Department of Pharmacology and Toxicology, Medical College of Georgia, and Medical Research Service, Veterans Administration Medical Center, Augusta, Georgia 30912-2300
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ABSTRACT |
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Wang, Xueyong and Nevin A. Lambert. GABAB Receptors Couple to Potassium and Calcium Channels on Identified Lateral Perforant Pathway Projection Neurons. J. Neurophysiol. 83: 1073-1078, 2000. Activation of presynaptic GABAB receptors inhibits neurotransmitter release at most cortical synapses, at least in part because of inhibition of voltage-gated calcium channels. One synapse where this is not the case is the lateral perforant pathway synapse onto dentate granule cells in the hippocampus. The current study was conducted to determine whether the neurons that make these synapses express GABAB receptors that can couple to ion channels. Perforant pathway projection neurons were labeled by injecting retrograde tracer into the dorsal hippocampus. The GABAB receptor agonist baclofen (10 µM) activated inwardly rectifying potassium channels and inhibited currents mediated by voltage-gated calcium channels in retrogradely labeled neurons in layer II of the lateral entorhinal cortex. These effects were reversed by coapplication of the selective GABAB receptor antagonist CGP 55845A (1 µM). Equivalent effects were produced by 100 µM adenosine, which inhibits neurotransmitter release at lateral perforant pathway synapses. The effects of baclofen and adenosine on inward currents were largely occlusive. These results suggest that the absence of GABAB receptor-mediated presynaptic inhibition at lateral perforant pathway synapses is not simply due to a failure to express these receptors and imply that GABAB receptors can either be selectively localized or regulated at terminal versus somatodendritic domains.
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INTRODUCTION |
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Activation of presynaptic G protein-coupled
receptors (GPCRs) inhibits neurotransmitter release at many synapses
(Miller 1998; Thompson et al. 1993
;
Wu and Saggau 1997
). This inhibition is often mediated
by a decrease in presynaptic calcium influx resulting from inhibition
of presynaptic voltage-gated calcium channels (Dittman and
Regehr 1996
; Wu and Saggau 1997
). The large
majority of central presynaptic terminals contain G protein subunits
that can inhibit the calcium channels responsible for neurotransmitter release. This leaves a limited number of mechanisms whereby neurons can
avoid presynaptic inhibition mediated by GPCRs. A neuron can simply
fail to express a particular GPCR protein, or selective targeting or
regulation could allow neurons to use receptors at some membrane
domains, but not at presynaptic terminals. The former mechanism appears
to be widely used. GPCRs are often expressed in distinct populations of
neurons, and inhibition of calcium channels in neuron cell bodies is
often predictive of inhibition in presynaptic terminals. In contrast,
there are relatively few instances of subcellular utilization of GPCRs
in neurons. For example, structural and functional evidence suggests
that metabotropic glutamate receptors (mGluRs) can be selectively
targeted to or used at subcellular membrane domains (Scanziani
et al. 1998
; Shigemoto et al. 1996
;
Stowell and Craig 1999
).
GABAB receptors are the G protein-coupled
metabotropic receptors for GABA (Bettler et al. 1998;
Möhler and Fritschy 1999
; Mott and Lewis
1994
). Similar to other GPCRs that couple to pertussis toxin-sensitive G proteins, activation of GABAB
receptors activates inwardly rectifying K+ (GIRK)
channels and inhibits voltage-gated calcium channels. These receptors
are ubiquitously expressed in cortical neurons, and activation of
presynaptic GABAB receptors inhibits
neurotransmission at most cortical synapses (Mott and Lewis
1994
). However, a few cortical synapses are unaffected by
GABAB receptor agonists. One such synapse is the
lateral perforant pathway (LPP) synapse made by neurons in layer II of
the lateral entorhinal cortex on the distal dendrites of granule cells
in the dentate gyrus (Ault and Nadler 1983
;
Brunner and Misgeld 1993
; Lanthorn and Cotman
1981
; Mott and Lewis 1994
). Dentate granule
cells also receive input from the medial entorhinal cortex through the
medial perforant pathway (MPP). These synapses, unlike LPP synapses,
are inhibited by activation of GABAB receptors.
