Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada
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ABSTRACT |
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Chéry, Nadège and Yves De Koninck. GABAB Receptors Are the First Target of Released GABA at Lamina I Inhibitory Synapses in the Adult Rat Spinal Cord. J. Neurophysiol. 84: 1006-1011, 2000. We have previously provided functional evidence that glycine and GABA are contained in the same synaptic vesicles and coreleased at the same synapses in lamina I of the rat spinal dorsal horn. However, whereas both glycine receptors (GlyRs) and GABAA receptors (GABAARs) are expressed on the postsynaptic target, under certain conditions inhibitory events appeared to be mediated by GlyRs only. We therefore wanted to test whether GABAB receptors could be activated in conditions where GABA released was insufficient to activate GABAARs. Focal stimulation in the vicinity of visually identified lamina I neurons elicited monosynaptic IPSCs in the presence of ionotropic glutamate receptor antagonists. Pairs of stimuli were given at different interstimulus intervals (ISI), ranging from 25 ms to 1 s to study the depression of the second of evoked IPSCs (paired pulse depression; PPD). Maximal PPD of IPSCs was 60 ± 14% (SE) (of the conditioning pulse amplitude), at ISI between 150 and 200 ms. PPD was observed with IPSCs evoked at stimulus intensities where they had no GABAAR component. PPD of small evoked IPSCs was not affected by the GABAAR antagonist bicuculline but significantly attenuated by 10-30 µM CGP52432, a specific GABAB receptor antagonist. These data indicate that, under conditions where GABA released is insufficient to affect postsynaptic GABAARs at lamina I inhibitory synapses, significant activation of presynaptic GABAB receptors can occur.
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INTRODUCTION |
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We have recently
shown that GABA and glycine are coreleased from the same synaptic
vesicles at inhibitory synapses on lamina I neurons of the spinal cord.
However, we have also shown that miniature (action potential
independent) or small evoked IPSCs (mIPSCs) involve activation of
glycine receptors (GlyRs) only, even though the postsynaptic neurons
expressed both GlyRs and GABAA receptors
(GABAARs). Activation of postsynaptic
GABAA receptors could be detected only by
enhancing the affinity of GABAARs with a
benzodiazepine or following stimuli of sufficient intensity to allow
synchronous activation of a sufficient number of terminals, presumably
because under such conditions GABA could spillover from synapses to
reach extrasynaptic GABAARs (Chéry
and De Koninck 1999a). We therefore wanted to test whether
presynaptic GABAB autoreceptors could be
activated at stimulus intensities where GlyR-mediated IPSCs are
elicited, but GABAAR activation is not detectable. This is because GABAB receptors
(GABABRs) may display a greater affinity for GABA
than do GABAARs (Isaacson et al.
1993
; Yoon and Rothman 1991
). In many regions of
the CNS GABABRs are often found localized on
axonal endings (Bowery 1993
), indicating that they might
play a role in the modulation of neurotransmitter release. A classical
test of GABABRs activation on synaptic release of
GABA in the brain is the study of paired-pulse depression (PPD) of
inhibitory synaptic events where one analyzes the response to a test
stimulus following a conditioning stimulus at different interstimulus
intervals (ISIs) (Davies et al. 1990
; Otis et al. 1993
). Thus we studied PPD of evoked IPSCs by using focal
electrical stimuli conditions whereby the phasic release of GABA is
insufficient to activate GABAARs on spinal lamina
I neurons. Our findings indicate that, under those circumstances, the
amount of GABA released from synaptic terminals first serves to
activate GABAB receptors. Preliminary accounts of
this study have been reported in abstract form (De Koninck and
Chéry 1999
).
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METHODS |
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Slicing procedure
Adult male Sprague-Dawley rats (weighing 150-250g) were
anesthetized with pentobarbital sodium (30 mg/kg), and spinal cord slices were obtained as described previously (Chéry and De
Koninck 1999a; Chéry et al. 2000
).
Briefly, rats were perfused with ice-cold sucrose artificial
cerebrospinal fluid (ACSF; in which 126 mM NaCl was replaced with 252 mM sucrose; see below for a description of normal ACSF) and rapidly
decapitated. The spinal cord was removed by hydraulic extrusion, and
the cervical and lumbar segments (2 cm long) were isolated and glued,
lateral side down, on a brass platform with cyanoacrylate cement, in a
chamber filled with oxygenated ice-cold sucrose ACSF. Parasagittal
400-µm-thick slices were cut, incubated in sucrose-ACSF at room
temperature (23-28°C) for 30 min, and transferred to normal ACSF for
at least one hour prior to electrophysiological recordings. Next, the
slices were transferred to a recording chamber under a Zeiss Axioscope
equipped with infrared differential interference contrast (IR-DIC) and
water immersion-objectives for visualization of neurons in thick live
tissue. The slices were perfused at ~2 ml/min with oxygenated ACSF
containing (in mM) 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, 26 NaHCO3, 1.25 NaH2PO4 (pH 7.35; 300-310
mOsm), and the glutamate receptor antagonists
6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 10 µM; Tocris Cookson)
and D
2-amino-5-phosphonovaleric acid (D-AP5, 40 µM; Tocris Cookson).
