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J Neurophysiol 94: 3893-3902, 2005. First published August 17, 2005; doi:10.1152/jn.00211.2005
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Nicotinic Acetylcholine Receptor Subtypes Involved in Facilitation of GABAergic Inhibition in Mouse Superficial Superior Colliculus

Toshiaki Endo1, Yuchio Yanagawa2,3,4, Kunihiko Obata5 and Tadashi Isa1,3,6

1Department of Developmental Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki; 2Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi; 3Core Research for Evolutional Science and Technology and 4Solution Oriented Research for Science and Technology of the Japan Science and Technology Corporation, Kawaguchi; 5Neuronal Circuit Mechanisms Research Group, RIKEN Brain Science Institute, Wako; and 6The Graduate University for Advanced Studies, Hayama, Kanagawa, Japan

Submitted 28 February 2005; accepted in final form 10 August 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The superficial superior colliculus (sSC) is a key station in the sensory processing related to visual salience. The sSC receives cholinergic projections from the parabigeminal nucleus, and previous studies have revealed the presence of several different nicotinic acetylcholine receptor (nAChR) subunits in the sSC. In this study, to clarify the role of the cholinergic inputs to the sSC, we examined current responses induced by ACh in GABAergic and non-GABAergic sSC neurons using in vitro slice preparations obtained from glutamate decarboxylase 67-green fluorescent protein (GFP) knock-in mice in which GFP is specifically expressed in GABAergic neurons. Brief air pressure application of acetylcholine (ACh) elicited nicotinic inward current responses in both GABAergic and non-GABAergic neurons. The inward current responses in the GABAergic neurons were highly sensitive to a selective antagonist for {alpha}3{beta}2- and {alpha}6{beta}2-containing receptors, {alpha}-conotoxin MII ({alpha}CtxMII). A subset of these neurons exhibited a faster {alpha}-bungarotoxin-sensitive inward current component, indicating the expression of {alpha}7-containing nAChRs. We also found that the activation of presynaptic nAChRs induced release of GABA, which elicited a burst of miniature inhibitory postsynaptic currents mediated by GABAA receptors in non-GABAergic neurons. This ACh-induced GABA release was mediated mainly by {alpha}CtxMII-sensitive nAChRs and resulted from the activation of voltage-dependent calcium channels. Morphological analysis revealed that recorded GFP-positive neurons are interneurons and GFP-negative neurons include projection neurons. These findings suggest that nAChRs are involved in the regulation of GABAergic inhibition and modulate visual processing in the sSC.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The superior colliculus (SC) is a key station in the extrageniculate visual pathway and involved in the sensory processing related to visual salience and control of orienting behaviors to novel visual stimuli (Dean et al. 1989Go; Sparks 1986Go; Wurtz and Albano 1980Go). The superficial layer of the SC (sSC) is exclusively concerned with visual processing. The sSC receives retinotopically organized projections from the retina and the visual cortex and project to the thalamus and the deeper layers of the SC. A prominent feature of the cytoarchitecture of the sSC is the high proportion of GABAergic neurons. The proportion of GABAergic neurons is estimated at ~45% of all neurons in the cat sSC (Mize 1988Go). It has been shown that GABAergic inhibition underlies fundamental visual response properties of the sSC neurons, such as surround inhibition and response habituation (Binns and Salt 1997Go). Therefore it is important to characterize the properties and the control mechanisms of the GABAergic circuit in the sSC.

The sSC projects to the ipsilateral parabigeminal nucleus (PBN), and the PBN sends dense cholinergic projections to the sSC bilaterally (Baizer et al. 1991Go; Feig and Harting 1992Go; Graybiel 1978Go; Hall et al. 1989Go; Jiang et al. 1996Go; Mufson et al. 1986Go; Sefton and Martin 1984Go; Sherk 1979bGo; Stevenson and Lund 1982Go; Watanabe and Kawana 1979Go). The PBN neurons respond to visual stimuli presented in the receptive field aligned in a retinotopic manner (Sherk 1978Go, 1979aGo,bGo). Based on the tight connection with the sSC, the PBN has been regarded as a satellite system of the SC (Graybiel 1978Go). Despite the close relationship with the SC, the functional roles of the parabigeminocollicular projection has not been studied extensively. It is well known that the nucleus isthmi, the non-mammalian homologue of the PBN, plays important roles in the modulation of visual receptive field properties of tectal cells and visual orientation behaviors (reviewed in Wang 2003Go). Two recent studies in rats investigated the effects of cholinergic agonists on the responses to visual stimuli and electrical stimulations of the optic fibers in the sSC (Binns and Salt 2000Go; Lee et al. 2001Go). These studies demonstrated that nicotinic agonists inhibited the responses of projection neurons through GABAB receptor-mediated inhibition, which would be achieved by an excitation of inhibitory interneurons. However, the functions of nicotinic receptors in individual sSC neurons are poorly understood.

