1Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland; and 2General Zoology and Neurobiology, Ruhr-University Bochum, D-44780 Bochum, Germany
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ABSTRACT |
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Pasternack, Michael, Mathias Boller, Belinda Pau, and Matthias Schmidt. GABAA and GABAC Receptors Have Contrasting Effects on Excitability in Superior Colliculus. J. Neurophysiol. 82: 2020-2023, 1999. We have recently found that GABAC receptor subunit transcripts are expressed in the superficial layers of rat superior colliculus (SC). In the present study we used immunocytochemistry to demonstrate the presence of GABAC receptors in rat SC at protein level. We also investigated in acute rat brain slices the effect of GABAA and GABAC receptor agonists and antagonists on stimulus-evoked extracellular field potentials in SC. Electrical stimulation of the SC optic layer induced a biphasic, early and late, potential in the adjacent superficial layer. The late component was completely inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione or CoCl2, indicating that it was generated by postsynaptic activation. Muscimol, a potent GABAA and GABAC receptor agonist, strongly attenuated this postsynaptic potential at concentrations >10 µM. In contrast, the GABAC receptor agonist cis-aminocrotonic acid, as well as muscimol at lower concentrations (0.1-1 µM) increased the postsynaptic potential. This increase was blocked by (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid, a novel competitive antagonist of GABAC receptors. Our findings demonstrate the presence of functional GABAC receptors in SC and suggest a disinhibitory role of these receptors in SC neuronal circuitry.
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INTRODUCTION |
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GABAC receptors are a novel
class of ionotropic GABA receptors with distinct pharmacological and
biophysical properties (Feigenspan et al. 1993;
Lukasiewicz 1996
). In particular,
GABAC receptors show high affinity for GABA and
muscimol and are specifically activated by the GABA analogue
cis-aminocrotonic acid (CACA) (Feigenspan and Bormann
1994
; Johnston et al. 1975
; Shimada et
al. 1992
). GABAC receptors are not
blocked by the GABAA receptor antagonist bicuculline and are insensitive to benzodiazepines and barbiturates, which modulate GABAA receptors (Feigenspan
and Bormann 1994
; Shimada et al. 1992
). Recently
a specific competitive inhibitor of GABAC receptors, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), has been described (Ragozzino et al. 1996
).
The characteristics of GABAC receptors are
faithfully reproduced in heterologous expression studies by GABA
receptor -subunits cloned from the retina of a number of species
(Cutting et al. 1991
, 1992
;
Ogurusu et al. 1995
; Ogurusu and Shingai
1996
; Wegelius et al. 1996
; Zhang et al.
1995
). In addition to functional distinctions mentioned above,
recent molecular evidence strengthens the classification of
-subunits to a separate receptor group:
-subunits do not combine
(Hackam et al. 1998
) nor co-localize (Koulen et
al. 1998
) with GABAA receptor subunits.
Finally, GABAC but not
GABAA receptors are specifically linked to the
cytoskeleton by MAP-1B protein (Hanley et al. 1999
).
Although the pharmacological and functional properties of
heterologously expressed GABAC receptors have
been studied in detail, not much is known about their synaptic
function. Bicuculline-insensitive GABA responses have been reported
earlier in frog tectal neurons (Nistri and Sivilotti
1985) and in guinea pig superior colliculus (SC)
(Arakawa and Okada 1988
; Platt and Withington
1998
). In view of recent findings demonstrating
-subunit
expression in rat SC (Boué-Grabot et al. 1998
;
Wegelius et al. 1998
), it is conceivable that these
bicuculline-resistant GABA responses could be mediated by functional
GABAC receptors, particularly because they were achieved with agonist concentrations that could selectively activate the high-affinity GABAC receptors (Arakawa
and Okada 1988
). The aim of the present study was to
demonstrate the presence of functional GABAC
receptors in superficial gray layer (SGL) of rat SC, and to investigate
the functional role of these receptors.
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METHODS |
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Long-Evans rats (6-8 wk old, 200-350 g) were anesthetized with halothane (2-4% in room air) and ketamine (100 mg/kg body wt) and transcardially perfused with ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 MgSO4, 2 CaCl2, and 10 glucose, continuously gassed with 5% CO2-95% O2. After decapitation, 400-µm-thick transverse slices of SC were made using either a disk slicer (SPI, Oppenheim, Germany) or a vibratome (Rhema, Hofheim, Germany). The slices were allowed to recover in ACSF at room temperature for at least 1 h before use.