Drug application
Bicuculline methiodide (10-20 µM; RBI), strychnine
hydrochloride (100 nM 0.5 µM; RBI) and CGP52432
(([3-[[3,4-dichlorophenyl)methyl] amino]propyl] (diethoxymethyl)
phosphinic acid; 10-30 µM, Ciba-Geigy) were used to block
GABAARs, GlyRs, and
GABABRs, respectively. The glutamate receptor
antagonists CNQX and D-AP5 were used to isolate
monosynaptic IPSCs. The action potential blocker tetrodotoxin (TTX, 1 µM; RBI) was used to record miniature IPSCs and the benzodiazepine flunitrazepam (1 µM; Sigma) was used to potentiate
GABAARs and thus unmask the
GABAAR components of mIPSCs.
Whole cell recordings and data analysis
For whole cell voltage-clamp recordings of IPSCs, patch pipettes
were pulled from borosilicate glass capillaries (with an inner
filament, WPI) using a two-stage vertical puller (Narishige PP-83). To
record mIPSCs and evoked IPSCs, the pipettes were filled with an
intracellular solution composed of (in mM) 110 CsCl, 10 HEPES, 2 MgCl2, 2 mM ATP (Sigma), 0.4 mM GTP (Sigma), 11 mM BAPTA (Sigma), 1 mM CaCl2 and 0.5% Lucifer
yellow (Sigma). The pH was adjusted to 7.2 with CsOH and the osmolarity
ranged from 260 to 280 mOsm (pipette resistance 3 M). For the paired
pulse experiments, 110 mM CsCl was replaced with 110 mM Cs-gluconate
and 5 mM CsCl, and the membrane was held at 0 mV to avoid the
confounding effect of action potential generation. Recordings were
obtained by lowering the patch electrode onto the surface of visually
identified neurons in lamina I. While monitoring current responses to 5 mV pulses, a brief suction was applied to form >G
seals. An
Axopatch 200B amplifier (Axon Instruments) with >80% series
resistance compensation was used for the recording. The access
resistance was monitored throughout each experiment. Only recordings
with access resistance between 7-20 M
were considered acceptable
for analysis of evoked IPSCs and only recordings with stable access
throughout the entire administration of antagonists were used for
further analysis. Monosynaptic IPSCs were evoked by focal electrical
stimulation using a patch micropipette. Square-wave constant
paired-pulses (200-300 µs duration) were applied at a frequency of
<0.3 Hz, at different interstimulus intervals, ranging from 25 ms to
1 s. The electrode was placed within 20-50 µm of cell body of
lamina I neurons. For analysis of the data, traces were low-pass
filtered at 10 kHz and stored on a videotape, using a digital data
recorder (VR-10B, Instrutech). Offline, the recordings were low-pass
filtered at 2-3 kHz and sampled at 10-20 kHz on an Intel
Pentium-based computer and analyzed using software designed by Y. De
Koninck (Chéry and De Koninck 1999a
; De
Koninck and Mody 1994
).
Statistical analysis
Student t-tests were used to analyze the differences between the kinetic and amplitude parameters of the IPSCs. The critical value for statistical significance was set at P < 0.05. All the data are expressed as mean ± SE, unless otherwise indicated.
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RESULTS |
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Whole cell patch-clamp recordings from identified lamina I neurons
were performed using a previously described parasagittal spinal slice
preparation, which provides optimal conditions for systematic
identification of neurons in this layer (Chéry et al.,
2000). With this approach, we have shown that lamina I neurons receive exclusively GlyR-mediated mIPSCs (Chéry and De
Koninck 1999a
) although GABA coexists with glycine in
superficial dorsal horn neurons (Todd and Spike 1993
).
Failure to detect a GABAAR-mediated component to
mIPSCs in lamina I neurons was due to a subthreshold activation of
GABAARs (Chéry and De Koninck
1999a
). We confirmed that at these synapses both GlyRs and
GABAARs can be activated during individual mIPSCs
by adding flunitrazepam to enhance the sensitivity of
GABAARs. Figure 1
illustrates that, while under normal conditions, all mIPSCs are
antagonized by 100 nM strychnine, in the presence of flunitrazepam, an
additional slowly rising and slowly decaying
GABAAR-mediated component appeared in the large
majority of events. Following the application of flunitrazepam, >85%
of the mIPSCs had a dual kinetic with a very prolonged second component
(rise time, 4.1 ± 0.9 ms; decay, 52.8 ± 8.9 ms). Given that
mIPSCs represent the activation of postsynaptic receptors by single
vesicles of transmitter (Edwards et al. 1990
), these results indicate corelease of GABA and glycine from the same synaptic vesicles and thus from the same terminals. This evidence is consistent with previous reports indicating that GABA and glycine are taken up by
the same vesicular transporter (Burger et al. 1991
;
Chaudhry et al. 1998
; Dumoulin et al.
