made from wild-type (n = 4 recordings in KA, 2 in
ATPA), GluR5/ (n = 5 KA, 3 ATPA), or GluR6/ (n = 11 KA, 5 ATPA) mice plotted relative to the magnitude of KA action in wild-type
cocultures. In some experiments, LY382884 (LY; 10 µM) was present continuously (n = 3).
Some cocultures contained GluR5/ DRG neurons and
GluR6/ dorsal horn neurons (n = 4 KA, 2 ATPA). *Significantly different from KA action in wild-type
cultures. Significantly different from baseline responses
in the absence of KA or ATPA. Dotted line indicates
100% of normalized depression in wild type.
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An alternative hypothesis explaining why GluR5 and
GluR6 deletions each partially reduced KA action is that the
extracellular stimulating electrode may have activated not only a
presynaptic DRG neuron but also nearby excitatory dorsal horn neuronal
cell bodies or axons. In this scenario, KA, which suppresses both
DRG-to-spinal and spinal-to-spinal excitatory transmission (Kerchner et
al., 2001b), may inhibit composite EPSCs by activating presynaptic GluR5-containing KA receptors on DRG neurons as well as presynaptic GluR6-containing KA receptors on dorsal horn neurons. This hypothesis would account for the observed reduction in KA action in both GluR5/ and GluR6/ cocultures relative to
wild type. Supporting this hypothesis, in mixed cocultures of
GluR5/ DRGs and GluR6/ dorsal horn
neurons, KA had no effect on EPSCs (Fig. 4). Further establishing that
GluR5 is required for KA modulation of DRG-to-spinal transmission, ATPA
strongly suppressed DRG to spinal transmission in wild-type and
GluR6/ cultures, but had no significant effect in
GluR5/ cocultures and in mixed
GluR5/ DRG/GluR6/ spinal cocultures (Fig.
4B). Finally, LY382884 blocked the action of KA in
cocultures from GluR6/ mice (Fig.
4B).
| |
DISCUSSION |
The GluR5 and GluR6 subunits make distinct contributions to KA
receptors expressed by DRG neurons and dorsal horn neurons. In DRG
cells, ATPA produced strong, long-lived desensitization (Fig. 3)
(Kerchner et al., 2001b), and functional KA receptors were eliminated
by GluR5 deletion. These data confirm that receptors in
wild-type DRG cells must contain GluR5 (Mulle et al., 2000) and that
the pharmacology of wild-type receptors is dominated by features
associated with the GluR5 subunit. Although peak KA-evoked current
densities in DRG neurons were not affected by GluR6
deletion, the differences in desensitization kinetics between wild-type and GluR6/ cells (Fig. 3D,E) suggest a
possible contribution of GluR6 to KA receptors in DRG cells that has
hitherto been discounted (Sommer et al., 1992; Swanson et al., 1998).
Other KA receptor subunits may contribute as well (Partin et al., 1993;
Petralia et al., 1994).
In contrast to DRG cells, the density of KA-evoked currents in dorsal
horn neurons was unaffected by GluR5 deletion but was significantly reduced in GluR6/ neurons. Approximately
one-half of the GluR6/ cells recorded did not respond to
KA; cells that did respond exhibited smaller current densities than
wild-type or GluR5/ cells, all of which were sensitive
to KA (Fig. 1B,D). Consistent with the direct
measurements of KA currents, the effects of presynaptic KA receptor
activation were preserved in spinal neurons lacking GluR5
but were diminished with GluR6 deletion. Thus, the GluR6
subunit is clearly an important component of KA receptors in most
dorsal horn neurons.
The present study focuses on the properties of cultured neurons.
Although the possibility exists that subunit expression in culture
might differ from that in vivo, our previous studies in rats
(Kerchner et al., 2001a,b) have documented good agreement between cell
culture and acute slice preparations in the properties of KA
receptor-mediated regulation of transmission.
Dorsal horn neurons: evidence for GluR5 expression
Although deletion of GluR5 did not affect the amplitude
or time course of KA receptor-mediated currents in dorsal horn neurons, our pharmacological data provide evidence for GluR5 expression in a
significant proportion of cells from wild-type and
GluR6/ mice. The GluR5-selective antagonists LY382884
and LY293558 produced a substantial block in all of the wild-type and
GluR6/ neurons tested but had virtually no effect in
GluR5/ cells (Fig. 1C), confirming the
selectivity of these compounds at native receptors (Bleakman et al.,
1996). Compounds selective for GluR5 had a somewhat larger effect on
dorsal horn neurons from GluR6/ animals than on cells
from wild-type mice or rats. In previous work on spinal neurons from
rats (Kerchner et al., 2001b; Wilding and Huettner, 2001), fewer than
half of the cells responded to ATPA; in those cells the ATPA-evoked
currents were small compared with currents elicited by KA. Similarly,
in dorsal horn neurons from wild-type mice, ATPA-evoked currents were
small in proportion to KA; likewise, ATPA produced only a modest
increase in mIPSC frequency in wild-type cultures (Fig.