In the mammalian brain, 11 neuronal nicotinic acetylcholine receptor (nAChR) subunits ({alpha}2-{alpha}7, {alpha}9-{alpha}10, {beta}2-{beta}4) have been cloned, and nAChRs with a specific subunit combination exhibit unique pharmacological and physiological properties (Gotti and Clementi 2004Go). In the sSC, the presence of multiple types nAChRs have been reported by a number of in situ hybridization, immunohistochemical, and receptor autoradiographic studies (Clarke et al. 1985Go; Cui et al. 2003Go; Dominguez del Toro et al. 1994Go; Prusky and Cynader 1988Go; Seguela et al. 1993Go; Swanson et al. 1987Go; Wada et al. 1989Go; Whiteaker et al. 2000Go, 2002Go). However, there have been no available physiological studies regarding the functions of nAChRs expressed in individual sSC neurons. In the present study, we examined current responses of the sSC neurons induced by an activation of nAChRs with special interest in the nicotinic control of GABAergic circuits. We took advantage of glutamate decarboxylase (GAD) 67-green fluorescent protein (GFP) knock-in mice (Tamamaki et al. 2003Go). In these mice, GABAergic neurons can be reliably identified with GFP fluorescence in slice preparations (Endo et al. 2003Go). Here we show that both GABAergic interneurons and non-GABAergic projection neurons express functional nAChRs. Furthermore, we found that presynaptic nAChRs would contribute to the regulation of GABAergic synaptic transmission mediated by GABAA receptors. We also investigated the possible subunit combinations of nAChRs expressed in the sSC.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present experiments were approved by the Animal Research Committee of the Okazaki National Research Institutes. All efforts were made to minimize both the suffering and number of animals used in this study.

Animals

The procedures for the generation and genotyping of GAD67-GFP knock-in mice are described elsewhere (Tamamaki et al. 2003Go). Mice heterozygous for the GAD67-GFP allele were mated with ICR or C57BL/6 wild-type mice to obtain the heterozygous mice. Sixteen- to 71-day-old heterozygous mice were used in the experiments.

Slice preparations

Frontal slices (250–300 µm thick) of the SC were prepared. The animals were killed with ether and decapitated. The depth of anesthesia was carefully confirmed by absence of reflexes to toe pinches. The brains were quickly removed and submerged in ice-cold modified Ringer solution for 5–10 min. The modified Ringer solution contained (in mM) 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 11 glucose and was bubbled with 95% O2-5% CO2 (pH 7.4). Then slices were cut with a Microslicer (DTK-2000, Dosaka EM, Kyoto, Japan) and incubated in standard Ringer solution at room temperature for >1 h before recording. The standard Ringer solution contained (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 25 glucose and was bubbled with 95% O2-5% CO2 (pH 7.4).

Recordings and analysis

Whole cell patch-clamp recordings were obtained from the neurons in the stratum zonale (SZ) and the stratum griseum superficiale (SGS) by visual control of patch pipettes. Slices were mounted in a recording chamber on an upright microscope (DM LFS, Leica, Germany or BX61WI, OLYMPUS, Tokyo, Japan) and continuously superfused with the standard Ringer solution. GFP-positive and -negative neurons were selected using fluorescent optics, and then a whole cell configuration was obtained using bright field optics. Patch pipettes were prepared from borosilicate glass capillaries and were filled with either of the following internal solutions. One contained (in mM) 160 K-gluconate, 2 MgCl2, 2 Na2ATP, 0.5 Na3GTP, 0.2 EGTA, 10 HEPES, and 0.1 spermine (pH 7.3). The other contained (in mM) 120 Cs-gluconate, 10 CsCl, 2 MgCl2, 4 Na2ATP, 10 EGTA, 10 HEPES and 0.1 spermine (pH was adjusted to 7.3 with CsOH). Using these solutions, the estimated equilibrium potential for Cl– (ECl) was –88 and –57 mV, respectively. To stain the recorded neurons, biocytin (5 mg/ml; Sigma, St. Louis, MO) was dissolved in the solutions. The resistance of the electrodes was 3–7 M{Omega} in the Ringer solutions. The actual membrane potentials were corrected by the liquid junction potential of –10 mV. When Cd2+ was added to the extracellular solution, the Ringer solution contained (in mM) 145 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose and was continuously bubbled with 100% O2 (pH 7.4). In this solution, the ECl was –92 mV for the K-gluconate intracellular solution. All recordings were performed at 30–34°C unless otherwise stated. Data were acquired by using an EPC9 patch-clamp amplifier and a software PULSE (Heka, Lambrecht, Germany).

In most of the experiments, acetylcholine chloride (ACh; 1 mM; Sigma) was applied with air pressure pulses (5–20 psi, 10- to 50-ms duration) through a micropipette identical to the patch pipette. The micropipette was placed within 50 µm of the recorded neurons. Tetrodotoxin (TTX) (0.25–1 µM; Sankyo, Tokyo, Japan) was routinely bath perfused. Depending on the requirement for each experiment, atropine(1 µM), bicuculline methobromide (5–10 µM), picrotoxin (50 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) and D-2-amino-5-phosphonovaleric acid (APV; 50 µM) (all from Sigma) were also bath perfused. To examine the effects of antagonists for nAChRs and GABA receptors, ACh was repeatedly applied at 40- to 60-s intervals. After control current response became stable, the antagonists were applied by bath perfusion for ≥10 min in which the effect reached maximum for the typical case. Antagonists for nAChRs were mecamylamine, {alpha}-bungarotoxin ({alpha}Btx), dihydro-{beta}-erythroidine (DH{beta}E) (all from Sigma), and {alpha}-conotoxin MII ({alpha}CtxMII) (Tocris Cookson, Ballwin, MO). Antagonists for GABA receptors were bicuculline methobromide, picrotoxin and 2-[3-carboxypropyl]-3-amino-6-[4-methoxyphenyl] pyridazinium bromide (SR-95531; all from Sigma).