1999
) and with evidence at the motoneuron synapse that
stimulation of single inhibitory interneurons produces mixed GlyRs and
GABAAR-mediated IPSCs (Jonas et al.
1998
).
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While under certain conditions, the GABA coreleased with glycine may be
subthreshold to activation of GABAARs, it may
still be sufficient to activate another receptor subtype, namely
GABAB receptors. GABAB
autoreceptors are found predominantly in laminae I-II of the dorsal
horn (Bowery 1993) and may have a greater affinity for
the inhibitory transmitter than GABAARs. To test
this hypothesis, we sought to detect activation of
GABAB receptors under conditions where
GABAARs are not activated (i.e., conditions in
which inhibitory currents are mediated by GlyRs only). Figure
2 illustrates that IPSCs evoked by focal
stimuli at low intensity (<100 µA for 200 µs) in the vicinity of
identified lamina I neurons were completely blocked by strychnine (Fig.
2A; n = 12). In the presence of strychnine, GABAAR-mediated evoked IPSCs were only obtained
on increasing the stimulus intensity (Fig. 2B).
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Using such stimuli that resulted in pure GlyR-mediated IPSCs, we
studied the paired-pulse depression (PPD) of IPSCs evoked in lamina I
neurons. Paired-pulse depression is typically associated with
activation of presynaptic GABABRs (Davies
et al. 1990). A conditioning current and a test current were
applied focally at different interstimulus intervals (ISIs; see Fig.
3). The ISIs ranged from 25 ms to 1 s. When the ISI was shorter than the decay of the conditioning IPSCs,
an overlap in time of the conditioning and test currents was observed.
Thus a digital subtraction was used to obtain accurate values for the
peak of the test IPSCs, as previously described (Otis et al.
1993
). Figure 3 illustrates PPD of evoked IPSCs in a lamina I
neuron. The maximal depression of the test IPSCs (60 ± 14% of
the amplitude of the conditioning IPSC; P < 0.01) was
observed at 150-200 ms ISIs (n = 6).
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Up to 20 µM bicuculline failed to affect the amplitude of the conditioning pulse (Fig. 4B; nor the PPD ratio), indicating that IPSCs evoked by minimal stimuli do not involve activation of postsynaptic GABAARs. PPD of small evoked IPSCs was reversed following bath application of 10-30 µM CGP52432 (Fig. 4C), a specific GABAB receptors antagonist. This suggests that GABAB autoreceptors appear to be the first target of GABA released at inhibitory synapses in lamina I neurons. The evoked IPSCs were completely abolished by strychnine (Fig. 4B), confirming that they are selectively mediated by GlyRs.
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DISCUSSION |
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Our findings indicate that, while GABA and glycine appear to be released from the same synaptic terminals in lamina I, the amount of GABA released on activation of a few synaptic terminals may be subliminal to the activation of postsynaptic GABAA receptors, yet the released GABA may be sufficient to significantly activate presynaptic GABAB receptors.
The predominant localization of GABAB receptors
in superficial laminae of the spinal cord (Malcangio et al.
1993) and their preferential occurrence on synaptic terminals
in many CNS regions (Bowery 1993
) indicate that they may
have an important role in the modulation of GABA release in the dorsal horn.
Given the evidence of both GABA and glycine are contained in the same
terminals and most likely in the same synaptic vesicles (this study and
Burger et al. 1991; Chaudhry et al. 1998
;
Chéry and De Koninck 1999a
; Dumoulin et al.
1999
; Jonas et al. 1998
) and that small evoked
IPSCs were mediated exclusively by glycine provided an ideal setting to
test whether activation of GABAB receptors occurs
in conditions where GABAAR activation may not be
detectable, because the release event could be measured independent of
a GABAAR component using the GlyR-mediated event.
In this study we were able to show GABAB-mediated
PPD of GlyR IPSCs. Thus our results provide evidence that
GABAB autoreceptors are present on glycinergic
interneurons that also contain GABA (Todd and Spike 1993
), where they modulate the release of both inhibitory
transmitters from interneurons terminals. This evidence is consistent
with the recent demonstration of presynaptic inhibition of both GABA and glycine release at spinal interneuron-motoneuron synapses by the
GABABR agonist baclofen (Jonas et al.
1998
).