2B). In contrast, relative peak current amplitude triggered by ATPA in cells from GluR6/ mice was
substantial (more than half of that triggered by KA) (Fig.
1D,E). In addition, brief exposure to ATPA produced
significant cross desensitization of spinal KA receptors only in
GluR6/ cells (Fig. 1E). ATPA reliably
triggered the quantal release of GABA and glycine in GluR6/ dorsal horn neurons (Fig.
2A,B), further supporting the expression of GluR5.
These data clearly suggest that GluR5 and GluR6 both contribute to KA
receptor-mediated currents in dorsal horn neurons in mice; however, it
is more difficult to determine whether individual receptors are
heteromeric or whether there exist distinct populations of homomeric
receptors. The ability of GluR5 antagonists to block instantaneous
current at the onset of agonist exposure in wild-type cells indicates
that most surface receptors were affected by the drug and are therefore
likely to include a GluR5 subunit. This result, together with the
evidence discussed above implicating GluR6 as a component in most
wild-type KA receptors, suggests that many dorsal horn neurons express
heteromeric receptors that include both GluR5 and GluR6. The relative
lack of effectiveness of ATPA to activate responses or to cross
desensitize KA responses in wild-type neurons is not necessarily
inconsistent with this reasoning, if the effect of this agonist depends
on the stoichiometry of GluR5 within a heteromeric complex (Vignes et
al., 1998). In other words, dorsal horn neurons may express KA
receptors with a low ratio of GluR5 to other subunits sufficient to
confer sensitivity to LY382884 but not to ATPA. This hypothesis might
explain the finding that GluR5 deletion had little to no
effect on overall KA receptor-mediated current density. It could also
explain why ATPA appears more effective in GluR6/ cells
than in wild type, because GluR6 deletion might increase
GluR5 stoichiometry at the level of individual receptors.
In addition to GluR5 and GluR6, other subunits may contribute to KA
receptors in dorsal horn neurons. Tölle et al. (1993), using
in situ hybridization to map the mRNA distribution for all five KA receptor subunits in adult rats, observed a prominent expression of KA2 in the superficial dorsal horn and substantially lower expression of KA1. Weak but widespread labeling was also observed
for the GluR7 subunit and, in significantly fewer cells, for GluR5
(Furuyama et al., 1993). Tölle et al. (1993) reported that GluR6
mRNA was undetectable by in situ labeling in adult rat
spinal cord; however, a more recent developmental in situ hybridization study (Stegenga and Kalb, 2001) suggests that spinal KA
receptor subunit expression, including expression of GluR6, may be
significantly higher in newborn animals. Our physiological results from
both mice (Figs. 1, 2) and rats (Kerchner et al., 2001b; Wilding and
Huettner, 2001) clearly highlight an important role for the GluR6
subunit and a less prominent role for GluR5 in the assembly of
functional somatodendritic and presynaptic terminal KA receptors in
dorsal horn neurons. In preliminary reverse transcription PCR
experiments, we detected strong expression of GluR6 in both cultured
and freshly isolated newborn rat dorsal horn (J. E. Huettner,
unpublished observations). In future work, it will be of interest to
test for production of functional KA receptors in GluR5/ × GluR6/ double knock-out mice, as well as in mice deficient in
other KA receptor subunits, when they become available.
Finally, the prevalence of KA receptor subunits may vary among
different subpopulations of dorsal horn neurons. Some cells may express
heteromeric receptors that include both GluR6 and GluR5, whereas other
cells may express GluR6 without GluR5. If this were true, then
GluR6 deletion should abolish KA currents in some dorsal
horn neurons but not in others; this was indeed the case (Fig.