To examine the effect of an activation of nicotinic receptors on miniature inhibitory postsynaptic currents (mIPSCs), 1,1-dimethyl-4-phenyl piperazinium iodide (DMPP; 1 µM; Sigma), a specific nAChR agonist, was bath applied for 30–60 s in the presence of TTX (0.5 µM), CNQX (10 µM), and APV (50 µM). When DMPP was applied repeatedly, each application was interspaced by ≥15 min. Data were analyzed with the Igor Pro program (WaveMetrics, Lake Oswego, OR). IPSCs were detected as events which have a rising slope and an amplitude above manually set thresholds. The thresholds were determined from traces which were apparently free from mIPSCs.

Mean values are given as the means ± SE. Statistical significance was examined with a two-tailed paired t-test or Kolmogorov-Smirnov test, and the difference was considered significant if P < 0.05.

Histological procedures

After recording, the slices were fixed with 4% paraformaldehyde in 0.12 M phosphate buffer (pH 7.4) for more than a day at 4°C. After fixation, biocytin-filled neurons were visualized by the ABC method. Details are described elsewhere (Isa et al. 1998Go).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Current responses induced by ACh in sSC neurons

We used GAD67-GFP knock-in mice throughout this study. The feasibility of our identification of GABAergic neurons with GFP fluorescence was confirmed in preceding studies (Endo et al. 2003Go; Tamamaki et al. 2003Go). In the first series of experiments, current responses to ACh were obtained from 18 GABAergic (GFP-positive) and 72 non-GABAergic (GFP-negative) neurons using K-gluconate intracellular solution. ACh (1 mM) was applied with a brief air pressure pulse (10–50 ms, 5–20 psi) to the somatodendritic region, and current responses were routinely recorded at a holding potential of –60 mV in the presence of TTX (0.25–1 µM). In recordings from 8 GFP-positive and 50 GFP-negative neurons, atropine (1 µM) was also dissolved in the bath solution to remove any possible contamination of muscarinic responses; however, no apparent difference was observed.

In general, current responses to ACh were divided into two different types. In 17 of 18 GFP-positive and 30 of 72 GFP-negative neurons, ACh elicited inward current responses at –60 mV (mean amplitude, 62.0 ± 5.7 pA; Fig. 1, A and B). In contrast, 41 GFP-negative neurons showed noisy outward current responses, which were composed of a barrage of postsynaptic current (PSC)-like events, at the same holding potential (Fig. 1C). Among these neurons, 25 neurons also showed inward current responses that preceded the outward current responses (e.g., Fig. 2B). In these 25 neurons, the inward responses were overcome by the outward responses and the net charge transfer was outward (mean net charge, 12.6 ± 2.2 pC). The inward and outward responses were suppressed by a broad-spectrum nAChR antagonist mecamylamine (10 µM) (n = 3 for inward only and 3 for inward and outward, Fig. 2), indicating that these responses were mediated by nAChRs. Some GFP-positive neurons also showed similar noisy currents around the peak and the decay phase of the inward current responses (data not shown). However, outward responses in GFP-positive neurons were inconspicuous, and the net charge transfer was outward in only two neurons (1.8 and 3.8 pC, respectively). One GFP-positive and one GFP-negative neuron did not show detectable responses.



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  		FIG. 1. Drawings of the superficial superior colliculus (sSC) neurons and their current responses induced by brief pressure applications of acetylcholine (ACh; 1 mM). A: green fluorescent protein (GFP)-positive neuron with inward current responses to ACh. The dorsal surface of the sSC is visible because the slice was cut obliquely to the surface; - - -, cutting edge of the slice. B: GFP-negative neuron with inward current responses. C: GFP-negative neuron with outward current responses. All recordings were obtained at the holding potential of –60 mV and in the presence of 0.25–0.5 µM TTX. {blacksquare}, the duration of the pressure pulse for ACh application.

 


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  		FIG. 2. The inward and outward current responses to ACh in the sSC neurons are dependent on the nAChRs. A: broad-spectrum nAChR antagonist, mecamylamine (Mec; 10 µM), inhibited the inward current response recorded in a GFP-positive neuron. B: outward current response that was preceded by an inward current response recorded in a GFP-negative neuron. Mecamylamine inhibited both responses.

 
Morphology of the recorded neurons

Figure 1 shows examples of the morphology of the recorded neurons and their current responses to ACh. Among all the recorded neurons, the somatodendritic morphology was recovered in 26 GFP-positive and 20 GFP-negative neurons. The morphological characteristics of GFP-positive neurons were described in a previous study from our laboratory (Endo et al. 2003Go). Most of the GFP-positive neurons had horizontally elongated dendritic field (200–600 µm diameter in the horizontal direction) and include horizontal cells described in the rat by Langer and Lund (1974)Go (Fig. 1A). The dendrites and the axons did not enter the intermediate layer, indicating that they are GABAergic interneurons. GFP-negative neurons showed heterogeneous morphological properties, including neurons with vertically or horizontally oriented dendrites, and neurons with stellate-like morphology. The horizontal extent of dendritic field of most GFP-negative neurons was much smaller than GFP-positive neurons (<200 µm). Some of these neurons resembled retrogradely labeled colliculothalamic projection neurons (Lee et al. 2001Go), corresponding to narrow field vertical cells in the rat sSC (Langer and Lund 1974Go) (Fig. 1, B and C). A part of the axon was stained in 12 GFP-positive neurons, and 4 of them reached stratum opticum and 2 reached the deep collicular layer. These results indicate that GFP-negative neurons recorded in this study include projection neurons of the sSC. Two GFP-negative neurons had long horizontal dendrites similar to GFP-positive neurons. It is not clear whether these neurons were judged in error due to low intensity of GFP fluorescence or represent another subclass of non-GABAergic neurons. We failed to find any apparent correlation between the morphology and the properties of ACh responses.