The released GABA is likely originating from the same terminal as the
released glycine because results from immunocytochemical studies
indicate that virtually all of the glycinergic neurons and terminals in
the superficial dorsal horn also contain GABA (Mitchell et al.
1993; Todd and Sullivan 1990
) and evidence
indicate that in these terminals, glycine and GABA are packaged in the same synaptic vesicles (Burger et al. 1991
;
Chéry and De Koninck 1999a
; Dumoulin et al.
1999
; Jonas et al. 1998
). It remains however that some GABAergic neurons do not contain glycine. Thus it is possible
that some of the GABA released may originate from separate terminals
from those releasing the glycine. In such case, our results indicate
that the amount of GABA released would still be subliminal to
activation of postsynaptic GABAARs leaving
GABAB receptors as the primary target of the
released GABA.
The PPD ratio reported here is in general agreement with previous
studies of PPD of GABA release in other regions of the CNS (Davies et al. 1990; Otis et al. 1993
).
Consistent with these results, maximal reductions of the test pulse
amplitude (up to 48% of the conditioning pulse) have been reported to
occur at ISIs between 100-200 ms.
Manipulating GABA release at lamina I inhibitory synapses may be an
important means to control excitability in this area and thus the relay
of nociceptive input to the brain. Interestingly, the results of
binding studies in dorsal horn slices and spinal cord synaptosomes
indicate that GABAB receptors on GABAergic
terminals may be distinct from the heteroreceptors present on
glutamate-releasing terminals (Bonanno et al. 1998;
Teoh et al. 1996
). Increasing the release of GABA may be
particularly useful to enhance inhibition in lamina I, given our recent
evidence that GABAARs are only activated under
conditions that promote spillover of GABA from synapses in lamina I
(Chéry and De Koninck 1999a
). Thus it may be
possible to selectively antagonize GABAB
autoreceptors to favor inhibitory transmission mediated by both GABA
and glycine in lamina I without significantly affecting
GABAB heteroreceptors (present on
glutamate-containing synaptic terminals). Selective targeting of
GABAB autoreceptors may prove particularly useful
for the treatment of chronic pain states (Henry 1982
).
The issue of whether distinct interneurons are responsible for
GABAA- and GABAB-mediated
inhibition has been discussed for quite some time and several lines of
evidence indicate that these two classes of receptors may be
differentially activated at some synapses (Benardo 1994;
Newberry and Nicoll 1984
; Nurse and Lacaille 1997
; Otis and Mody 1992
; Segal
1990
; Solis and Nicoll 1992
; Sugita et
al. 1992
). In cases of mixed GABAA and
GABAB responses, recruitment of few inhibitory
interneurons appear to often preferentially activate
GABAARs, while activation of
GABAB autoreceptors often requires recruitment of
a larger number of afferents as it more likely promotes GABA spillover
from synapses (Dutar and Nicoll 1988
; Isaacson et
al. 1993
; Nurse and Lacaille 1997
; Otis
and Mody 1992
; Ouardouz and Lacaille 1997
). The
converse scenario appears to apply to lamina I inhibitory synapses and
could be attributed to the fact that in this case
GABAARs, like GABABRs, are
likely located at a distance from the release site [i.e., a
preferential extrasynaptic distribution of
GABAARs (Chéry and De Koninck
1999a
)]. Differential subsynaptic distribution of
GABAARs may thus be an important determinant of
the pharmacology of GABAergic synapses. This is of particular interest
in light of evidence that such synaptic arrangement can be altered
under certain conditions. For example, we have observed a recruitment
of GABAARs to synaptic junctions at lamina I
synapses following peripheral nerve injury (Chéry and De
Koninck 1999b
).
Thus GABAergic inhibition appears to be modulated in a selective manner in lamina I, whereby GABAB autoreceptors may be the first target of GABA released in this spinal area. Such regulation of GABA release may have important physiological implications, notably under conditions that favor hyperexcitability in the dorsal horn.
In summary, these data indicate that under conditions where GABA release is insufficient to significantly affect postsynaptic GABAARs on lamina I neurons, it may rather serve to activate GABAB receptors to regulate the release of both glycine and GABA.
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ACKNOWLEDGMENTS |
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We thank Hoffman-La Roche for the generous donation of flunitrazepam and A. Constantin for expert technical assistance.
This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-34022 and by Canadian Medical Research Council (MRC) Grant MT 12942 to Y. De Koninck. Y. De Koninck is a scholar of the Canadian MRC. N. Chéry was the recipient of a Faculty of Medicine Graduate Award and an Eileen Peters McGill Major Fellowship.
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FOOTNOTES |
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Address for reprint requests: Y. De Koninck, Dept. of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, #1317, Montreal, Quebec H3G 1Y6, Canada (E-mail: ydk{at}SpinalCord.McGill.CA).
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 9 March 2000; accepted in final form 25 May 2000.
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REFERENCES |
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