1D). Also supporting this hypothesis is the indirect evidence that GluR6 deletion in dorsal horn neurons
apparently prevented KA-induced suppression of spinal-to-spinal
excitatory transmission in mixed GluR5/
DRG/GluR6/ spinal cocultures (Fig. 4) (also see below),
suggesting that GluR6 may be required for KA receptor expression by
glutamatergic dorsal horn neurons. In contrast, presynaptic KA
receptors on inhibitory dorsal horn neurons were eliminated by neither
GluR5 nor GluR6 deletion (Fig. 2) (also see
above), indicating that inhibitory neurons likely contain heteromeric
receptors that include both subunits, either of which is sufficient for
the production of functional receptors. Mulle et al. (2000) reached a
similar conclusion concerning the subunit contribution to KA receptors
in hippocampal CA1 inhibitory interneurons. They showed that deletion
of either GluR5 or GluR6 alone was not sufficient
to eliminate KA receptors; however, receptors were abolished by the
combined deletion of both subunits (Mulle et al., 2000).
DRG neurons
Unlike dorsal horn neurons, functional KA receptors on DRG neurons
exhibited an absolute requirement for GluR5. GluR5 may form homomeric
receptors on some DRG cells (Swanson et al., 1998) or it may combine
with GluR6, GluR7, KA1, or KA2 (Partin et al., 1993). However, in
GluR5/ DRG cells, KA receptor-mediated currents were not
detected, indicating that other subunits, if present at all, did not
contribute to functional receptors in the absence of GluR5.
The involvement of both GluR5 and GluR6 in the KA-induced inhibition of
excitatory transmission (Fig. 4) likely reflects activation by the
extracellular stimulating electrode of presynaptic elements derived
from both DRG and dorsal horn neurons. Even when the electrode is
placed against the cell body of a DRG cell, the electrical field
generated by the stimulating pulse may extend to include nearby dorsal
horn neuronal cell bodies or axons; thus, excitatory dorsal horn
neurons could contribute to a composite NMDA receptor-mediated EPSC.
The slow kinetics of these EPSCs (Fig. 4A) would
easily hide the presence of multiple responses with minor variations in
latency. Supporting the notion that GluR5 is required for receptors regulating DRG to spinal transmission and that GluR6 is important for
spinal-to-spinal excitatory transmission, neither KA nor ATPA affected
EPSCs in mixed cocultures containing GluR5/ DRG cells and GluR6/ dorsal horn neurons (Fig. 4).
Although our results indicate that many DRG cells and spinal neurons
are likely to express heteromeric KA receptors that include both the
GluR5 and GluR6 subunits, we also confirmed in wild-type and
subunit-deficient mice the profound difference in cross desensitization of KA receptors by ATPA between DRG cells and dorsal horn neurons. Additional experiments will be needed to determine whether this difference in pharmacology reflects differential KA receptor subunit expression or stoichiometry, expression of alternative splice variants
of GluR5 and/or GluR6, or different interactions with cytoplasmic
proteins or enzymes that may be unique to DRG or spinal cells. A
molecular distinction between KA receptors on DRG neurons and those on
dorsal horn neurons, underlying the potential for pharmacological
selectivity, is particularly attractive from a clinical perspective.
It is predicted that selective manipulation of presynaptic KA receptors
at primary afferent synapses would alter pain transmission with fewer
side effects than might be apparent using nonselective agents.
Consistent with the ability of ATPA to inhibit DRG to spinal
transmission, some evidence already suggests that in rats, selective
activation of GluR5-containing KA receptors reduces nociceptive spinal
reflexes in vitro (Procter et al., 1998) and nociceptive
behavioral responses in vivo (Mascias et al., 2001). Additional in vivo studies have shown that systemic
administration of GluR5-selective antagonists reduces hyperalgesia
(Sang et al., 1998; Simmons et al., 1998), implicating GluR5-containing
receptors in nociceptive processing more generally. Although the
location of receptors responsible for these behavioral effects remains to be established, these studies highlight GluR5-containing KA receptors as a possible therapeutic target. Elucidation of the pathways
underlying these effects, and the development and testing of agents
selective for other KA receptor subunits, represent important areas for
future work.
| |
FOOTNOTES |
Received Dec. 14, 2001; revised June 26, 2002; accepted July 3, 2002.
This work was supported by National Institutes of Health Grants
DA10833, NS38680, and NS30888. We thank Stephen F. Heinemann for
providing GluR5/ and GluR6/ mice
and David Bleakman at Eli Lilly and Company for the gifts of LY293558
and LY382884.
Correspondence should be addressed to Dr. James E. Huettner, Department of Cell Biology and Physiology, Washington
University School of Medicine, Campus Box 8228, 660 South Euclid
Avenue, St. Louis, MO 63110, E-mail: huettner{at}cellbio.wustl.edu, or to Dr. Min Zhuo, Department of Anesthesiology, Washington University School of Medicine, Campus Box 8054, 660 South Euclid Avenue, St.
Louis, MO 63110, E-mail: zhuom{at}morpheus.wustl.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