The purpose of this study was to ask whether lateral perforant path
projection neurons express functional GABAB
receptors. The results indicate that these neurons express
GABAB receptors that couple to potassium channels
and voltage-gated calcium channels. Coupling to ion channels in these neurons by GABAB receptors is as effective as
coupling by adenosine receptors, activation of which inhibits
neurotransmitter release at LPP synapses (Kahle and Cotman
1993
). These neurons thus possess the signal transduction
machinery required for presynaptic inhibition mediated by
GABAB receptors but avoid using this mechanism at their synapses in the dentate gyrus. This suggests that
GABAB receptors, similar to other G
protein-coupled receptors, are selectively targeted to or regulated at
subcellular membrane domains.
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METHODS |
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Retrograde labeling
Sprague-Dawley rats (10-14 days old) were placed in a
stereotaxic frame under barbiturate anesthesia. Approximately 0.5-1.0 µl of a 5% solution of 3,3'-dioctadecyloxacarbocyanine perchlorate (diO; dissolved in dimethylformamide) or of a 5% suspension of fluorescent polystyrene microspheres (40-nm diam; Molecular Probes) was
injected into the dorsal hippocampi bilaterally through a glass
pipette. Wounds were closed with sutures, and animals were allowed to
recover from anesthesia (body temperature maintained with a heating
pad) and then returned to their home cages. Fluorescently labeled
neurons were evident as early as 24 h after the injection. Neurons
labeled with diO contained punctate fluorescent inclusions (Fig.
1). Relatively fast, punctate labeling
suggests that diO-labeled membrane was internalized and transported to
the cell body by retrograde transport (Honig and Hume
1989).
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Electrophysiology
Between 3 and 14 days after injection, coronal slices of entorhinal cortex (400 µm) were prepared and maintained at 22°C in artificial cerebrospinal fluid (ACSF) that contained (in mM) 125 NaCl, 25 NaHCO3, 3.3 KCl, 2 MgCl2, 2 CaCl2, and 20 D-glucose and was oxygenated with 95% O2-5% CO2. This solution was used for recording with various additions or substitutions as indicated: 2 mM BaCl2, 0.5 µM TTX, and 100 µM CdCl2. Slices containing the lateral entorhinal area were placed on the stage of an Olympus BX50WI microscope and epi-illuminated with a fluorescein filter set (XF22, Omega Optical). Retrogradely labeled neurons were imaged using a ×40 water-immersion objective, a cooled charge-coupled device (CCD) camera (LSR Olympix) and a CCD video camera (Dage-MTI). Images taken with fluorescence illumination and near-infrared transillumination were superimposed to ensure that labeled neurons were selected for recording (Fig. 1). In control experiments we found that prolonged illumination with the excitation wavelength (475-500 nm) had no obvious effect on the physiological properties of labeled neurons. Nonetheless, we illuminated neurons only briefly (a few seconds) before recording. Whole-cell recordings were made using pipettes containing (in mM) either 120 potassium gluconate, 20 KCl, 10 HEPES, 10 EGTA, 14 phosphocreatine, 4 MgATP, and 0.3 tris GTP, or the same solution with cesium salts substituted for potassium gluconate and KCl (pH adjusted with KOH or CsOH, respectively). Series resistance compensation was not used. Voltage commands were delivered and data were acquired using a multifunction input-output board (National Instruments) and WCP software (John Dempster, Strathclyde University, Strathclyde, UK). Slices were constantly perfused with ACSF (~2 ml/ min). Drugs were added directly to the perfusing solution. All chemicals were from Sigma, with the exception of TTX (Calbiochem) and CGP 55845A, which was a gift from Novartis Pharma.
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RESULTS |
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Identification of lateral perforant pathway projection neurons
Injection of diO or fluorescent polystyrene microspheres into the
dorsal hippocampus reliably labeled a distinct band of neurons in the
superficial layers of the entorhinal cortex. As previously reported
(Dolorfo and Amaral 1998;
Hjörth-Simonsen and Jeune 1972
; Steward
and Scoville 1976
), two groups of retrogradely labeled neurons
could be identified with this injection site: one group just ventral to
the rhinal sulcus in the lateral entorhinal area (Fig. 1) and a second
group in the medial entorhinal area, which were most apparent when
slices were made in the horizontal plane (not shown). Anterograde
tracing studies indicate that the labeled neurons in the lateral
entorhinal cortex project to the outer molecular layer of the dentate
gyrus (Tamamaki 1997
) and are thus the cells of origin
of the lateral perforant pathway (LPP).