Immunocytochemistry
The slices were washed briefly in 0.1 M phosphate buffer (PB; pH
7.4), fixed in 4% paraformaldehyde in 0.1 M PB for 20 min, cryoprotected in PB with 30% sucrose, and cryosectioned in 30-µm sections. The sections were incubated overnight with an affinity purified rabbit polyclonal anti- antibody (1:100 in PB), kindly donated by H. Wässle, Max-Planck Institut für
Hirnforschung, Frankfurt, Germany (see also Enz et al.
1996
). Following washes, the sections were incubated in
secondary antibody (Cy3-conjugated donkey anti-rabbit, DAKO, Hamburg,
Germany) for 2 h, and photomicrographs were taken on a photo
microscope (Axiophot, Zeiss, Germany) using 400 ASA black and white
film (Kodak T-Max 400).
Electrophysiology
For electrophysiological recordings, slices were placed in a
submerged-type slice chamber and continuously superfused with ACSF at
3.5 ml/min. Extracellular field potentials (FPs) were evoked by
electrical stimulation (1-2 mA, 25 µs, 0.1 Hz) with a concentric
bipolar stimulation electrode (SNEX-100X, Rhodes, Woodland Hills,
CA) placed in the optic layer. FP were recorded from SGL using
glass microelectrodes with a tip resistance of 1-2 M, when filled
with 165 mM NaCl. FPs were amplified, low-pass filtered at 3 kHz, and
digitized at 40 kHz (1401 laboratory interface, CED, Cambridge, UK).
The peak amplitude of the postsynaptic FP component was analyzed
off-line using commercial software (Spike2, CED, UK). GABA, muscimol,
lidocaine (Sigma Aldrich, Germany), CACA, TPMPA, and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Cookson, Bristol,
UK) were delivered via bath perfusion.
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RESULTS |
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Immunocytochemistry
Staining with -subunit antibody revealed a punctate expression
pattern, which was stronger in the SGL than in other SC layers, whereas
deeper SC layers remained unlabeled (Fig.
1B). Throughout SGL, numerous
puncta were found that frequently surrounded unstained putative cell
bodies (Fig. 1C). As has been demonstrated for the retina,
these immunopositive puncta most likely represent high-density clusters
of
-subunits at GABAC receptor-rich synaptic
sites (Enz et al. 1996
; Koulen et al.
1998
).
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Electrophysiology
Extracellular FPs evoked in SGL by optic layer stimulation (Fig.
2A) consisted of an early
phase (delay 1.1 ms) and a late phase (delay 2.1 ms). Both early and
late phases were reversibly abolished by 200 µM lidocaine (Fig.
2B). The late phase was abolished by blockade of
voltage-activated calcium channels when cobalt (1 mM
CoCl2) was added to the ACSF, and by the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor
antagonist CNQX (40 µM; Fig. 2C), indicating that it was
generated by postsynaptic activation (Fig. 1B). The presynaptic potential showed great variability in amplitude between experiments, whereas the postsynaptic potential was fairly constant with a peak amplitude of 0.3-0.5 mV under control conditions.
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We observed a dual effect of GABA on FP amplitudes (Fig.
3A): at low concentrations
(<500 µM) GABA induced an increase in FP amplitude by 26 ± 15% (mean ± SD; n = 7), whereas at higher concentrations (1 mM) GABA reduced the amplitude by 55 ± 18% (n = 6). The amplitude increase observed at low GABA
concentrations could be mediated by GABAC
receptors because GABAC receptors have higher
affinity to GABA and muscimol than GABAA
receptors (Bormann and Feigenspan 1995
). In line with
this, the GABAC receptor antagonist TPMPA only
blocked the effect of low GABA concentrations but had no effect on the
amplitude decrease induced by higher GABA concentrations (Fig.