Activation of presynaptic nAChRs induces release of GABA

As shown in Fig. 3A, the outward current responses disappeared when the membrane potential was clamped at –90 mV, which was close to the Cl– equilibrium potential in our experimental conditions. The outward currents were completely abolished by a GABAA receptor antagonist bicuculline (10 µM; Fig. 3B). A similar effect was observed in all cells challenged with bicuculline (n = 3) or SR95531 (10 µM; n = 6, e.g., Fig. 4B4), indicating that the outward current responses induced by ACh were mediated by GABAA receptors. Because all experiments were performed in the presence of a voltage-dependent Na+ channel blocker TTX, we have concluded that the activation of the presynaptic nAChRs in the GABAergic synapses leads to the release of GABA, which in turn activates the GABAA receptors in the recorded neurons.



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  		FIG. 3. The outward current responses are mediated by GABAA receptors. A: current responses to ACh were recorded at different holding potentials in a GFP-negative neuron. Large outward current responses followed small inward currents. The outward responses disappeared at –90 mV, which was close to the equilibrium potential of Cl– (ECl = –88 mV). B: GABAA receptor antagonist, bicuculline (Bic; 10 µM), markedly reduced the outward current response, while a small inward peak remained intact. Recordings in A and B were obtained from the same neuron in the presence of 0.25 µM TTX.

 


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  		FIG. 4. The ACh-induced GABA release was dependent on extracellular calcium and the activation of voltage-dependent Ca2+ channels. A: outward current response completely disappeared in the Ca2+-free extracellular solution. B: extracellular Cd2+ inhibited the outward response without noticeable effects on the preceding inward peak (2). After recovery from the blockade by Cd2+ (3), a GABAA receptor antagonist, SR95531 (10 µM), was tested in the same cell. SR95531 inhibited the outward current and had no effects on the preceding inward current (4). All recordings were obtained in the presence of 1 µM atropine and 1 µM TTX.

 
We next investigated the mechanisms underlying the ACh-induced GABA release. The outward current responses to ACh were eliminated when Ca2+ was removed from the extracellular solution (n = 3, Fig. 4A), indicating that Ca2+ influx is necessary for the ACh-induced GABA release. Then we investigated whether the activation of voltage-dependent Ca2+ channels accounts for the Ca2+ influx. As exemplified in Fig. 4B, a voltage-dependent Ca2+ channel blocker Cd2+(100–200 µM), which does not block nAChRs (Gray et al. 1996Go; Khiroug et al. 1997Go; Kristufek et al. 1999Go; Rathouz and Berg 1994Go), suppressed the outward current responses in all neurons tested (n = 4). The reduction by Cd2+ was 89–102% of that by GABAA receptor antagonists applied after complete recovery from the blockade by Cd2+ (n = 3, measured as a current amplitude at the time point of the peak of the outward responses). Thus Cd2+ almost completely abolished the GABA release induced by ACh. These results suggest that the presynaptic nAChRs depolarize the presynaptic membrane and activate voltage-dependent Ca2+ channels to induce the GABA release.

To further confirm the presynaptic location of nAChRs, we examined effect of a nicotinic agonist DMPP on the frequency and the amplitude of GABAergic mIPSCs. In the presence of TTX (0.5 µM), CNQX (10 µM), and APV (50 µM), mIPSCs were recorded at room temperature (about 25°C) from 12 GFP-negative neurons. SR95531 (10 µM) completely blocked the mIPSCs (n = 3; Fig. 5B). Bath application of DMPP (1 µM) increased the frequency of mIPSCs 3.97 ± 0.52 times (range, 1.86–7.48) in 11 of 12 neurons (Fig. 5A), and the distribution of inter-mIPSC interval shifted significantly toward smaller value (Kolmogorov-Smirnov test, P < 0.005; Fig. 5C). The distribution of the amplitude did not change in most neurons (n = 9/11; Fig. 5C) except two neurons that showed significant increase (P < 0.05). Application of Cd2+ (200 µM) in the bath solution decreased the frequency and the amplitude of mIPSCs in the control condition (before DMPP application). In the presence of Cd2+, DMPP significantly increased the frequency in one of five neurons, decreased in one neuron, and had no effect on three neurons (Fig. 5D). The effect of DMPP on the frequency was reduced in all neurons tested (3.40 ± 0.59 times before Cd2+ application and 1.18 ± 0.32 times in the presence of Cd2+, n = 5). Thus DMPP virtually had no effect in the presence of Cd2+. These results verify that presynaptic nAChRs are involved in the GABA release through an activation of voltage-dependent Ca2+ channels.



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  		FIG. 5. 1,1-Dimethyl-4-phenyl piperazinium iodide (DMPP) increases the frequency of miniature inhibitory postsynaptic currents (mIPSCs) without altering the distribution of their amplitude. A: example of current recording from a GFP-negative neuron (top, middle), and the histogram of the number of mIPSCs (bottom) corresponding to the top. The middle traces are expanded traces of the top trace. The frequency of mIPSCs was increased during the application of DMPP (1 µM). The duration of DMPP application is indicated by the solid line above the top trace. B: Recording from the neuron in A in the presence of SR95531 (10 µM). SR95531 blocked mIPSCs completely, and DMPP did not induce any detectable responses. C: cumulative probability distribution of the amplitude (left) and the inter-mIPSC interval (right) derived from the recording in A. The plots were constructed from 50-s period before and 15 s during DMPP application. mIPSCs overlapping the decay of the preceding events were not included in the analysis of the amplitude. The difference of the distribution of the interval is statistically significant (Kolmogorov-Smirnov test, P < 0.0001), whereas the amplitude distribution is not (P > 0.9999). D: DMPP increased the frequency of mIPSCs under the control condition (1) but had no effect in the presence of 200 µM Cd2+ (2) in the same neuron. All recordings were obtained at +10 mV in the presence of 0.5 µM TTX, 10 µM CNQX, and 50 µM APV.