GABAB receptors couple to potassium channels on LPP projection neurons
We first asked whether lateral perforant pathway projection
neurons express GABAB receptors that couple to
GIRK channels. With 3.3 mM extracellular K+ (140 mM
internal K+), bath application of the selective
GABAB receptor agonist baclofen (10 µM) reversibly
induced an outward current in every neuron tested (46 ± 16 pA;
mean ± SE; n = 6) at a holding potential
of 70 mV. During steps to a potential of
100 mV, baclofen induced an inward current (
195 ± 37 pA; Fig.
2A). These effects were reversed by washing with control solution, or by coapplication of the
selective GABAB receptor antagonist CGP 55845A (1 µM).
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We further characterized the GABAB receptor-activated
current in these cells by applying voltage ramps (140 to +40 mV, 500 ms) in the presence of 30 mM extracellular K+, TTX (0.5 µM), and cadmium (100 µM). Under these conditions baclofen induced
a current that displayed pronounced inward rectification and reversed
polarity near the calculated equilibrium potential of -39 mV for a
current carried by K+ ions (Fig. 2B). In six
of six neurons, baclofen induced a 12.9 ± 2.4-nS increase in
chord conductance (measured at
140 mV). Again, the changes produced
by baclofen were reversed by 1 µM CGP 55845A. These results are
consistent with activation of GIRK channels by GABAB
receptors in LPP projection neurons.
In most cortical neurons several GPCRs couple to pertussis
toxin-sensitive (Gi or Go) G proteins
(Nicoll et al. 1990). In many cases GABAB
receptors and A1 adenosine receptors activate a common
population of GIRK channels (Sodickson and Bean 1998
). Knowing that activation of adenosine receptors inhibits
neurotransmitter release at LPP synapses, we tested the relative
effects of baclofen and adenosine on GIRK channels in LPP projection
neurons. In three neurons 100 µM adenosine activated a current that
was nearly identical with that activated by 10 µM baclofen (97 ± 11% of the baclofen-induced current at
140 mV; mean ± SD;
Fig. 2).
GABAB receptors couple to calcium channels on LPP projection neurons
In all known instances in which GABAB
receptors activate GIRK channels, these receptors also inhibit
voltage-gated calcium channels through similar G proteins in the same
neurons. However, selective coupling to GIRK channels or calcium
channels (but not both) has been reported for other receptors in some
neurons (Fernandez-Fernandez et al. 1999; Li and
Bayliss 1998
), and activation of GIRK channels alone may not
produce presynaptic inhibition (Lüscher et al. 1997
). Thus if GABAB receptors coupled
only to GIRK channels in LPP projection neurons, this could explain the
resistance of LPP synapses to GABAB receptor activation.
We therefore tested the ability of baclofen to inhibit currents
mediated by voltage-gated calcium channels in LPP projection neurons.
Labeled neurons were recorded in voltage-clamp mode using cesium-filled
pipettes in the presence of TTX (0.5 µM), and 2 mM barium was
substituted for calcium as a charge carrier. Barium was used because it
effectively blocks GIRK channels as well as several other potassium
channels. Neurons were held at 80 mV and were stepped to a positive
potential (
10 or 0 mV). These commands evoked large inward currents
that were completely blocked by external cadmium (100 µM; not shown).
Because these recordings were made from fairly intact LPP projection
neurons, voltage clamp was clearly compromised. Barium currents were
slow to activate and deactivate compared with currents in
electrotonically compact cells. In some cases small, steplike current
components appeared during a command, suggesting the presence of
uncontrolled distal compartments. Despite this problem, we were able to
qualitatively assess coupling of GABAB receptors
to calcium channels in these neurons. Application of 10 µM baclofen
inhibited peak inward current by 21 ± 3% in 18 of 18 neurons
(Fig. 3A). In the same cells
baclofen inhibited current at the end of 200-ms commands by 15 ± 3%. This degree of inhibition is similar to that which has been
observed in other relatively intact neuronal preparations
(Lambert and Wilson 1996
; Scholz and Miller
1991
). Baclofen consistently produced a small outward shift in
the holding current (7 ± 2 pA; n = 12), and a
small in change in leak currents (Fig. 3B,
bottom). Linear leak subtraction indicated that these
changes were too small to account for the observed change in inward
current (Fig. 3C). Moreover, changes in leak appear to have
been restricted to negative potentials; baclofen produced no change in
currents evoked by large positive square commands (data not shown).