3A). To exclude a possible activation of
GABAB receptors by GABA or TPMPA
(Ragozzino et al. 1996
), we used low concentrations of
muscimol (<1 µM) or CACA (10-20 µM) in the presence of the specific GABAB receptor antagonist CGP 35348 (kindly provided by Novartis AG, Basel, CH) to selectively activate
GABAC receptors. As shown in Fig. 3, B
and C, the increase in FP amplitude induced by a low
concentration of muscimol or by CACA was completely blocked by TPMPA,
strengthening the conclusion that these excitatory effects were
mediated by GABAC receptors. As expected, higher
concentrations of muscimol (10-100 µM) decreased FP amplitudes in a
TPMPA-insensitive manner (Fig. 3D).
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DISCUSSION |
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Our findings clearly demonstrate the presence of functional
GABAC receptors in SGL of rat SC, and that
GABAA and GABAC receptors have contrasting roles in the modulation of excitability in SC. First,
GABAC receptor -subunits are present at high
density in the SGL, and the clustered appearance of the
immunoreactivity suggests that GABAC receptors
are localized at synaptic sites (Enz et al. 1996
;
Koulen et al. 1998
). Second, the
GABAC receptor agonist CACA specifically
facilitated FP amplitudes, and the GABAC receptor
antagonist TPMPA blocked this enhancement. Finally, GABA and muscimol
mimicked the CACA effect only at low concentrations, when they
primarily, if not exclusively, act on GABAC
receptors, but they strongly decreased FP amplitudes at high
concentrations, when they also activate GABAA
receptors. As expected, TPMPA blocked the presumably
GABAC receptor-mediated facilitatory effects of GABA and muscimol at low concentrations but not the presumably GABAA receptor-mediated inhibitory effects of
agonists at higher concentrations.
The present observations on the functional role of
GABAC receptors in SC differ from what has been
shown in retina, where GABAC receptors inhibit
transmitter release from bipolar cells, like
GABAA receptors (Koulen et al.
1998), only with different temporal properties
(Lukasiewicz and Shields 1998
). Increased excitability,
on the contrary, has also been reported in guinea pig SC where GABA
application can induce a form of LTP. Because this LTP induction is
insensitive to bicuculline and baclofen, a contribution of
GABAC receptors has been assumed (Platt
and Withington 1998
). The increase in excitability induced by
GABAC receptor activation in SC could either
arise from direct neuronal excitation or from presynaptic
disinhibition. Because the ionic selectivities of
GABAA and GABAC receptors
are similar (Bormann and Feigenspan 1995
), the
electrochemical gradient of chloride or bicarbonate would have to
differ in cells possessing GABAC receptors to
explain a GABAC receptor-mediated direct
excitation. Because there is no evidence for this, we propose that the
FP amplitude increases following GABAC receptor
activation could be mediated by an indirect pathway. The functional
presence of GABAC receptors is supported by the
localization of both
-subunit mRNA (Boué-Grabot et al.
1998
; Wegelius et al. 1998
) and protein in the
SGL, which suggests that the receptors are expressed in locally
sprouting cells, like interneurons. If GABAC
receptors are mainly, or exclusively, expressed in GABAergic SGL
interneurons, GABAC receptor activation would
decrease their activity, leading to disinhibition and increased FP
amplitudes. About 40-55% of SC neurons are GABAergic, and the density
of GABAergic neurons is highest in the superficial layers (Mize
1992
). Because the SC receives GABAergic input from several
sources, including the contralateral SC and pretectum (Appell
and Behan 1990
; Nunes Cardozo et al. 1994
), the
neuronal circuitry necessary for the proposed disinhibitory mechanism
is well established. Intracellular recordings combined with
immunocytochemical identification of the cells involved would be needed
to identify the synaptic circuitry underlying the
GABAC receptor-mediated mechanisms in SC.
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ACKNOWLEDGMENTS |
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This research was supported by the Deutsche Forschungsgemeinschaft (Schm 734/4-1), the Deutscher Akademischer Austauschdienst (313/SF-PPP), and the Academy of Finland.
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FOOTNOTES |
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Address for reprint requests: M. Schmidt, Allgemeine Zoologie and Neurobiologie, Ruhr-Universität Bochum, ND 7/74, D-44780 Bochum, Germany.
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 March 1999; accepted in final form 2 June 1999.
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REFERENCES |
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