 
We also recorded the response to pressure applied ACh using intracellular solution which contain 20 mM BAPTA in nine neurons, and six neurons showed ACh-induced GABAergic currents (data not shown). Therefore it seems that Ca2+-dependent processes in the postsynaptic neurons do not involved in the GABA release.

Pharmacological properties of somatic nAChRs in GABAergic neurons

The above results suggest that nAChRs in the sSC are involved in the control of GABAergic inhibition by activating GABAergic neurons postsynaptically and modulating GABA release presynaptically. In the second series of experiments, we examined pharmacological properties of nAChRs in the sSC, focusing on the postsynaptic receptors in GABAergic neurons and presynaptic receptors that would induce the GABA release to non-GABAergic neurons. We used Cs-gluconate intracellular solution in these experiments. APV (50 µM), CNQX (10 µM), TTX (0.5–1 µM), and atropine (1 µM) were contained in the extracellular solution. For recordings of postsynaptic nicotinic currents in the GABAergic neurons, the GABAA receptor antagonist picrotoxin (50 µM) or bicuculline (5–10 µM) was also perfused.

We applied ACh (1 mM) to 62 GFP-positive neurons, and 59 neurons showed detectable postsynaptic current responses at a holding potential of –60 mV (mean amplitude, 32.5 ± 2.6 pA). First we examined the effects of {alpha}-bungarotoxin ({alpha}Btx), a specific antagonist for {alpha}7 nAChRs, on postsynaptic nAChRs in the GABAergic neurons. Figure 6 shows an example of the effects of {alpha}Btx. In this neuron, {alpha}Btx (50 nM) considerably suppressed the current responses to ACh, and the current component suppressed by {alpha}Btx showed a faster activation and deactivation than the remaining component. Nineteen of 30 systematically tested GFP-positive neurons had similar {alpha}Btx-sensitive and -resistant components to a varying degree. The amplitude of the {alpha}Btx-sensitive component was 20.1 ± 3.0 pA, and the relative peak amplitude to the {alpha}Btx-resistant component was 1.6 ± 0.4. In three neurons, the concentration of {alpha}Btx was switched from 50 to 200 nM, and a clear additional inhibition was not observed (83.1, 90.1, and 116.0% of responses in 50 nM {alpha}Btx), indicating that the antagonism by {alpha}Btx was maximum, and the remaining component was mediated by pharmacologically and kinetically different nAChRs from {alpha}Btx-sensitive component. Eleven of 30 GFP-positive neurons showed {alpha}Btx-resistant component only. These results indicate that most of the GABAergic neurons express non-{alpha}7 nAChRs, and a subset of these neurons express {alpha}7-containing receptors simultaneously.



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  		FIG. 6. {alpha}Btx inhibits the rapidly activating component of current responses to an activation of postsynaptic nAChRs in GFP-positive neurons. The inward current response induced by ACh (1 mM; 1) in a GFP-positive neuron was suppressed by 50 nM {alpha}Btx (2). Switching the concentration of {alpha}Btx to 200 nM (3) did not result in further inhibition. Subtraction (5) of the 50 nM {alpha}Btx trace (2) from the control trace (1) shows the faster current component inhibited by {alpha}Btx. The holding potential was –60 mV. Traces 1–4 are averages of 5 consecutive recordings.

 
To characterize the {alpha}Btx-resistant nicotinic currents, we examined effects of three other nAChR antagonists in the presence of 50 nM {alpha}Btx. A broad-spectrum nAChR antagonist mecamylamine (10 µM) almost completely suppressed the remaining currents (4.8 ± 2.2% of control, P < 0.01, n = 6; Fig. 7, A and D), ensuring that the {alpha}Btx-resistant component was mediated by nicotinic receptors. Low concentrations of {alpha}-conotoxin MII ({alpha}CtxMII), an {alpha}3{beta}2- and {alpha}6{beta}2-containing nAChR-specific antagonist (Cartier et al. 1996Go; Champtiaux et al. 2002Go; Dowell et al. 2003Go; Kuryatov et al. 2000Go), significantly inhibited the {alpha}Btx-resistant currents in a dose-dependent manner (10 nM, 49.5 ± 11.7% of control, P < 0.05, n = 6; 50 nM, 14.5 ± 4.5% of control, P < 0.01, n = 10; 200 nM, 12.4 ± 8.5% of control, P < 0.01, n = 6; Fig. 7, B and D). A relatively selective antagonist for {alpha}4{beta}2-containing nAChR, dihydro-{beta}-erythroidine (DH{beta}E), also inhibited the {alpha}Btx-resistant currents but was much less effective than {alpha}CtxMII (100 nM, 67.6 ± 3.1% of control, P < 0.01, n = 7; 1 µM, 52.6 ± 4.8% of control, P < 0.05, n = 6) (Fig. 7C and D). We could not find differences in the sensitivity to {alpha}CtxMII, DH{beta}E, and mecamylamine between neurons with and without the {alpha}Btx-sensitive component, although we did not systematically examine them.