Similarly, baclofen inhibited inward currents evoked by depolarizing
ramp commands (
90 to +50 mV, 175 ms), but produced no change in
whole-cell current at potentials positive to +10 mV (Fig.
3D). These results are consistent with voltage-dependent
inhibition of calcium channels (Bean 1989
), without
significant changes in passive properties. Inhibition of barium
currents in LPP projection neurons by baclofen was completely reversed
by washing or coapplication of 1 µM CGP 55845A (Fig. 3A).
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Because adenosine inhibits transmission at LPP synapses, we also tested the effect of adenosine (100 µM) on barium currents in LPP projection neurons. Adenosine reversibly inhibited peak inward currents by 26 ± 3% (n = 16; Fig. 3A). In nine cells baclofen and adenosine were both applied sequentially. In these cells baclofen inhibited peak inward current by 23 ± 2%, and adenosine inhibited peak current by 26 ± 4%. These values were not significantly different (P = 0.57; paired t-test). To determine whether adenosine and GABAB receptors couple to common effector mechanisms in LPP projection neurons, we applied saturating concentrations of baclofen and adenosine in succession. In seven neurons 50 µM baclofen inhibited peak inward current by 29 ± 1%. Addition of 100 µM adenosine in the continued presence of baclofen produced only an additional 2 ± 4% inhibition (Fig. 3B). These results suggest that GABAB and adenosine receptors couple to a common effector mechanism in LPP projection neurons.
To control for the possibility that the injection of retrograde tracer
in the hippocampus induced the expression of
GABAB receptors in LPP projection neurons, we
made recordings from a large number of neurons in layer II of the
lateral entorhinal cortex of uninjected control animals. Although some
of these neurons may not have projected to the dentate gyrus, the
density of labeled neurons in slices from injected animals suggested
that a substantial number were, in fact, LPP projection neurons (see
also Schwartz and Coleman 1981). Activation of
GABAB receptors activated GIRK currents
(n = 5) or inhibited barium currents (n = 32) in every neuron tested (data not shown). This result suggests
that the retrograde labeling procedure did not induce expression of
GABAB responses in neurons that would not
otherwise have them. This experiment cannot rule out the possibility,
however, that labeling changed the amount of response observed in
individual LPP projection cells.
Medial perforant pathway projection neurons
Unlike LPP synapses, MPP synapses on dentate granule cells are
susceptible to presynaptic inhibition by activation of
GABAB receptors (Ault and Nadler
1983; Lanthorn and Cotman 1981
; Mott and
Lewis 1994
). We therefore tested the ability of baclofen to activate GIRK channels and inhibit barium currents in retrogradely labeled MPP projection neurons in the medial entorhinal cortex (Fig.
4). Under conditions identical with those used for studying GIRK
channels in LPP projection neurons (30 mM extracellular
K+, TTX, and cadmium), baclofen produced a
12.6 ± 2.0-nS (mean ± SD; n = 4) increase in chord
conductance at
140 mV. This value was not significantly different
from that observed in LPP projection neurons (P = 0.93;
Student's t-test). Under conditions identical with those
used for studying barium currents in LPP projection neurons (TTX, 2 mM
barium), baclofen inhibited peak inward current by only 9 ± 1%
(mean ± SE; n = 5) in MPP projection neurons. This was
significantly less inhibition than was observed in a comparable population of LPP projection cells (P < 0.0001;
Student's t-test).
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DISCUSSION |
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A wide variety of GPCRs couple to
Gi-Go proteins in neurons.
