GABAA and GABAC Receptors Have Contrasting Effects on Excitability in Superior Colliculus

Michael Pasternack,1 Mathias Boller,2 Belinda Pau,2 and Matthias Schmidt2

 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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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.


    INTRODUCTION
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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 rho -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 rho -subunits to a separate receptor group: rho -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 rho -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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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-rho 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 MOmega , 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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Immunocytochemistry

Staining with rho -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 rho -subunits at GABAC receptor-rich synaptic sites (Enz et al. 1996; Koulen et al. 1998).



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Fig. 1. A: line drawing of a frontal section through the rat midbrain. The box indicates the area shown at higher power in the fluorescence micrograph in B. B: immunocytochemical localization of GABAC receptor rho -subunits in the rat superior colliculus (SC). The highest density of rho -subunits was found in the superficial gray layer (SGL) of the SC, whereas deeper SC layers remained unlabeled. C: throughout SGL, numerous puncta were found that frequently surrounded unstained putative cell bodies (stars). Scale bar, 2 mm in A, 0.5 mm in B, and 25 µm in C. CG, central gray; MGB, medial geniculate body; SN, substantia nigra.

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 alpha -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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Fig. 2. Characterization of SC SGL field potential components. A: position of stimulation and recording electrodes shown in a representative SC frontal section micrograph (left) and reconstruction (right). B: field potentials under control condition and in the presence of lidocaine, CoCl2, or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Whereas lidocaine strongly decreased both pre- (open arrow) and postsynaptic (filled arrow) components, CNQX and CoCl2 had depressive effects mainly on the postsynaptic component.

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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Fig. 3. Differential effect of GABAA and GABAC receptor activation on SGL field potentials. A: bath application of 200 µM GABA increased field potential (FP) amplitude, whereas application of 5 mM GABA strongly decreased it. Only the increase was blocked by coapplication of 50 µM (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA). Bottom panel: FP peak amplitudes (in % of control value) vs. time. Black bars indicate presence of drugs in the bath. Top panel: averaged FPs before and during drug applications taken at times indicated by numbers in the bottom panel. B: application of muscimol at 1 µM, where it specifically activates GABAC receptors, strongly increased the FP amplitude. In the presence of 50 µM TPMPA, the enhancement induced by muscimol was completely blocked. C: similarly, application of the specific GABAC receptor agonist cis-aminocrotonic acid (CACA) increased SC field potentials, this effect again was completely blocked by coapplication of 50 µM TPMPA. D: in contrast to that, the FP amplitude decrease induced by application of 10 µM muscimol was unaffected by coapplication of TPMPA. Because TPMPA could act as a weak GABAB agonist at the concentration used, data in all experiments shown were obtained in the presence of the GABAB receptor antagonist CGP 35348. All FP plots are averages of 10 consecutive records. Scale bars for FPs indicate 0.2 mV and 1 ms.


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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 rho -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 rho -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.


    ACKNOWLEDGMENTS

This research was supported by the Deutsche Forschungsgemeinschaft (Schm 734/4-1), the Deutscher Akademischer Austauschdienst (313/SF-PPP), and the Academy of Finland.


    FOOTNOTES

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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