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  		FIG. 7. Effects of nAChR antagonists on the {alpha}-bungarotoxin ({alpha}Btx)-resistant current component induced by an activation of postsynaptic nAChRs in GFP-positive neurons. A: mecamylamine (Mec; 10 µM) inhibited the ACh-induced current. B: {alpha}-conotoxin MII ({alpha}CtxMII; 50 nM) inhibited the ACh-induced current. C: dihydro-{beta}-erythroidine (DH{beta}E; 100 nM) slightly inhibited the ACh-induced current. D: summary of the effect of the nAChR antagonists on the currents induced by an activation of postsynaptic nAChRs in GFP-positive neurons. The concentrations of {alpha}CtxMII and DH{beta}E are indicated at the bottom of the panel in nanomole. Recordings in A–C were obtained from three different GFP-positive neurons in the presence of 50 nM {alpha}Btx and other drugs (see text). The holding potential was –60 mV. All Traces are averages of 5 consecutive recordings.

 
Pharmacological properties of presynaptic nAChRs in presynaptic GABAergic neurons

To examine the properties of presynaptic nAChRs underlying the ACh-induced GABA release, we recorded current responses to ACh from GFP-negative neurons in the presence of TTX (0.5–1 µM) and other drugs (see preceding text). Recordings were obtained at 0 mV, which was presumed to be the reversal potential of nAChR-mediated current responses. In these experiments, we evaluated the magnitude of the current responses by their charge transfer. We applied ACh (1 mM) to 60 neurons, and 46 showed clear outward current responses composed of bursts of miniature IPSCs (mean charge transfer, 115.1 ± 16.9 pC), which were completely abolished in the presence of 50 µM picrotoxin (n = 7; Fig. 8, A and G). Figure 8, B and C, shows examples of the effects of nAChR antagonists on the outward current responses induced by ACh. Application of {alpha}CtxMII (50 nM) considerably suppressed the ACh responses (range, 7.2–26.0% of control; mean, 15.1 ± 2.8% of control; P < 0.01; n = 7; Fig. 8, B and G), whereas DH{beta}E (100 nM) was less effective but clearly reduced the ACh responses in all neurons tested (range 50.9–86.9% of control; mean, 70.1 ± 5.2%; P < 0.01; n = 7; Fig. 8, C and G). In contrast, the effect of {alpha}Btx varied among cells. The response in the presence of {alpha}Btx (50 nM) ranged from 59.4 to 117.1% of control (mean, 91.3 ± 7.6% of control; P = 0.34; n = 8). To verify that the activation of {alpha}7 nAChRs can induce GABA release, we investigated whether choline could induce the release. Previous studies demonstrated that choline selectively activated {alpha}7 receptors (Alkondon et al. 1997Go). As exemplified in Fig. 8, E and F, application of choline (10 mM) induced a burst of mIPSCs in four of five neurons tested. The response to choline was abolished in the presence of 50 µM picrotoxin (n = 2; Fig. 8E) or 50 nM {alpha}Btx (n = 2; Fig. 8F). These results suggest that {alpha}7-containing receptors are also involved in the GABA release, however, non-{alpha}7 nAChRs play a predominant role in the GABA release.



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  		FIG. 8. Effects of nAChR antagonists on the ACh-induced outward current responses in GFP-negative neurons. A: outward current response was not observed in the presence of 50 µM picrotoxin (Ptx). B–D: {alpha}CtxMII (50 nM) considerably inhibited the outward current response (B), whereas 100 nM DH{beta}E (C) and 50 nM {alpha}Btx (D) only slightly inhibited the outward current. E and F: choline (10 mM) induced outward current responses. The outward current responses were abolished in the presence of Ptx (E) or {alpha}Btx (F). G: summary of the effect of the antagonists on the ACh-induced inhibitory postsynaptic currents (IPSCs) in GFP-negative neurons. Recordings in A–F were obtained from 5 different GFP-negative neurons in the presence of 0.5 µM TTX and other drugs (see text) at the holding potential of –0 mV.

 

   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Expression of functional nicotinic receptors in the sSC neurons

In the sSC, previous in situ hybridization studies have reported the presence of nAChR mRNAs including {alpha}3, {alpha}4, {alpha}7, {beta}2, and {beta}3 subunits (Cui et al. 2003Go; Seguela et al. 1993Go; Wada et al. 1989Go; Whiteaker et al. 2000Go). Receptor autographic studies and immunohistochemical studies have also demonstrated presence of multiple types of nAChRs (Clarke et al. 1985Go; Cui et al. 2003Go; Dominguez del Toro et al. 1994Go; Prusky and Cynader 1988Go; Swanson et al. 1987Go; Whiteaker et al. 2000Go, 2002Go). However, there has been no available physiological study regarding the functions and the subunit combinations of nAChRs expressed in the sSC neurons. In the present study, we were able to record current responses through nAChRs in the sSC neurons. Morphological analysis revealed that recorded GFP-negative neurons include projection neurons. On the other hand, the GFP-positive neurons are regarded as GABAergic interneurons. These results indicate that both GABAergic interneurons and non-GABAergic projection neurons express functional nAChRs in the mouse sSC.