Activation of these receptors liberates G protein dimers, which
directly interact with calcium channel
1A,
1B, or
1E subunits,
inhibiting channel function (Zamponi and Snutch 1998
). This modulation is thought to underlie presynaptic inhibition mediated
by GPCRs at many synapses (Wu and Saggau 1997
), and
parallel inhibition of somatic calcium currents and nerve terminal
calcium influx is commonly observed in individual neurons. Several
different Gi-Go-coupled
GPCRs can inhibit neurotransmission at individual synapses. Therefore,
at synapses where one such receptor inhibits calcium channels, other
Gi-Go-coupled receptors
must either be absent or selectively uncoupled to avoid inhibiting
neurotransmitter release.
One possible mechanism of regulating presynaptic inhibition mediated by
GPCRs is subcellular localization of these receptors. Recent studies
have indicated that mGluRs can be selectively localized at
somatodendritic and axonal sites
(Shigemoto et al. 1996; Stowell and Craig
1999
) and can selectively inhibit neurotransmitter release at a
subset of synapses along a single axon (Scanziani et al. 1998
). This prompted us to ask whether
GABAB receptors, which are structurally related
to mGluRs, can also be used at discrete subcellular domains. We
examined lateral perforant pathway projection neurons, which are known
to make synapses in the dentate gyrus that are insensitive to
activation of GABAB receptors (Lanthorn and Cotman 1981
) but are sensitive to activation of other
Gi-Go-coupled receptors
(Kahle and Cotman 1993
). We found that LPP projection neurons express functional GABAB receptors that
activate GIRK channels and appear to inhibit voltage-gated calcium
channels. Identical effects were produced by activation of adenosine
receptors. This suggests that GABAB receptors
couple to somatodendritic voltage-gated calcium channels in these
neurons, but presumably do not couple to presynaptic calcium channels
at terminals.
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The experiments presented here are not without limitations. For
example, it is possible that the somatodendritic channels inhibited by
baclofen in LPP projection neurons are not involved in neurotransmitter
release at LPP terminals. However,
Gi-Go proteins typically
inhibit calcium channels that participate in neurotransmission (i.e.,
channels that contain 1A,
1B, or
1E subunits)
(Zamponi and Snutch 1998
). In addition, occlusion of
adenosine inhibition of barium current by baclofen suggests that this
possibility is unlikely, unless adenosine receptors couple to different
calcium channels in terminals and cell bodies. Direct measurements of presynaptic calcium transients in LPP terminals will be required to
confirm that calcium influx is unaffected by activation of GABAB receptors (and inhibited by activation of
adenosine receptors). It should also be emphasized that the limited
space clamp of intact neurons prevented us from documenting
characteristic features of
Gi-Go inhibition of calcium
channels, such as kinetic slowing and voltage-dependent relief of
inhibition. Recordings from acutely isolated LPP projection neurons are
necessary to demonstrate these features, which would make a more
convincing case for direct inhibition of calcium channels.
Whatever the mechanism that prevents inhibition of neurotransmitter
release by activation of GABAB receptors at LPP
terminals, it will be of interest to determine whether a similar
mechanism is used at other excitatory and inhibitory synapses that do
not respond to activation of these receptors (Colbert and Levy
1992; Gil et al. 1997
; Lambert and Wilson
1993
; Tang and Hasselmo 1994
). The finding that
GABAB receptors are present on LPP projection neurons raises a number of questions. Which
GABABR subunits are expressed in LPP projection
neurons? Are GABAB receptors excluded from LPP
terminals, or are they present and in some way selectively uncoupled?
Do the local collaterals of LPP projection neurons (Lingenhöhl and Finch 1991
; Tamamaki and
Nojyo 1993
) make synapses that are sensitive to activation of
GABAB receptors? What provides the signal that
ultimately prevents GABAB modulation of calcium channels at LPP terminals? If this signal is provided by the
postsynaptic granule cell, is it restricted to the distal dendrites?
Answering these questions should lead to a greater understanding of how neurons use GPCRs to modulate synaptic function.
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ACKNOWLEDGMENTS |
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We thank J. Dempster for providing data acquisition and analysis software, the Imaging Core Facility of the Institute for Molecular Medicine and Genetics for use of their facilities, and Novartis Pharma AG for supplying CGP 55845A.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-36455 and a Merit Award from the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests: Nevin A. Lambert, Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300.
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 1 June 1999; accepted in final form 8 October 1999.
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REFERENCES |
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