We also found that at least two pharmacologically and kinetically different nAChR subtypes are expressed in the GABAergic neurons. The suppression of the faster current component by {alpha}Btx suggests the expression of {alpha}7-containing nAChRs in a subset of GABAergic neurons. In agreement with present results, presence of {alpha}7 mRNAs (Seguela et al. 1993Go), immunostaining (Dominguez del Toro et al. 1994Go), and {alpha}Btx binding sites (Clarke et al. 1985Go) are reported in the sSC. It should be pointed out that repetitive application of high concentration of ACh would desensitize nAChRs, especially {alpha}7-containing nAChRs significantly. Therefore we cannot exclude the possibility that the number of neurons with {alpha}Btx-sensitive responses is underestimated.

The current responses that remained in the presence of {alpha}Btx were considerably suppressed by low concentrations of {alpha}CtxMII, which is reported to be specific for {alpha}3{beta}2- and {alpha}6{beta}2-containing nAChRs (Cartier et al. 1996Go; Champtiaux et al. 2002Go; Dowell et al. 2003Go; Kuryatov et al. 2000Go). Autoradiographic studies demonstrated the presence of dense {alpha}CtxMII binding sites in the sSC and the optic tract (Champtiaux et al. 2002Go; Whiteaker et al. 2000Go). In {alpha}6 nAChR knock-out mice, whereas high-affinity {alpha}CtxMII binding sites were completely eliminated in the sSC, a smaller amount of low-affinity sites, which would correspond to {alpha}3{beta}2-containing receptors, still remained (Champtiaux et al. 2002Go). The expression of {alpha}6 mRNAs is restricted to the retina and the catecholamineregic nucleus (Champtiaux et al. 2002Go; Le Novere et al. 1996Go). On the other hand, {alpha}3 mRNAs are detected in the SC (Wada et al. 1989Go; Whiteaker et al. 2000Go). Although {alpha}CtxMII binding is not affected in {alpha}3 nAChR knock-out mice (Whiteaker et al. 2002Go), it seems conceivable that strong binding to {alpha}6{beta}2-containing receptors obscured the change in weak binding to {alpha}3{beta}2-containing receptors. Therefore {alpha}6 subunits would be located on the retinal axons, and {alpha}3{beta}2-containing receptors are the most likely candidate for the {alpha}CtxMII-sensitive current observed in the present study. DH{beta}E also inhibited the {alpha}Btx-resistant component although it was much less effective than {alpha}CtxMII. DH{beta}E is a relatively selective antagonist for {alpha}4{beta}2-containing receptors; however, {alpha}3{beta}2 receptors also show some sensitivity to DH{beta}E (Chavez-Noriega et al. 1997Go; Harvey and Luetje 1996Go). Taken together, we conclude that the {alpha}Btx-resistant current is mediated mainly by {alpha}3{beta}2-containing nAChRs.

Presynaptic facilitation of GABA release

One major finding of the present study is that ACh could induce the release of GABA, which elicits GABAA receptor-mediated current responses especially in the GFP-negative, non-GABAergic neurons in the sSC. The GABA release was induced in the presence of TTX, indicating that the ACh-induced GABA release is independent of voltage-gated Na+-channels and the conduction of sodium action potentials. In addition, a low concentration of DMPP increased the frequency of GABAergic mIPSCs without affecting their amplitude in the presence of TTX. These results indicate that nAChRs are expressed at presynaptic site of GABAergic synapses, and an activation of these receptors increases the release of GABA.

Application of ACh was not able to induce GABA release when extracellular Ca2+ was removed, indicating that the release is dependent on the Ca2+ influx from the extracellular space. The high Ca2+ permeability of nAChRs (Seguela et al. 1993Go; Vernino et al. 1992Go) raises the possibility that Ca2+ influx through nAChRs can cause a sufficient elevation of Ca2+ concentration in presynaptic terminals to induce a transmitter release. On the other hand, depolarization induced by the activation of nAChRs would lead to the activation of voltage-dependent Ca2+ channels and an increase of Ca2+ concentration in presynaptic terminals. Both mechanisms have been shown to work in different areas and preparations (e.g., Dolezal et al. 1996Go; Gray et al. 1996Go; Lena and Changeux 1997Go; Soliakov and Wonnacott 1996Go). In the present study, ACh did not induce the release of GABA, and DMPP had virtually no presynaptic effect in the presence of Cd2+. These results suggest that the presynaptic nAChRs and the release machinery are not directly coupled, and the activation of voltage-dependent Ca2+ channels is necessary to the presynaptic action of nicotinic receptors (Fig. 9). Commonly in the CNS nAChRs are located at the preterminal region rather than the presynaptic terminal (Vizi and Lendvai 1999Go). However, considering that the action of ACh agonists were observed in the presence of TTX, the present results suggest that nAChRs are located at the terminal region.



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  		FIG. 9. Schematic drawing of hypothetical circuit. GABAergic interneurons make dendrodendritic synaptic contact on non-GABAergic projection neurons. Cholinergic axons from PBN make synaptic contact on interneuron dendrites including presynaptic dendrites. These inputs activate {alpha}3{beta}2-containing and {alpha}7-containing nAChRs, and depolarize dendritic membrane. This depolarization results in Ca2+ influx through voltage-dependent Ca2+ channels, then induce GABA release from the presynaptic dendrites, which in turn activate GABAA and GABAB receptors on projection neurons. Thus PBN afferents inhibit projection neurons. The PBN afferent axons also terminate on the projection neurons and facilitate the activity of these neurons through nAChRs.

 
The ACh-induced GABA release showed high sensitivity to {alpha}CtxMII and much lower sensitivity to {alpha}Btx and DH{beta}E. On the other hand, application of choline was able to induce GABA release. These results suggest that {alpha}3{beta}2- or {alpha}6{beta}2-containing nAChRs predominantly contribute to the GABA release, and {alpha}7-containing receptors are also involved to some extent. The nicotinic current recorded in GFP-positive neurons showed similar pharmacological profiles to the ACh-induced GABA release, suggesting that GABAergic interneurons in the sSC have the same receptor subtypes as those responsible for the GABA release. Therefore considering the abundance of GABAergic interneurons in the sSC, it seems reasonable that the GABA release resulted from the activation of nAChRs located at the presynaptic site of GABAergic interneurons in the sSC.

Previous studies in carnivores and primates have shown that major targets of choline acetyltransferase immunoreactive synaptic endings and parabigeminocollicular axon terminals are dendrites including presumptive GABAergic presynaptic dendrites (PSDs) (Feig et al. 1992Go; Hall et al. 1989Go; Henderson 1989Go; Jeon et al. 1993Go). These observations raise a possibility that the cholinergic inputs directly modulate dendrodendritic inhibitory transmissions through nAChRs expressed at the PSDs (Feig et al. 1992Go) (Fig. 9). This is supported by the present results that show similarities in pharmacological characteristics between the somatodendritic receptors in the GABAergic neurons and the presynaptic receptors responsible for the GABA release. However, besides interneurons, the sSC receives GABAergic projections from the pretectum and the ventral lateral geniculate nucleus (Appell and Behan 1990Go; Nunes Cardozo et al. 1994Go), and we cannot role out the possibility that ACh activated nAChRs on these GABAergic terminals. Precise ultrastructural localization of nAChR subunits might provide an important clue as to the target of the presynaptic modulation of GABAergic transmission by nAChRs.

Functional considerations

Recent in vivo and in vitro studies have demonstrated that the application of nicotinic agonists reduced the responses of sSC neurons to visual stimuli (Binns and Salt 2000Go) and optic fiber stimulations (Lee et al. 2001Go). These inhibitory effects were reversed by GABAB receptor antagonists but not by a GABAA receptor antagonist bicuculline. Because nAChRs in the sSC have been associated with retinal axons (Prusky and Cynader 1988Go; Swanson et al. 1987Go), these authors proposed that presynaptic facilitation of retinal inputs to the GABAergic interneurons might lead to a reduction of the activity of the projection neurons through GABAB receptor-mediated inhibition. The present results showed that the functional nAChRs are expressed in GABAergic and non-GABAergic sSC neurons. Furthermore, the present results suggest that nAChRs are involved in the modulation of GABAA receptor-mediated inhibition. Although we did not make a special attempt, obvious GABAB receptor-like responses were not observed in our experiments. A possible explanation is that the excitation caused by ACh might be too weak to induce GABAB receptor-mediated responses. Postsynaptic GABAB receptor-mediated responses require a strong excitation of afferent fibers that would be achieved by sodium action potentials (e.g., Dutar and Nicoll 1988Go; Saitoh et al. 2004Go). In our experiments, TTX was routinely contained in the bath solution. Application of ACh did not induce sodium action potentials and, consequently, did not induce sufficient amount of GABA release. In any case, nicotinic receptors participate in the regulation of the functions of the sSC in multiple ways.

The principle source of cholinergic inputs to the sSC is the PBN (Hall et al. 1989Go; Mufson et al. 1986Go). The PBN receives inputs mainly from the ipsilateral sSC and sends outputs back to the sSC bilaterally, and the mutual connection is retinotopically organized (Baizer et al. 1991Go; Feig and Harting 1992Go; Graybiel 1978Go; Hall et al. 1989Go; Jiang et al. 1996Go; Mufson et al. 1986Go; Sefton and Martin 1984Go; Sherk 1979bGo; Stevenson and Lund 1982Go; Watanabe and Kawana 1979Go). The functions of the nucleus isthmi, the non-mammalian homologue of the PBN, have been studied extensively. Unilateral ablation of the nucleus isthmi results in a scotoma in the contralateral monocular field (Caine and Gruberg 1985Go; Gruberg et al. 1991Go; Weber et al. 1996Go). The isthmic input exerts both excitatory and inhibitory influences on tectal neurons (George et al. 1999Go; Wang and Matsumoto 1990Go) and modulates the receptive field properties of tectal neurons (Wang et al. 2000Go). The positive and negative feedback loops are thought to compose a winner-take-all network (Wang 2003Go). Although the functions of the projections from the PBN are not clear, the previous (Binns and Salt 2000Go; Lee et al. 2001Go) and the present results suggest that the cholinergic projection is involved in the regulation of inhibitory circuits, which underlie fundamental properties of the sSC neurons (Binns and Salt 1997Go). Moreover, the present results showed that ACh elicited inward current responses that would depolarize the projection neurons, suggesting that the cholinergic inputs do not simply inhibit the activity of the projection neurons. Rather such a dual action could enhance the contrast of the visual images and the signal-to-noise ratio of the network activity or provide a substrate of a winner-take-all network similar to the isthmo-tectal network. However, many more physiological and anatomical studies will be necessary to elucidate the function of the PBN-SC network.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan Grants 15700310 to T. Endo, 15500220 and 16015315 to Y. Yanagawa, and 13041057 and 13854029 to T. Isa.


   FOOTNOTES
 
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.

Address for reprint requests and other correspondence: T. Endo, Dept. of Developmental Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan (E-mail: tendo{at}nips.ac.jp)


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